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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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Articles by Hermann Lage in JoVE

 JoVE General

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi


JoVE 2099 8/18/2010

,

Institute of Pathology, Charité Campus Mitte

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

Other articles by Hermann Lage on PubMed

Identification of Differentially Expressed Genes in Classical and Atypical Multidrug-resistant Gastric Carcinoma Cells

Anticancer Research. Nov-Dec, 2002  |  Pubmed ID: 12530067

The phenomenon of multidrug resistance (MDR) in human cancers is the major cause of failure of chemotherapy. To better understand the molecular events associated with the development of different types of MDR, a classical MDR P-glycoprotein expressing gastric carcinoma cell line and an atypical MDR P-glycoprotein-negative variant were analyzed by cDNA array hybridization. Of 588 cDNAs spotted on the array, a total of 9 genes showed differences in mRNA expressions. An enhanced expression level of 3 genes could be detected in both different MDR models (HLH, IR21, CCT5, Tx P-1), 4 genes were overexpressed solely in classical MDR cells (Hsp27, Rcl, aldehyde dehydrogenase 1, vimentin), whereas the expression of one gene was increased in atypical MDR cells (ProTa). Furthermore, the mRNA expressions of 3 genes were down-regulated in atypical MDR cells (Hsp27, vimentin, JNK2), whereas a decrease in mRNA expression in classical MDR cells could be determined for none of them. These differences were confirmed by a semiquantitative RT-PCR approach. Further characterization of these factors may provide more insight into the biology and development of different types of MDR in human gastric carcinomas. Moreover, these genes may be potential candidate factors for the diagnostics and/or prognosis of clinical drug resistance.

YB-1 Relocates to the Nucleus in Adenovirus-infected Cells and Facilitates Viral Replication by Inducing E2 Gene Expression Through the E2 Late Promoter

The Journal of Biological Chemistry. Mar, 2002  |  Pubmed ID: 11788582

The adenovirus early proteins E1A and E1B-55kDa are key regulators of viral DNA replication, and it was thought that targeting of p53 by E1B-55kDa is essential for this process. Here we have identified a previously unrecognized function of E1B for adenovirus replication. We found that E1B-55kDa is involved in targeting the transcription factor YB-1 to the nuclei of adenovirus type 5-infected cells where it is associated with viral inclusion bodies believed to be sites of viral transcription and replication. We show that YB-1 facilitates E2 gene expression through the E2 late promoter thus controlling E2 gene activity at later stages of infection. The role of YB-1 for adenovirus replication was demonstrated with an E1-minus adenovirus vector containing a YB-1 transgene. In infected cells, AdYB-1 efficiently replicated and produced infectious progeny particles. Thus, adenovirus E1B-55kDa protein and the host cell factor YB-1 act jointly to facilitate adenovirus replication in the late phase of infection.

Impact of BCRP/MXR, MRP1 and MDR1/P-Glycoprotein on Thermoresistant Variants of Atypical and Classical Multidrug Resistant Cancer Cells

International Journal of Cancer. Journal International Du Cancer. Feb, 2002  |  Pubmed ID: 11857350

The impact of the ABC transporters breast cancer resistance protein/mitoxantrone resistance associated transporter (BCRP/MXR), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance gene-1/P-glycoprotein (MDR1/PGP) on the multidrug resistance (MDR) phenotype in chemoresistance and thermoresistance was investigated in the parental human gastric carcinoma cell line EPG85-257P, the atypical MDR subline EPG85-257RNOV, the classical MDR subline EPG85-257RDB and their thermoresistant counterparts EPG85-257P-TR, EPG85-257RNOV-TR and EPG85-257RDB-TR. Within the atypical MDR subline EPG85-257RNOV expression of BCRP/MXR and of MRP1 were clearly enhanced (vs. parental and classical MDR lines). MDR1/PGP expression was distinctly elevated in the classical MDR subline EPG85-257RDB (vs. parental and atypical MDR sublines). In all thermoresistant counterparts basal expression of BCRP/MXR, MRP1 and MDR1/PGP was increased relative to thermosensitive sublines. Although it could be shown that the overexpressed ABC transporters were functionally active, however, no decreased drug accumulations of doxorubicin, mitoxantrone and rhodamine 123 were observed. Thus, expression of BCRP/MXR, MRP1 and MDR1/PGP was found to be dependent on the appropriate type of chemoresistance; correlating with a classical or atypical MDR phenotype. Within the thermoresistant variants, however, the increase in ABC transporter expression did obviously not influence the MDR phenotype.

Study of Therapy Resistance in Cancer Cells with Functional Proteome Analysis

Clinical Chemistry and Laboratory Medicine : CCLM / FESCC. Mar, 2002  |  Pubmed ID: 12005211

Different types of cancer are naturally resistant to many anticancer drugs. Additionally, these tumours develop acquired drug resistance, which includes the classical multidrug resistance (MDR) accompanied by the synthesis of P-glycoprotein, a member of the superfamily of ATP-binding cassette (ABC) transporters. Furthermore, atypical MDR is mediated by several different, some unknown, mechanisms. To overcome chemoresistance problems, antineoplastic drugs are often combined with other modes of therapy, e.g. hyperthermia, where good response has been reported in several experimental tumour models and in advanced cancer patients. The success of this combined anticancer treatment may be limited by an increase in chemoresistance and thermoresistance. A model system to study resistance phenomena is the use of chemoresistant and thermoresistant cancer cell lines. We have established chemoresistant cancer cell lines (gastric and pancreatic carcinoma, fibrosarcoma, melanoma) and now thermoresistant cell lines derived from gastric and pancreatic carcinoma cells and their counterparts that were resistant towards daunorubicin (classical MDR) and mitoxantrone (atypical MDR). Using proteomics, in this paper we evaluate the drug resistance of chemoresistant melanoma cells (parental cell line MeWo and sublines exhibiting drug resistance towards etoposide, cisplatin, fotemustine and vindesine) as a paradigm for analysis of drug resistance phenomena. Additionally, we investigate heat resistance and the interaction of chemoresistance and thermoresistance to identify common pathways using the parental and drug resistant stomach cancer cell lines EPG85-257, EPG85-257RNOV, EPG85-257RDB and their thermoresistant counterparts. Possible implications of differential protein expression will be discussed.

Transport of Phosphatidylserine Via MDR1 (multidrug Resistance 1)P-glycoprotein in a Human Gastric Carcinoma Cell Line

The Biochemical Journal. Jul, 2002  |  Pubmed ID: 12071854

The ATP-binding cassette transporter multidrug resistance 1 P-glycoprotein (MDR1 Pgp) has been implicated with the transport of lipids from the inner to the outer leaflet of the plasma membrane. While this has been unambigously shown for the fluorescent lipid analogues [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (C6-NBD)-phosphatidylcholine, -phosphatidylethanolamine, -sphingomyelin and -glucosylceramide, by using a novel approach we have now found significantly increased outward transport also for C6-NBD-phosphatidylserine (C6-NBD-PS) in EPG85-257 human gastric carcinoma cells overexpressing MDR1 (coding for MDR1 Pgp). The increased transport of C6-NBD-PS is mediated by MDR1 Pgp, shown by transport reduction nearly to the level of controls in the presence of MDR1 Pgp inhibitors [PSC 833, cyclosporin A and dexniguldipine hydrochloride (Dex)]. Addition of MK 571, a specific inhibitor of the MDR protein MRP1, does not decrease transport in either of the two cell lines. The plasma-membrane association of FITC-annexin V, a fluorescent protein conjugate binding PS, is significantly increased in MDR1-overexpressing cells as compared with controls, and can be reduced by an MDR1 Pgp inhibitor. This suggests that MDR1 Pgp transports endogenous PS, the lipid exhibiting the most pronounced transverse asymmetry in the plasma membrane.

Modulation of the Atypical Multidrug-resistant Phenotype by a Hammerhead Ribozyme Directed Against the ABC Transporter BCRP/MXR/ABCG2

Cancer Gene Therapy. Jul, 2002  |  Pubmed ID: 12082458

The phenomenon of multidrug resistance (MDR) in human cancers is one of the major causes of failure of chemotherapy. A recently identified new member of the superfamily of ATP-binding cassette transporters, breast cancer resistance protein (BCRP), was demonstrated to confer an atypical multidrug-resistant phenotype to tumor cells. To overcome the BCRP-mediated drug resistance, a specific anti-BCRP hammerhead ribozyme was introduced into the human gastric carcinoma cell line, EPG85-257RNOV, exhibiting an atypical MDR phenotype. By this approach, the expression levels of the targeted BCRP-encoding mRNA and the BCRP transport protein were decreased to the low constitutive expression level that was observed in highly drug-sensitive parental gastric carcinoma cells. In addition, in the anti-BCRP ribozyme-treated cells, the cellular drug accumulation was dramatically increased to the level measured in drug-sensitive cells. These effects were accompanied by an extensive reversal of the drug-resistant phenotype of more than 80%. Because additional mechanisms contribute to the multimodal-mediated MDR phenotype exhibited by this gastric carcinoma cell line, the data suggest that the BCRP-mediated contingent to the drug resistance was overcome nearly completely. Moreover, the data indicate that ribozyme-based gene therapy may be clinically applicable in preventing and reversing BCRP-mediated atypical MDR.

Multiple Mechanisms Confer Different Drug-resistant Phenotypes in Pancreatic Carcinoma Cells

Journal of Cancer Research and Clinical Oncology. Jul, 2002  |  Pubmed ID: 12136248

Drug-resistant phenotypes of cancer cells may be caused by complex multimodal mechanisms of resistance. In order to gain further insighte into these mechanisms, a P-glycoprotein-mediated multidrug-resistant phenotype induced by daunorubicin-selection and an alternative drug resistance due to treatment with mitoxantrone were investigated in pancreatic carcinoma-derived cells.

Association of Genomic Imbalances with Drug Resistance and Thermoresistance in Human Gastric Carcinoma Cells

International Journal of Cancer. Journal International Du Cancer. Mar, 2003  |  Pubmed ID: 12516094

Therapy resistance is the major obstacle to advances in successful cancer treatment. To characterize chromosomal alterations associated with different types of acquired MDR and thermoresistance, we applied CGH to compare a unique panel of human gastric carcinoma cells consisting of the parental, drug-sensitive and thermosensitive cancer cell line EPG85-257P, the atypical MDR variant EPG85-257RNOV, the classical MDR subline EPG85-257RDB and their thermoresistant counterparts EPG85-257P-TR, EPG85-257RNOV-TR and EPG85-257RDB-TR. CGH with genomic DNA prepared from these cell lines as probes successfully identified genomic gains and/or losses in chromosomal regions encoding putative genes associated with drug resistance and/or thermoresistance. These genes included various members of the families of ABC transporters and molecular chaperones. The importance of these cell variant-specific genomic imbalances in the development of MDR and thermoresistance is discussed and remains to be elucidated.

Modulation of the Classical Multidrug Resistance (MDR) Phenotype by RNA Interference (RNAi)

FEBS Letters. Jun, 2003  |  Pubmed ID: 12804765

For reversal of MDR1 gene-dependent multidrug resistance (MDR), two small interfering RNA (siRNA) constructs were designed to inhibit MDR1 expression by RNA interference. SiRNA duplexes were used to treat human pancreatic carcinoma (EPP85-181RDB) and gastric carcinoma (EPG85-257RDB) cells. In both cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels. Resistance against daunorubicin was decreased to 89% (EPP85-181RDB) or 58% (EPG85-257RDB). The data indicate that this approach may be applicable to cancer patients as a specific means to reverse tumors with a P-glycoprotein-dependent MDR phenotype back to a drug-sensitive one.

Overexpression of CMOAT (MRP2/ABCC2) is Associated with Decreased Formation of Platinum-DNA Adducts and Decreased G2-arrest in Melanoma Cells Resistant to Cisplatin

The Journal of Investigative Dermatology. Jul, 2003  |  Pubmed ID: 12839578

Resistance to various anti-neoplastic agents is a common observation in clinical management of melanoma. The biologic mechanisms conferring these different drug-resistant phenotypes, including resistance against the commonly used anti-cancer drug cisplatin, are unclear. In order to elucidate the role of the membrane adenosine triphosphate binding cassette-transporter cMOAT (canalicular multispecific anion transporter) (MRP2/ABCC2) in cisplatin resistance of melanoma, the expression of this protein was analyzed in the platinum drug-resistant cell line MeWo CIS 1. Cisplatin-resistant melanoma cells showed a distinct overexpression of cMOAT on mRNA and protein level. This observation was accompanied by a reduced formation of platinum-induced intrastrand cross-links in the nuclear DNA measured by an immunocytologic assay. This decrease in DNA platination was accompanied by an accelerated re-entry into the cell cycle after the typical cisplatin-induced G2 arrest, and a resistance to undergo apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity for Pt-d(GpG) adducts was not elevated in platinum drug-resistant melanoma cells. The decrease in platinum-DNA adduct formation in cisplatin-resistant melanoma cells was rather a reflection of the protecting activity of the transporter cMOAT. In conclusion, the functional inhibition of cMOAT might be a promising strategy in the reversal of resistance to platinum-based anti-cancer drugs in human melanoma.

Study of the Development of Chemoresistance in Melanoma Cell Lines Using Proteome Analysis

Electrophoresis. Jul, 2003  |  Pubmed ID: 12874874

Malignant melanomas have poor prognosis since treatment with anti-neoplastic agents is mostly ineffective. The biological mechanisms of this strong intrinsic therapy resistance are unknown. In order to identify new molecular factors potentially associated with the drug-resistant phenotype of malignant melanoma, a panel of human melanoma cell variants exhibiting low and high levels of resistance to four commonly used anticancer drugs in melanoma treatment, i.e., vindesine, etoposide, cisplatin, and fotemustine, was characterized using proteomic tools (two-dimensional electrophoresis for protein fractionation and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry for protein identification). In the neutral and weak acidic milieu (pH 4.0-8.0) a total number of 14 proteins showed alterations in expression whereas 20 proteins were differentially expressed in the basic milieu (pH 8.0-11.0). Besides proteins with unknown physiologic function, several factors were identified that show chaperone activity. Moreover, proteins involved in drug detoxification, metabolism, and regulation of apoptotic pathways could be identified. The possible role of these proteins in the development of chemoresistance is discussed, although detailed functional tests with these proteins have still to be performed. Nevertheless, it is clear that this proteomic approach for studying chemoresistance phenomena is a prerequisite before further investigation can yield insight into the biology and development of drug resistance in malignant melanoma.

Dynamic Expression Profile of P21WAF1/CIP1 and Ki-67 Predicts Survival in Rectal Carcinoma Treated with Preoperative Radiochemotherapy

Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology. Sep, 2003  |  Pubmed ID: 12885834

We investigated p53 and its downstream effectors p21WAF1/CIP1, BAX, and hMSH2 as well as the proliferation marker Ki-67 (mki-67/MIB-1) in patients undergoing preoperative radiochemotherapy for rectal carcinoma to identify prognostic and predictive factors. The focus of this study was on the dynamics of these genetic markers in a longitudinal study-that is, before and after radiochemotherapy.

Homozygous Mutation Arg768Trp in the ABC-transporter Encoding Gene MRP2/cMOAT/ABCC2 Causes Dubin-Johnson Syndrome in a Caucasian Patient

Journal of Human Genetics. 2003  |  Pubmed ID: 12942343

Dubin-Johnson syndrome (DJS) is an autosomal recessive disorder characterized by conjugated hyperbilirubinemia and caused by mutations of the ATP-binding cassette (ABC) transporter encoding gene MRP2/cMOAT/ABCC2. Previous studies reported on mutations in DJS patients and polymorphisms in healthy human individuals. The genomic DNA sequence of a female Caucasian DJS patient was analyzed by DNA sequencing and revealed the identification of a homozygous missense mutation C2302T. This DJS-causing alteration results in an amino acid exchange Arg768Trp.

Enhanced Exposure of Phosphatidylserine in Human Gastric Carcinoma Cells Overexpressing the Half-size ABC Transporter BCRP (ABCG2)

The Biochemical Journal. Dec, 2003  |  Pubmed ID: 12946267

Members of the ABC (ATP-binding cassette) transporter super-family are emerging to be involved in lipid transport. In the present study, we studied the organization of phospholipids in the plasma membrane of EPG85-257 human gastric carcinoma cells overexpressing BCRP (breast cancer resistance protein, ABCG-2), a half-size transporter belonging to the ABCG subfamily. A significantly increased plasma membrane association of the PS (phosphatidylserine)-binding probe FITC-Annexin V in comparison with control cells was observed. Treatment of BCRP -overexpressing cells with the inhibitor Tryprostatin A decreased FITC-Annexin V binding almost to the control level. This suggests an enhanced exposure of PS in BCRP -overexpressing cells, which is dependent on functional BCRP. A role of BCRP in the transverse distribution of lipids in the plasma membrane is supported by the increased outward transport of the lipid analogue C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PS in BCRP -overexpressing EPG85-257 cells and MCF-7 human breast cancer cells. As shown for BCRP -overexpressing EPG85-257 cells, enhanced outward redistribution of the lipid analogue is inhibited by Tryprostatin A as well as by reduction of BCRP expression on transfection with an anti- BCRP -ribozyme. We also observed an enhanced outward transport of C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylcholine in BCRP -overexpressing EPG85-257 cells, suggesting that the influence of BCRP on transverse lipid organization is not highly specific.

ABC-transporters: Implications on Drug Resistance from Microorganisms to Human Cancers

International Journal of Antimicrobial Agents. Sep, 2003  |  Pubmed ID: 13678820

Resistance to chemotherapy is a common clinical problem in patients with infectious diseases as well as in patients with cancer. During treatment of infections or malignant tumors, the drug targets of prokaryotic or eukaryotic microorganisms and neoplastic cells are often found to be refractory to a variety of drugs that have different structures and functions. This phenomenon has been termed multidrug resistance (MDR). The mechanisms leading to MDR are frequently caused by trans-membrane xenobiotic transport molecules belonging to the superfamily of ATP-binding cassette (ABC) transporters. There is an urgent need to understand the structure-function relationships of these efflux pumps that underlie their transport mechanism and drug selectivity. This knowledge may allow the rational design of new drugs that can inhibit or circumvent the activity of these MDR transport molecules. Furthermore, the development of such chemosensitizing agents would help us learn more about the physiological functions and substrates of these pump proteins. This review will discuss the current state of knowledge of the functional and structural similarities among ABC-transporters in prokaryotic and eukaryotic cells and their impact on MDR.

Reversal of Breast Cancer Resistance Protein-mediated Drug Resistance by Tryprostatin A

International Journal of Cancer. Journal International Du Cancer. Dec, 2003  |  Pubmed ID: 14566821

MDR in human cancers is one of the major causes of failure of chemotherapy. A member of the superfamily of ABC transporters, BCRP, was demonstrated to confer an atypical MDR phenotype to tumor cells. To overcome the BCRP-mediated drug resistance, the fungal secondary metabolite TPS-A, a diketopiperazine, was analyzed with regard to its potency to reverse the BCRP-mediated drug-resistant phenotype. At concentrations of 10-50 microM, TPS-A reversed a mitoxantrone-resistant phenotype and inhibited the cellular BCRP-dependent mitoxantrone accumulation in the human gastric carcinoma cell line EPG85-257RNOV, the human breast cancer cell line MCF7/AdrVp (both exhibiting acquired BCRP-mediated MDR) and the BCRP cDNA-transfected breast cancer cell line MCF-7/BCRP clone 8. No cytotoxicity was seen at effective concentrations. These data indicate that TPS-A is a novel BCRP inhibitor.

Molecular Analysis of Therapy Resistance in Gastric Cancer

Digestive Diseases (Basel, Switzerland). 2003  |  Pubmed ID: 14752223

Therapy resistance is the main cause of therapeutic failure and death in patients suffering from gastric carcinoma. Clinical resistance against systemic chemotherapy of gastric cancer is likely to be multifactorial and heterogenous. So far, no significant resistance factor that predicts the clinical outcome of systemic treatment of gastric carcinoma has been identified. In order to gain further understanding of therapy resistance in gastric carcinoma, various in vitro model systems were established. One of these models consists of the parental, drug-sensitive and thermosensitive human gastric carcinoma cell line EPG85-257P, its classical multidrug-resistant variant EPG85-257RDB, its atypical multidrug-resistant subline EPG85-257RNOV and their thermoresistant counterparts EPG85-257P-TR, EPG85-257RDB-TR, and EPG85-257RNOV-TR. This panel of cells was analyzed using morphological, biochemical, cellular and molecular biological methods to identify potential new factors involved in therapy resistance of gastric carcinoma. Cellular alterations that could be identified in these models were evaluated by functional investigations. This review will discuss the current state of knowledge of these new therapy resistance-associated factors, e.g. glypican-3 (GPC3), as well as the impact of well-known drug resistance-associated factors, such as MDR1/P-glycoprotein, on therapy resistance of gastric carcinoma.

Proteomics in Cancer Cell Research: an Analysis of Therapy Resistance

Pathology, Research and Practice. 2004  |  Pubmed ID: 15237919

Proteomics, the global analysis of expressed cellular proteins, provides powerful tools for the detailed comparison of proteins from normal and neoplastic tissue. In particular, cancer cell culture models are suited for applying proteomics techniques, such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry, to identify specific protein expression profiles and/or proteins that may be associated with a defined phenotype of the cancer cells. As an instance of such an application of proteomics techniques, the detailed proteome analyses of different drug-resistant and thermoresistant cancer cell lines will be discussed. Finally, the potential roles of newly identified factors in a distinct biological mechanism have to be proven by functional studies. This experimental validation strategy will be discussed for two different factors identified by 2D-PAGE analyses of drug-resistant carcinoma cell lines, the "transporter associated with antigen presentation 1" (TAP1) and 14-3-3sigma.

RNA Expression of MDR1/P-glycoprotein, DNA-topoisomerase I, and MRP2 in Ovarian Carcinoma Patients: Correlation with Chemotherapeutic Response

Gynecologic Oncology. Jul, 2004  |  Pubmed ID: 15262134

Clinical drug resistance is the major obstacle in the successful treatment of ovarian cancer. Besides elevated expression of adenosine triphosphate binding cassette (ABC) transporters, such as MDR1/P-gp or MRP2/cMOAT/ABCC2, alterations in the expression of DNA topoisomerase I (TOP1) are associated with drug-resistant phenotypes in various model systems.

Effective Knock Down of Very High ABCG2 Expression by a Hammerhead Ribozyme

Anticancer Research. Jul-Aug, 2004  |  Pubmed ID: 15330166

Ribozymes are an effective tool to reduce the mRNA levels of specific target genes. Overexpression of the drug transport protein, ABCG2, has been associated with multidrug resistance in cancer cells.

Stable and Complete Overcoming of MDR1/P-glycoprotein-mediated Multidrug Resistance in Human Gastric Carcinoma Cells by RNA Interference

Cancer Gene Therapy. Nov, 2004  |  Pubmed ID: 15375376

Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp). For stable reversal of "classical" MDR by RNA interference (RNAi) technology, an H1-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed. By introduction of anti-MDR1/P-gp shRNA expression vectors into the extremely high drug-resistant human gastric carcinoma cell line EPG85-257RDB, the MDR phenotype was completely reversed. The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells. The data indicate that stable shRNA-mediated RNAi can be tremendously effective in reversing MDR1/P-gp-mediated MDR and is therefore a promising strategy for overcoming MDR by gene therapeutic applications.

Chromosomal Imbalances Associated with Drug Resistance and Thermoresistance in Human Pancreatic Carcinoma Cells

European Journal of Cell Biology. Oct, 2004  |  Pubmed ID: 15679104

Resistance to therapeutic treatment is the major obstacle to advances in the successful management of pancreatic cancer. To characterize chromosomal alterations associated with different phenotypes of acquired multidrug resistance (MDR) and thermoresistance, comparative genomic hybridization (CGH) was applied to compare human pancreatic carcinoma-derived cells. This panel of cell lines consists of the parental, drug- and thermosensitive pancreatic carcinoma cell line EPP85 - 181P, its atypical MDR variant EPP85-181RNOV, the classical MDR subline EPP85-181RDB, and their thermoresistant counterparts EPP85-181P-TR, EPP85-181RNOV-TR, and EPP85 - 181RDB-TR, respectively. CGH using genomic DNA prepared from these cell lines as probes successfully identified genomic gains and/or losses in chromosomal regions encoding putative genes associated with drug resistance and/or thermoresistance. These genes included 23 members of the family of ABC transporters, 27 members of the family of cytochrome P450 (CYP) monooxygenases, various molecular chaperones, DNA repair enzymes, and factors involved in the regulation of cell cycle and apoptosis. The importance of these cell variant-specific genomic imbalances in the development of MDR and thermoresistance is discussed and remains to be elucidated.

Glypican-3 is Involved in Cellular Protection Against Mitoxantrone in Gastric Carcinoma Cells

Oncogene. Jan, 2004  |  Pubmed ID: 14661052

Elevated expression of the heparan sulphate proteoglycan glypican-3 (GPC3) was found on mRNA and protein levels in the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV, which was established by in vitro selection against mitoxantrone. In order to elucidate a putative role of GPC3 in the drug-resistant phenotype, the mitoxantrone-resistant cell line EPG85-257RNOV was transfected with an expression vector construct carrying an anti-GPC3 hammerhead ribozyme. It could be demonstrated that in anti-GPC3 ribozyme-transfected cell clones, the GPC3-specific mRNA and corresponding protein expression levels were decreased to levels that are similar to those observed in nonresistant, parental cells. The anti-GPC3 ribozyme-containing clones reduced the mitoxantrone resistance level up to 21% of the original resistance and the crossresistance against etoposide to 33% of the original value. This reversal of drug resistance was accompanied by an increased cellular mitoxantrone accumulation in the anti-GPC3 ribozyme-expressing cells. In conclusion, it was verified that GPC3 is involved in the cellular protection against mitoxantrone in the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV.

Multidrug-resistant Cancer Cells Facilitate E1-independent Adenoviral Replication: Impact for Cancer Gene Therapy

Cancer Research. Jan, 2004  |  Pubmed ID: 14729641

Resistance to chemotherapy is responsible for a failure of current treatment regimens in cancer patients. We have reported previously that the Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug resistant-tumor phenotype. YB-1 predicts drug resistance and patient outcome in breast cancer. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In drug-resistant cancer cells and in adenovirus-infected cells YB-1 is found in the nucleus. Nuclear accumulation of YB-1 in adenovirus-infected cells is a function of the E1 region, and we have shown that YB-1 facilitates adenovirus replication. Here we report that E1A-deleted or mutant adenovirus vectors, such as Ad312 and Ad520, replicate efficiently in multidrug-resistant (MDR) cancer cells and induce an adenovirus cytopathic effect resulting in host cell lysis. Thus, replication-defective adenoviruses are a previously unrecognized vector system for a selective elimination of MDR cancer cells. Our work forms the basis for the development of novel oncolytic adenovirus vectors for the treatment of MDR malignant diseases in the clinical setting.

Study of the Development of Thermoresistance in Human Pancreatic Carcinoma Cell Lines Using Proteome Analysis

Electrophoresis. Jan, 2004  |  Pubmed ID: 14730581

In order to find candidate proteins that are potentially associated with the thermoresistant phenotype in combination with drug resistance, we analyzed the differential protein expression in vitro in the human pancreatic cancer cell line EPP85-181-P and classical and atypical multidrug-resistant variants and their thermoresistant counterparts using proteomics. This study identifies sets of proteins that may lead to the development of thermoresistance. These results provide a fundamental basis to elucidate the molecular mechanism of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers.

Protection of Platinum-DNA Adduct Formation and Reversal of Cisplatin Resistance by Anti-MRP2 Hammerhead Ribozymes in Human Cancer Cells

International Journal of Cancer. Journal International Du Cancer. Jun, 2005  |  Pubmed ID: 15688364

Resistance to platinum-containing antineoplastic drugs is the major limitation in their clinical use. To elucidate the role of the ABC transporter MRP2 in platinum drug resistance, its expression was analyzed in human cisplatin-resistant cell lines: the ovarian carcinoma line A2780RCIS, the adrenocortical carcinoma line D43/86RCIS and the melanoma line MeWoCIS1. All these cells showed overexpression of MRP2. For reversal of platinum resistance, 2 anti-MRP2 hammerhead ribozymes were introduced into A2780RCIS cells. Both ribozymes showed gene-silencing activities and reversed the drug-resistant phenotype. Moreover, formation of platinum-induced intrastrand cross-links was measured in DNA. The level of DNA platination corresponded inversely to the level of MRP2 expression and was accompanied by increased caspase-3-dependent apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity was not altered; the decrease in platinum-DNA adduct formation was rather a reflection of the protecting activity of MRP2. In conclusion, functional inhibition of MRP2 might be a promising strategy in the reversal of resistance to platinum-based anticancer drugs. This was reflected by the specific inhibition of MRP2 by ribozyme technology, indicating that this gene therapeutic approach may be applicable as a specific means to overcome platinum resistance in human neoplasms.

Reversal of Different Drug-resistant Phenotypes by an Autocatalytic Multitarget Multiribozyme Directed Against the Transcripts of the ABC Transporters MDR1/P-gp, MRP2, and BCRP

Molecular Therapy : the Journal of the American Society of Gene Therapy. Apr, 2005  |  Pubmed ID: 15771954

A "multitarget multiribozyme" (MTMR) was constructed. It consists of three trans-acting hammerhead ribozymes directed against the transcripts of the ABC transporters MDR1/P-gp, BCRP, and MRP2; three cis-acting MDR1/P-gp-specific ribozymes; and three MDR1/P-gp-homologous spacer sequences. The trans-acting hammerhead ribozymes are liberated from the MTMR through autocatalytic self-cleavage by the cis-acting ribozymes. The MTMR was characterized with regard to its kinetic parameters. Comparison of the MTMR-specific kinetic values with those of the corresponding monoribozymes demonstrated that MTMR fragments could cleave their specific substrates without loss of efficiency. The MTMR was applied to three cell models, each overexpressing another ABC transporter, i.e., the gastric carcinoma cell line EPG85-257RDB expresses MDR1/P-gp, the cell variant EPG85-257RNOV synthesizes BCRP, and the ovarian carcinoma line A2780RCIS produces MRP2. In all cellular systems, the MTMR could specifically decrease the expression of the respective ABC transporter at the mRNA level (97% decrease in the MDR1/P-gp mRNA, 80% decrease in the BCRP mRNA, 96% decrease in the MRP2 mRNA) and the protein level. Resistance against the selection drug was reversed completely (100% in EPG85-257RDB) or by 94 (EPG85-257RNOV) or 63% (A2780RCIS). Thus, the MTMR technology provides a novel tool for gene therapeutic applications to reverse different ABC-transporter-dependent drug-resistant phenotypes.

Overexpression of Cathepsin B in Gastric Cancer Identified by Proteome Analysis

Proteomics. Apr, 2005  |  Pubmed ID: 15789341

We aimed to validate an analytical approach based on proteomics on gastric cancer specimens for the identification of new putative diagnostic or prognostic markers. Primary screening was performed on gastrectomy specimens obtained from ten consecutive patients with gastric cancer. Gastric epithelial cells were obtained with an epithelial cell enrichment technique, homogenized and then separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The differential protein expression pattern was verified stepwise by Western blotting and immunohistochemistry on samples from 28 and 46 cancer patients, respectively. The putative clinical applicability and prognostic use were tested by an enzyme-linked immunoabsorbent assay on serum samples obtained from 149 cancer patients. One hundred-ninety-one differentially expressed protein spots were found by 2-D PAGE and identified by mass spectrometry, including cathepsin B, which was over-expressed in six (60%) patients. Western blotting confirmed that the active form of cathepsin B is over-expressed, while immunohistochemistry showed strong cytoplasmic staining in cancer tissues of 45 (98%) patients. The serum level of cathepsin B was increased in patients with gastric cancer compared to healthy controls (P = 0.0026) and correlated with T-category and the presence of distant metastases (P < 0.05). Serum levels above 129 pmol x L(-1) were associated with a reduced survival rate (P = 0.0297). Proteome analysis is a valuable tool for the identification of prognostic markers in gastric cancer: Increased cathepsin B serum levels are associated with advanced tumor stages and progressive disease, which enables the classification of some gastric cancer patients into a subgroup that should undergo aggressive therapy.

Transcriptome Analysis of Different Multidrug-resistant Gastric Carcinoma Cells

In Vivo (Athens, Greece). May-Jun, 2005  |  Pubmed ID: 15875780

Multidrug resistance (MDR) of human cancers is the major cause of failure of chemotherapy. To better understand the molecular events associated with the development of different types of MDR, two different multidrug-resistant gastric carcinoma cell lines, the MDR1/P-glycoprotein-expressing cell line EPG85-257RDB and the MDR1/P-glycoprotein-negative cell variant EPG85-257RNOV, as well as the corresponding drug-sensitive parental cell line EPG85-257P, were used for analyses of the mRNA expression profiles by cDNA array hybridization. Of more than 12,000 genes spotted on the arrays, 156 genes were detected as being significantly regulated in the cell line EPG85-257RDB in comparison to the non-resistant cell variant, and 61 genes were found to be differentially expressed in the cell line EPG85-257RNOV Seventeen genes showed a differential expression level in both multidrug-resistant gastric carcinoma variants. The impact of these alterations in gene expression levels in different multidrug-resistant gastric carcinoma cell variants is discussed.

Augmented Expression of Metallothionein and Glutathione S-transferase Pi As Unfavourable Prognostic Factors in Cisplatin-treated Ovarian Cancer Patients

Virchows Archiv : an International Journal of Pathology. Sep, 2005  |  Pubmed ID: 15968547

Resistance to cis- or carboplatin represents the principal cause of therapeutic failures in ovarian carcinoma. The phenomenon of resistance to platinum-based drugs is partly related to expression of metallothionein (MT) and of glutathione S-transferase pi (GST-pi), but opinion on the subject is discordant. Documentation of a negative predictive effect of MT and GST-pi expression for the therapy employing platinum-based drugs would permit to select resistant cases in which other therapeutic approaches could be employed. The present study aimed at examining the relation between intensities of MT and GST-pi expressions in ovarian carcinomas and dynamics of the clinical course in the neoplastic disease in a group of cisplatin-treated patients. The analyses were performed on samples of ovarian carcinoma originating from 43 first-look laparotomies (FLLs) and, in 30 cases, from second-look laparotomies (SLL) from the same patients. Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies to MT and GST-pi. The calculations showed that in cases with augmented expression of MT, mortality was higher. On the other hand, augmented expression of GST-pi predisposed to more frequent relapses, deaths and progression of the tumor. Kaplan-Meier analysis showed that a significantly shorter survival time was linked to cases of higher expression of MT at FLL and of higher expression of GST-pi at FLL, whereas a shorter progression-free time was manifested by cases with higher expression of GST-pi at FLL. The performed investigations indicate that augmented expressions of MT at FLL and GST-pi at FLL in ovarian cancer represent an unfavourable predictive factor in cisplatin-treated patients.

Prediction of Doxorubicin Sensitivity in Breast Tumors Based on Gene Expression Profiles of Drug-resistant Cell Lines Correlates with Patient Survival

Oncogene. Nov, 2005  |  Pubmed ID: 16044152

Up to date clinical tests for predicting cancer chemotherapy response are not available and individual markers have shown little predictive value. We hypothesized that gene expression patterns attributable to chemotherapy-resistant cells can predict response and cancer prognosis. We contrasted the expression profiles of 13 different human tumor cell lines of gastric (EPG85-257), pancreatic (EPP85-181), colon (HT29) and breast (MCF7 and MDA-MB-231) origin and their counterparts resistant to the topoisomerase inhibitors daunorubicin, doxorubicin or mitoxantrone. We interrogated cDNA arrays with 43 000 cDNA clones ( approximately 30 000 unique genes) to study the expression pattern of these cell lines. We divided gene expression profiles into two sets: we compared the expression patterns of the daunorubicin/doxorubicin-resistant cell lines and the mitoxantrone-resistant cell lines independently to the parental cell lines. For identifying predictive genes, the Prediction Analysis for Mircorarrays algorithm was used. The analysis revealed 79 genes best correlated with doxorubicin resistance and 70 genes with mitoxantrone resistance. In an independent classification experiment, we applied our model of resistance for predicting the sensitivity of 44 previously characterized breast cancer samples. The patient group characterized by the gene expression profile similar to those of doxorubicin-sensitive cell lines exhibited longer survival (49.7+/-26.1 months, n=21, P=0.034) than the resistant group (32.9+/-18.7 months, n=23). The application of gene expression signatures derived from doxorubicin-resistant and -sensitive cell lines allowed to predict effectively clinical survival after doxorubicin monotherapy. Our approach demonstrates the significance of in vitro experiments in the development of new strategies for cancer response prediction.

Prognostic Value of Immunohistochemical Estimation of CD24 and Ki67 Expression in Cisplatin and Paclitaxel Treated Ovarian Carcinoma Patients

Polish Journal of Pathology : Official Journal of the Polish Society of Pathologists. 2005  |  Pubmed ID: 16092668

CD24 is a small membranous protein which may participate in invasion of tumor cells. Present study aimed at evaluation of prognostic significance linked to immunohistochemical demonstration of CD24 expression and the proliferation index, Ki67 expression in ovarian cancers. The immunohistochemical reactions with monoclonal CD24- and Ki67-specific antibodies were performed in paraffin sections originating from 30 patients with ovarian cancer treated using cisplatin and paclitaxel. Results of the reactions and analysis of the clinical course of the patients were subjected to statistical analysis. Cases with cytoplasmic-membranous expression of CD24 (CD24c-m) were found to exhibit significantly shorter overall survival time (P = 0.0002) and progression-free period (P = 0.0005). Cases with membranous expression of CD24 (CD24m) manifested a longer overall survival time (P = 0.022). No relationship was disclosed between expression of Ki67 on the one hand and survival time and CD24 expression on the other. As documented using chi square test, expression of CD24c-m predisposed to relapses (P = 0.012), progression (P = 0.0362) and to death (P = 0.0034). Deaths were encountered significantly less frequently in cases with CD24m expression (P = 0.0465). The studies demonstrated that CD24c-m represented a strongly unfavorable prognostic indicator. The antigen represents an interesting target in the search for novel therapeutic methods. The more aggressive course of cases with CD24c-m expression was not linked to more intense proliferation of the tumor cells.

Relationship Between the Expression of Cyclooxygenase 2 and MDR1/P-glycoprotein in Invasive Breast Cancers and Their Prognostic Significance

Breast Cancer Research : BCR. 2005  |  Pubmed ID: 16168133

Recent reports suggest that expression of the cyclooxygenase 2 (COX-2) enzyme may up-regulate expression of MDR1/P-glycoprotein (MDR1/P-gp), an exponent of resistance to cytostatic drugs. The present study aimed at examining the relationship between the expression of COX-2 and of MDR1/P-gp in a group of breast cancer cases.

Potential Applications of RNA Interference Technology in the Treatment of Cancer

Future Oncology (London, England). Feb, 2005  |  Pubmed ID: 16555980

Inhibition of growth and progression of cancer cells is a challenge with major potential impact. RNA interference (RNAi) technology has been rapidly developed as a laboratory tool for the downregulation of the expression of a gene of interest. Moreover, RNAi offers a new potential for gene therapy of particular neoplasms by the specific inhibition of a cancer-associated target. This article will briefly describe the mechanism and application possibilities of RNAi, and illustrate the therapeutic potential in cancer gene therapy. The utilization of RNAi technology as a potential therapeutic tool for the treatment of cancer will be discussed in detail for two specific targets; the Bcr-Abl tyrosine kinase and the multidrug transporter MDR1/P-glycoprotein.

Analysis of Gene Expression Profiles in Melanoma Cells with Acquired Resistance Against Antineoplastic Drugs

Melanoma Research. Apr, 2006  |  Pubmed ID: 16567970

Resistance to various antineoplastic agents is common in the clinical management of malignant melanoma. The biological mechanisms conferring these different drug-resistant phenotypes are still unclear. To identify potential factors mediating drug resistance to melanoma cells, the mRNA expression profiles of the parental drug-sensitive human melanoma cell line MeWo and four derived drug-resistant sublines with acquired resistance against four commonly used drugs for melanoma treatment (cisplatin, etoposide, fotemustine and vindesine) were analysed. We investigated cDNA arrays with 43,000 cDNA clones ( approximately 30,000 unique genes) to study the expression patterns of these cell lines. We were able to simultaneously extract new candidate genes associated with drug resistance in malignant melanoma and to correlate the present findings with previously described resistance-associated genes. Using hierarchical clustering and analysing the overlap of genes with altered expression, we detected similarities between the expression signatures related to cisplatin and fotemustine resistance. The resistance against vindesine required a minimal set of changes in gene expression relative to the parental MeWo cell line. Our study provides new data that may be used to obtain further insight into the resistance characteristics of malignant melanoma.

Maspin Expression is Characteristic for Cisplatin-sensitive Ovarian Cancer Cells and for Ovarian Cancer Cases of Longer Survival Rates

International Journal of Gynecological Pathology : Official Journal of the International Society of Gynecological Pathologists. Apr, 2006  |  Pubmed ID: 16633061

High cytoplasmic expression of maspin was described in ovarian cancers of shorter survival rates. Until now, no relationship has been described between expression of maspin and sensitivity to cisplatin in ovarian cancers. This study aimed at examining the relationship between expression of maspin, detected by immunohistochemistry and clinical response to cisplatin in ovarian cancer cases as well as the in vitro sensitivity to cisplatin of 11 ovarian cancer cell lines. The analyzes were performed on 73 samples of ovarian cancer and on A2780P, A2780RCIS, CAOV-3, EFO 21, EFO 27, ES-2, Mdah 2774, OAW 42, OVCAR-3, PA-1, and SKOV-3 ovarian cancer cells. Cytoplasmic maspin expression in studied cells significantly correlated with cisplatin sensitivity. A significantly shorter overall survival and progression-free survival was associated with lower cytoplasmic maspin expression at first-look laparotomies and nuclear maspin expression and secondary cytoreductions. Higher nuclear maspin at first-look laparotomies expression was specific for cases of complete response. In the study, the elevated expression of maspin was shown to be typical for cisplatin-sensitive ovarian cancers.

CD24 Expression is Specific for Tamoxifen-resistant Ductal Breast Cancer Cases

Anticancer Research. Jan-Feb, 2006  |  Pubmed ID: 16739331

In breast cancer, the expression of CD24 represents a poorly recognised unfavourable prognostic factor. CD24 has been described to be potentially down-regulated by estrogen receptor alpha (ER). The present study was aimed at examining the predictive value of CD24 expression in tamoxifen-treated breast cancer cases.

MDR1/P-glycoprotein (ABCB1) As Target for RNA Interference-mediated Reversal of Multidrug Resistance

Current Drug Targets. Jul, 2006  |  Pubmed ID: 16842213

Resistance of tumor cells to multiple structurally unrelated cytotoxic drugs, multidrug resistance (MDR), is the major limitation to the successful chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype is the result from decreased cellular drug accumulation mediated by the adenosine triphosphate binding cassette (ABC)-transporter MDR1/P-glycoprotein (MDR1/P-gp, ABCB1) encoded by the human MDR1 gene. Inhibition of the drug extrusion activity of MDR1/P-gp by low-molecular weight pharmacologically active compounds as a method to reverse MDR in patients suffering on malignant diseases has been studied capaciously, but the clinical results have generally been disappointing. Thus, experimental therapeutic strategies to reverse MDR are under extensive investigation. These strategies included gene therapeutic approaches with antisense oligonucleotides (ODNs), ribozymes, or DNAzymes and, most recently, the application of the RNA interference (RNAi) technology. RNAi is a physiological double stranded RNA-triggered mechanism resulting in gene-silencing in a sequence-specific manner. Transient RNAi can be attained by application of small interferring RNAs (siRNAs), whereas a stable RNAi-mediated gene-silencing can be achieved by transfection of mammalian cells with short hairpin RNA (shRNA) encoding expression cassettes localized on plasmid or viral vectors. Transient and stable RNAi strategies were applied to overcome MDR1/P-gp-mediated MDR in different in vitro models derived from various neoplastic tissue and will be come up for discussion.

Inhibition of the Multidrug-resistant Phenotype by Targeting YB-1 with a Conditionally Oncolytic Adenovirus: Implications for Combinatorial Treatment Regimen with Chemotherapeutic Agents

Cancer Research. Jul, 2006  |  Pubmed ID: 16849566

Bearing in mind the limited success of available treatment modalities for the therapy of multidrug-resistant tumor cells, alternative and complementary strategies need to be developed. It is known that the transcriptional activation of genes, such as MDR1 and MRP1, which play a major role in the development of a multidrug-resistant phenotype in tumor cells, involves the Y-box protein YB-1. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In addition, it has been reported previously that YB-1 is an important factor in adenoviral replication because it activates transcription from the adenoviral E2-late promoter. Here, we report that an oncolytic adenovirus, named Xvir03, expressing the viral proteins E1B55k and E4orf6, leads to nuclear translocation of YB-1 and in consequence to viral replication and cell lysis in vitro and in vivo. Moreover, we show that Xvir03 down-regulates the expression of MDR1 and MRP1, indicating that recruiting YB-1 to the adenoviral E2-late promoter for viral replication is responsible for this effect. Thus, nuclear translocation of YB-1 by Xvir03 leads to resensitization of tumor cells to cytotoxic drugs. These data reveal a link between chemotherapy and virotherapy based on the cellular transcription factor YB-1 and provide the basis for formulating a model for a novel combined therapy regimen named Mutually Synergistic Therapy.

RNA Interference-triggered Reversal of ABCC2-dependent Cisplatin Resistance in Human Cancer Cells

Biochemical and Biophysical Research Communications. Sep, 2006  |  Pubmed ID: 16876126

The adenosine triphosphate binding cassette (ABC)-transporter ABCC2 (MRP2/cMOAT) can mediate resistance against the commonly used anticancer drugs cisplatin and paclitaxel. To overcome the ABCC2-depending drug resistance, two specific anti-ABCC2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS. Since both siRNAs showed biological activity, for stable inhibition of ABCC2 a corresponding short hairpin RNA (shRNA)-encoding expression vector was designed. By treatment of A2780RCIS cells with this construct, the expressions of the targeted ABCC2 encoding mRNA and transport protein were inhibited. These effects were accompanied by reversal of resistance against cisplatin and paclitaxel. Thus, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing and reversing ABCC2-depending drug resistance.

Gene Expression Profiling of 30 Cancer Cell Lines Predicts Resistance Towards 11 Anticancer Drugs at Clinically Achieved Concentrations

International Journal of Cancer. Journal International Du Cancer. Apr, 2006  |  Pubmed ID: 16217747

Cancer patients with tumors of similar grading, staging and histogenesis can have markedly different treatment responses to different chemotherapy agents. So far, individual markers have failed to correctly predict resistance against anticancer agents. We tested 30 cancer cell lines for sensitivity to 5-fluorouracil, cisplatin, cyclophosphamide, doxorubicin, etoposide, methotrexate, mitomycin C, mitoxantrone, paclitaxel, topotecan and vinblastine at drug concentrations that can be systemically achieved in patients. The resistance index was determined to designate the cell lines as sensitive or resistant, and then, the subset of resistant vs. sensitive cell lines for each drug was compared. Gene expression signatures for all cell lines were obtained by interrogating Affymetrix U133A arrays. Prediction Analysis of Microarrays was applied for feature selection. An individual prediction profile for the resistance against each chemotherapy agent was constructed, containing 42-297 genes. The overall accuracy of the predictions in a leave-one-out cross validation was 86%. A list of the top 67 multidrug resistance candidate genes that were associated with the resistance against at least 4 anticancer agents was identified. Moreover, the differential expressions of 46 selected genes were also measured by quantitative RT-PCR using a TaqMan micro fluidic card system. As a single gene can be correlated with resistance against several agents, associations with resistance were detected all together for 76 genes and resistance phenotypes, respectively. This study focuses on the resistance at the in vivo concentrations, making future clinical cancer response prediction feasible. The TaqMan-validated gene expression patterns provide new gene candidates for multidrug resistance.

Reversal of P-glycoprotein-mediated Multidrug Resistance in Cancer Cells by the C-Jun NH2-terminal Kinase

Cancer Research. Jan, 2006  |  Pubmed ID: 16397260

A significant impediment to the success of cancer chemotherapy is multidrug resistance (MDR). A typical form of MDR is attributable to the overexpression of membrane transport proteins, such as P-glycoprotein, resulting in an increased drug efflux. In this study, we show that adenovirus-mediated enhancement of the c-Jun NH2-terminal kinase (JNK) reduces the level of P-glycoprotein in a dose- and time-dependent manner. Protein turnover assay shows that the decrease of P-glycoprotein is independent of its protein stability. Instead, this occurs primarily at the mRNA level, as revealed by reverse transcription-PCR analysis. We find that P-glycoprotein down-regulation requires the catalytic activity of JNK and is mediated by the c-Jun transcription factor, as either pharmacologic inhibition of JNK activity or dominant-negative suppression of c-Jun remarkably abolishes the ability of JNK to down-regulate P-glycoprotein. In addition, electrophoretic mobility shift assay reveals that adenoviral JNK increases the activator protein binding activity of the mdr1 gene in the MDR cells. We further show that the decrease of P-glycoprotein level is associated with a significant increase in intracellular drug accumulation and dramatically enhances the sensitivity of MDR cancer cells to chemotherapeutic agents. Our study provides the first direct evidence that enhancement of the JNK pathway down-regulates P-glycoprotein and reverses P-glycoprotein-mediated MDR in cancer cells.

Novel Three-pronged Strategy to Enhance Cancer Cell Killing in Glioblastoma Cell Lines: Histone Deacetylase Inhibitor, Chemotherapy, and Oncolytic Adenovirus Dl520

Human Gene Therapy. Jan, 2006  |  Pubmed ID: 16409125

Resistance to radiation and chemotherapy remains an obstacle to the treatment of brain tumors. We have demonstrated that the replication-deficient adenovirus d1520, which lacks the E1A 13S protein, replicates efficiently and exhibits oncolytic potential in multidrug-resistant cells with nuclear localization of the human transcription factor YB-1. However, besides others, key factors regarding oncolytic virotherapy are limited tumor transduction rate and low replication efficiency. The objective of this study was to determine whether the chemotherapeutic agent irinotecan, by enhancing nuclear localization of YB-1, and the histone deacetylase inhibitor trichostatin A, by upregulating coxsackievirus-adenovirus receptor (CAR) expression, could augment replication of and cell lysis by adenovirus dl520 in glioblastomas in vitro. We found that trichostatin A upregulated CAR expression and that irinotecan caused increased nuclear localization of YB-1 in both glioblastoma cell lines. Irinotecan alone, and trichostatin A alone, enhanced replication of and cell lysis by dl520. Importantly, when combining both agents, the replication efficiency (maximum, 27-fold) and induction of cytopathic effect (maximum, 3.8-fold) of dl520 were further augmented significantly. These results support the hypothesis that the enhanced oncolytic effect of dl520, after incubation with chemotherapeutic agents, is mediated by an increased accumulation of YB-1 in the nucleus (due to irinotecan) and by upregulation of CAR (due to trichostatin A). Thus, therapy combining virotherapy, chemotherapy, and histone deacetylase inhibitor treatment is a novel approach to enhance the oncolytic efficacy of dl520.

High Antineoplastic Activity of New Heterocyclic Compounds in Cancer Cells with Resistance Against Classical DNA Topoisomerase II-targeting Drugs

International Journal of Cancer. Journal International Du Cancer. Jul, 2006  |  Pubmed ID: 16450374

Twenty previously synthesized fused heterocyclic DNA-topoisomerase II (Topo II)-inhibiting compounds were investigated for their potential efficacy in various human cancer cell lines that were derived from different tumor entities. Moreover, different multidrug-resistant variants of these cancer cell lines with decreased Topo II expression were investigated. In parental, drug-sensitive cells merely the compounds BD3 and G35 showed efficacies, in terms of microM, which were similar to that of the classical Topo II inhibitor etoposide. On the other hand, most of the tested heterocyclic compounds were found more effective in drug-resistant cells than in the parental, drug-sensitive ones, and some of the compounds showed high antineoplastic efficacy in several drug-resistant cell models. Compounds BD13, BD14 and BD16 exhibited high antineoplastic activities against the drug-resistant sublines EPG85-257RNOV and EPG85-257RDB derived from gastric carcinoma, EPP85-181RNOV and EPP85-181RDB derived from pancreatic carcinoma, MCF-7/Adr derived from breast cancer, D79/86RNOV derived from fibrosarcoma, and MeWoETO1 derived from melanoma. Furthermore, compound D23 was found highly efficient in the multidrug-resistant variants HT-29RNOV and HT-29RDB derived from colon carcinoma, and compound D24 exhibited the highest antineoplastic activity among the tested compounds in the drug-resistant subline MDA-MB-231ROV derived from breast cancer. In conclusion, compounds BD 13, BD 14, BD 16, D 23 and D 24 may be useful for the treatment of different multidrug-resistant cancer cells with cross resistance against "classical" Topo II-targeting drugs.

Significance of Cyclooxygenase 2 and MDR1/P-glycoprotein Coexpression in Ovarian Cancers

Cancer Letters. Apr, 2006  |  Pubmed ID: 15970376

Immunohistochemical analysis of prognostic significance of COX-2 and P-gp expression in ovarian cancers was performed on samples originating from 73 tumors. COX-2-positive cases were shown to demonstrate higher expression of P-gp. The studies demonstrated also that, higher P-gp expression was typical for cases which responded poorly to chemotherapy and for cases with shorter progression-free time. Expression of COX-2 predisposed to a more rapid disease progression. The study documented a relationship between COX-2 and P-gp suggesting that COX-2 inhibitors might investigated in clinical trials as a treatment supplementary to chemotherapy of ovarian cancers.

Complete Reversal of ABCG2-depending Atypical Multidrug Resistance by RNA Interference in Human Carcinoma Cells

Oligonucleotides. 2006  |  Pubmed ID: 16978089

In the chemotherapeutic treatment of patients with disseminated neoplasms, multidrug resistance (MDR) is a major obstacle. ABCG2 (BCRP/MXR), a member of the superfamily of adenosine triphosphate-binding cassette (ABC) transporters, was demonstrated to be associated with "atypical" forms of multidrug-resistant phenotypes of cancer cells. To overcome the ABCG2-depending MDR, two specific anti-ABCG2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the human gastric carcinoma cell line EPG85-257RNOV, exhibiting an atypical MDR phenotype. Because both siRNAs showed biological activity, for stable inhibition of ABCG2 corresponding short hairpin RNA (shRNA) expression vectors were constructed. By treatment of EPG85-257RNOV cells with these constructs, expression of the targeted ABCG2-encoding mRNA and transport protein was inhibited completely. Furthermore, anti-ABCG2 shRNA-treated cells increased cellular drug accumulation to the same level measured in drug-sensitive parental cells. These effects were accompanied by complete reversal of the drug-resistant phenotype. Thus, the data indicate that siRNA- and shRNA-mediated RNAi-based gene therapy may be applicable in preventing and reversing ABCG2-depending atypical MDR.

ABCC2 (MRP2, CMOAT) Can Be Localized in the Nuclear Membrane of Ovarian Carcinomas and Correlates with Resistance to Cisplatin and Clinical Outcome

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Dec, 2006  |  Pubmed ID: 17145840

Cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma. ABCC2 is commonly localized in apical cell membranes and could confer cisplatin resistance. Here, we show that ABCC2 can be localized in the cytoplasmic membrane as well as in the nuclear membrane of various human tissues including ovarian carcinoma cells.

CD46 Expression is Indicative of Shorter Revival-free Survival for Ovarian Cancer Patients

Anticancer Research. Nov-Dec, 2006  |  Pubmed ID: 17214367

The membrane cofactor protein CD46 represents a complement inhibitor, which protects autologous cells from complement - mediated cytotoxicity. CD46 may exhibit the potential to protect tumor cells from the immune responses of the host. The present study aimed to evaluate the prognostic significance of CD46 expression in ovarian cancers.

Taxol-resistance-associated Gene-3 (TRAG-3/CSAG2) Expression is Predictive for Clinical Outcome in Ovarian Carcinoma Patients

Virchows Archiv : an International Journal of Pathology. Feb, 2007  |  Pubmed ID: 17216190

An obstacle in chemotherapy of ovarian cancer is the development of drug resistance. Taxol (paclitaxel)-resistance-associated gene-3 (TRAG-3/CSAG2) was found to be overexpressed in a paclitaxel-resistant ovarian carcinoma cell line. However, clinical impact of TRAG-3 in ovarian carcinoma has not been demonstrated previously. For demonstration of potential clinical impact of TRAG-3, immunohistochemistry was applied to determine TRAG-3 protein expression in specimens obtained from ovarian carcinoma patients (n=37) who received a paclitaxel-based chemotherapy at two different time points, initial laparotomy before chemotherapy, and secondary cytoreduction after chemotherapy. The TRAG-3-specific immunohistochemical staining was correlated with clinical outcome. In ovarian carcinoma specimens obtained at the initial laparotomy, an advantage in overall (P < 0.001) and progression-free (P = 0.003) survival for patients with weak TRAG-3 expression could be demonstrated. Tumor specimens excised at secondary cytoreduction procedure were not predictive for clinical outcome. In summary, TRAG-3 was found to be a prognostic factor for the prediction of clinical outcome after the application of paclitaxel-based chemotherapy.

Nuclear Metallothionein Expression Correlates with Cisplatin Resistance of Ovarian Cancer Cells and Poor Clinical Outcome

Virchows Archiv : an International Journal of Pathology. Mar, 2007  |  Pubmed ID: 17235562

Elevated metallothionein (MT) expression in ovarian cancers treated with cisplatin-based schemes represents an unfavorable prognostic index. MT expression is significantly higher in tumor samples obtained after chemotherapy. The present study aimed at examining MT expression in ovarian carcinoma cells sensitive (A2780) or resistant (A2780RCIS) against platinum drug treatment as well as examining effects of exposure to cisplatin on MT expression. Subcellular expression of MT was evaluated also in samples originating from 73 ovarian tumors. Cisplatin-resistant A2780RCIS cells were exposed to increasing cisplatin concentrations, and the subcellular expression of MT was determined by immunocytochemistry. The studies demonstrated that cisplatin-resistant A2780RCIS cells exposed to cisplatin typically manifested a nuclear MT expression. The study demonstrated also that exposure to cisplatin was paralleled by growing MT expression in cell nuclei. The nuclear expression of MT was also found to be specific for ovarian cancers of poor clinical outcome. No relationship could be demonstrated between cytoplasmic expression of MT and clinical variables. Nuclear MT expression is induced by cisplatin and seems to protect DNA in the cells from toxic effects of the drug.

Expression of Factors Involved in Regulation of DNA Mismatch Repair- and Apoptosis Pathways in Ovarian Cancer Patients

Oncology Reports. Mar, 2007  |  Pubmed ID: 17273726

A major obstacle in treatment of ovarian cancer is intrinsic or acquired drug resistance causing failure of chemotherapy followed by a poor clinical outcome. Drug resistance of ovarian carcinoma can be caused by dysregulation of cellular factors involved in regulation of apoptosis and DNA repair pathways. In this study, 73 ovarian carcinoma specimens obtained before and after chemotherapy were analysed by immunohistochemistry for expression of seven proteins playing an important role in regulation of DNA mismatch repair and apoptosis. The prognostic significance of these proteins in the meaning of overall and progression-free survival was evaluated in univariate and multivariate analysis. Bcl-xL, hMSH2, caspase-3, p21 and p53 displayed prognostic importance in univariate analysis. Furthermore, it was demonstrated that caspase-3 and p21 were also independent prognostic markers for both, overall and progression-free survival. In conclusion, these data indicate that analysis of proteins involved in DNA mismatch repair and apoptosis can be useful for prediction of clinical outcome in ovarian carcinoma patients.

Regulation of MDR1 Gene Expression in Multidrug-resistant Cancer Cells is Independent from YB-1

Biochemical and Biophysical Research Communications. May, 2007  |  Pubmed ID: 17418094

The MDR1 gene encoded transmembrane ABC-transporter MDR1/P-glycoprotein can mediate the phenotype of multidrug resistance (MDR), a major obstacle in the clinical management of cancer patients. It was hypothesized that YB-1 is a fundamental regulatory factor of the MDR1 gene in tumor cells and can therewith enhance drug resistance. To analyze the potential impact of YB-1 in MDR cancer cells, two specific anti-YB-1 small interfering RNAs (siRNAs) were designed for transient triggering the gene-silencing RNA interference (RNAi) pathway in the MDR cell lines EPG85-257RDB and EPP85-181RDB as well as in their drug-sensitive counterparts EPG85-257P and EPP85-181P. Since both siRNAs showed biological activity, for stable inhibition of YB-1 corresponding tetracycline-inducible short hairpin RNA (shRNA)-encoding expression vectors were designed. By treatment of the cancer cells with these constructs, the expression of the targeted YB-1 encoding mRNA and protein was completely inhibited following tetracycline exposure. These gene-silencing effects were not accompanied by modulation of the MDR1 expression or by reversal of the drug-resistant phenotype. In conclusion, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that YB-1 is not involved in the regulation of the MDR1 gene or the development of the drug-resistant phenotype in MDR cancer cells.

The Gene Polymorphism of the Angiotensin I-converting Enzyme Correlates with Tumor Size and Patient Survival in Colorectal Cancer Patients

Neoplasia (New York, N.Y.). Sep, 2007  |  Pubmed ID: 17898867

We studied the putative significance of angiotensin I-converting enzyme (ACE) in colorectal cancer (CRC) biology. Local expression of ACE was investigated by quantitative reverse transcription-polymerase chain reaction and by immunohistochemistry in CRCs and adenomas. ACE insertion (I)/deletion (D) polymorphism was studied in 141 CRC patients and 189 controls. ACE mRNA was upregulated in CRCs compared to corresponding nonlesional tissues (2.5-fold; P = .009). ACE protein was more commonly expressed in adenomas [17 (81%)] and cancer epithelial cells [22 (100%)] than in corresponding non-neoplastic crypt and surface epithelium [2 (10%) and 2 (9%), respectively]. Thirty-seven CRC patients (26%) carried II genotype, 69 (49%) carried ID genotype, and 35 (25%) carried DD genotype. The distribution of the genotypes did not differ from that of controls. Female CRC patients more commonly carried the ID genotype and less frequently the II and DD genotypes compared with male patients (P = .033). Men heterozygous or homozygous for the D-allele had larger tumors compared to carriers of the II genotype (P < .01). Women homozygous for the D-allele lived longer than carriers of the ID and II genotypes. Our study shows that ACE is differentially expressed in CRCs and that gene polymorphism is associated with gender-specific differences in primary tumor size and patient survival.

A Web-based Data Warehouse on Gene Expression in Human Malignant Melanoma

The Journal of Investigative Dermatology. Feb, 2007  |  Pubmed ID: 16946712

The identification of melanoma-specific dysregulated genes could identify new molecular markers. By applying bioinformatic tools for screening of biomedical databases, a melanoma-specific gene expression profile "data warehouse" was constructed. Utilizable data sets of global gene expression analyses were available from nine studies that applied different technology platforms. A single study used cell lines, five investigations analyzed cell lines and tissues obtained from patients, two studies used exclusively specimens obtained from patients, and one study analyzed blood cells prepared from patients. The total number of investigated patients was 116. From 815 differential-regulated genes, 772 (95%) were identified merely in a single study, 37 in at least two studies, five (RAB33A, ERBB3, ADRB2, MERTK, SNF1LK, and ITPKB) in at least three studies, and a single gene, RAB33A, in four studies. These data show that the accuracy, reproducibility, and comparability among different gene expression profile studies are low in melanoma. In conclusion, the study demonstrates the high diversity of gene expression profiles associated with melanoma, the necessity to include a sufficient number of samples regarding clinical standards, for the design of standardized sample collecting and preparation, for the development of common standards for microarray data processing, and for developing standardized bioinformatic tools.

Resistance to Chemotherapy in Ovarian Carcinoma

Recent Results in Cancer Research. Fortschritte Der Krebsforschung. Progrès Dans Les Recherches Sur Le Cancer. 2007  |  Pubmed ID: 17607916

Resistance to cytotoxic chemotherapy is the main cause of therapeutic failure and death in women suffering from ovarian carcinoma. The standard first-line chemotherapy of ovarian cancer consists of a combination of a taxane and a platinum-containing drug. Thus, the cellular and molecular mechanisms involved in resistance against these compounds are of vital importance in the context of chemotherapy of ovarian cancer. This review will discuss the current state of knowledge of drug resistance-associated factors and their impact on clinical chemotherapy response in ovarian carcinoma as well as different strategies for reversal of drug resistance.

Overcoming the Classical Multidrug Resistance Phenotype by Adenoviral Delivery of Anti-MDR1 Short Hairpin RNAs and Ribozymes

International Journal of Oncology. Aug, 2007  |  Pubmed ID: 17611700

Simultaneous resistance of cancer cells to multiple cytotoxic drugs, multidrug resistance (MDR), is the major limitation to the successful chemotherapeutic treatment of disseminated neoplasms. The 'classical' MDR phenotype is conferred by MDR1/P-glycoprotein (MDR1/P-gp) that is expressed in almost 50% of human cancers. Recent developments in the use of small interfering RNAs for specific inhibition of gene expression have highlighted their potential use as therapeutic agents. DNA cassettes encoding RNA polymerase III promoter-driven siRNA-like short hairpin RNAs (shRNAs) allow long-term expression of therapeutic RNAs in targeted cells. A variety of viral vectors have been used to deliver such cassettes to mammalian cells. In this study, the construction of different adenoviruses for anti-MDR1/P-gp shRNA delivery in different human multidrug-resistant cancer cells was investigated. The efficiency of the shRNAs was compared to adenoviral delivery of an anti-MDR1/P-gp ribozyme construct. It could be demonstrated that MDR1/P-gp mRNA and protein expression could be completely inhibited by adenoviral delivery of anti-MDR1/P-gp shRNAs. This downregulation in mRNA and protein expression was accompanied by a complete inhibition of the pump activity of MDR1/P-gp and a reversal of the multidrug-resistant phenotype. By application of adenoviral encoded anti-MDR1/P-gp ribozyme construct merely weak effects on gene expression were observed. In conclusion, the data demonstrate that adenoviral delivery of shRNAs can chemosensitize human cancer cells, that adenoviral delivery of shRNAs is much more effective than adenoviral delivery of ribozymes, and that adenovirus-based vectors can be very effective agents for efficient delivery of therapeutic RNA molecules.

Antineoplastic Activity of 2-methoxyestradiol in Human Pancreatic and Gastric Cancer Cells with Different Multidrug-resistant Phenotypes

Journal of Gastroenterology and Hepatology. Sep, 2007  |  Pubmed ID: 17645459

Chemoresistance often leads to loss of the last treatment option for cancer. 2-Methoxyestradiol (2-ME2) has been shown to inhibit tumor growth. The aim was to examine the efficacy of 2-ME2 on multidrug-resistant human cells from pancreatic and gastric cancer.

Occurence of Stromal Myofibroblasts in the Invasive Ductal Breast Cancer Tissue is an Unfavourable Prognostic Factor

Anticancer Research. Jul-Aug, 2007  |  Pubmed ID: 17695471

Numerous experimental studies have described the capacity of myofibroblasts to stimulate mammary cancer cells in a paracrine manner. Until now, the prognostic significance of myofibroblasts present in breast cancer has not been examined.

Complete in Vivo Reversal of the Multidrug Resistance Phenotype by Jet-injection of Anti-MDR1 Short Hairpin RNA-encoding Plasmid DNA

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jan, 2008  |  Pubmed ID: 17878902

Triggering the RNA interference (RNAi) pathway by inducing the expression of short hairpin RNA (shRNA) molecules has become a promising tool for efficient silencing of a given gene in gene therapy applications. In this study, shRNA encoding DNA was utilized to reverse the classical MDR1/P-glycoprotein (MDR1/P-gp)-mediated multidrug resistance (MDR) phenotype in vivo. For the first time, the nonviral jet-injection technology was applied for delivering naked shRNA-vector constructs for direct intratumoral in vivo transfer. The highly efficient anti-MDR1 shRNA expression vectors were applied twice in the human MDR1/P-gp overexpressing MaTu/ADR cancer xenograft-bearing mice, and twice in the corresponding drug-sensitive parental MaTu tumor xenograft bearing mice as well. Two days after anti-MDR1 shRNA vector injection, the expression level of the MDR1 messenger RNA (mRNA) was decreased by more than 90% and the corresponding MDR1/P-gp protein was no longer detectable in the tumors. Two jet-injections of anti-MDR1 shRNA vectors into the tumors, combined with two intravenous (IV) administrations of doxorubicin, were sufficient to achieve complete reversal of the drug-resistant phenotype. The data show that jet-injection delivery of shRNA-expressing vectors is effective in reversing MDR1/P-gp-mediated MDR in vivo, and is therefore a promising strategy for making tumors with an MDR1/Pgp-dependent MDR phenotype revert to a drug-sensitive state.

Impact of Radiation Therapy on the Oncolytic Adenovirus Dl520: Implications on the Treatment of Glioblastoma

Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology. Mar, 2008  |  Pubmed ID: 17967494

Viral oncolytic therapy is emerging as a new form of anticancer therapy and has shown promising preclinical results, especially in combination with radio- and chemotherapy. We recently reported that nuclear localization of the human transcription factor YB-1 in multidrug-resistant cells facilitates E1-independent adenoviral replication. The aim of this study was to evaluate the combined treatment of the conditionally-replicating adenovirus dl520 and radiotherapy in glioma cell lines in vitro and in human tumor xenografts. Furthermore, the dependency of YB-1 on dl520 replication was verified by shRNA directed down regulation of YB-1.

Three New Jatrophane Polyesters and Antiproliferative Constituents from Euphorbia Tuckeyana

Planta Medica. Jan, 2008  |  Pubmed ID: 18176909

Three new jatrophane diterpene polyesters named tuckeyanols A and B and euphotuckeyanol, as well as ten known compounds, helioscopinolides A, B, D and E, naringenin, aromadendrin, coniferaldehyde, 4,20-dideoxy-5-hydroxyphorbol 12,13-diisobutyrate, 4,20-dideoxy-5-hydroxyphorbol 12-benzoate-13-isobutyrate and dehydrodiconiferyl diacetate, were isolated from the methanolic extract of Euphorbia tuckeyana. Their structures were elucidated by physical and spectroscopic methods including 1 D and 2 D homo- and heteronuclear NMR techniques (COSY, HMQC, HMBC and NOESY), and HR-mass spectrometry. Four of the isolated compounds were investigated for their antiproliferative activity in three human gastrointestinal cancer cell lines: gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29) carcinomas. Three of them have showed to be moderate inhibitors of the growth of gastric and pancreatic tumor cell lines.

Decreased Expression of P16 in Ovarian Cancers Represents an Unfavourable Prognostic Factor

Histology and Histopathology. May, 2008  |  Pubmed ID: 18283637

Decreased expression of p16 may result from hypermethylation of the promoter or from deletion of the gene. It can lead to intensified proliferation of neoplastic cells and to cytostatic drug resistance. The study was aimed at the examination of prognostic value of p16 expression in relation to Ki67 and caspase-3 in ovarian cancers using immunohistochemistry. The immunohistochemical studies were performed on 73 paraffin-embedded samples of ovarian cancers from 43 patients and samples from 6 healthy ovaries. We have used monoclonal antibodies against p16. ABC method and DAB were used for antigens visualisation. The intensity of the immunohistochemical reactions was appraised using the semi-quantitative IRS scale. In healthy ovaries we have shown strong reaction in the nuclei of surface epithelium. In the case of studied ovarian cancers, the reaction of a nuclear and cytoplasmic localization was obtained. The mean overall immunoreactivity score of nuclear p16 expression amounted to 5.30+/-3.44 SD in primary laparotomy material and 6.61+/-4.34 SD in secondary cytoreduction material. Statistical analysis demonstrated that lower p16 expression was typical of the younger patients and the patients who died. Kaplan-Meier's analysis proved that lower expression of p16 was characteristic of cases with shorter overall survival. In the present study we have demonstrated that lowered p16 expression represented an unfavourable prognostic index in ovarian cancer. Lowered p16 expression was also typical for chemotherapy-resistant ceases (cases of lower caspase-3 and higher Ki67 at secondary cytoreduction expression).

Improved Cellular Pharmacokinetics and Pharmacodynamics Underlie the Wide Anticancer Activity of Sagopilone

Cancer Research. Jul, 2008  |  Pubmed ID: 18593931

Sagopilone (ZK-EPO) is the first fully synthetic epothilone undergoing clinical trials for the treatment of human tumors. Here, we investigate the cellular pathways by which sagopilone blocks tumor cell proliferation and compare the intracellular pharmacokinetics and the in vivo pharmacodynamics of sagopilone with other microtubule-stabilizing (or tubulin-polymerizing) agents. Cellular uptake and fractionation/localization studies revealed that sagopilone enters cells more efficiently, associates more tightly with the cytoskeleton, and polymerizes tubulin more potently than paclitaxel. Moreover, in contrast to paclitaxel and other epothilones [such as the natural product epothilone B (patupilone) or its partially synthetic analogue ixabepilone], sagopilone is not a substrate of the P-glycoprotein efflux pumps. Microtubule stabilization by sagopilone caused mitotic arrest, followed by transient multinucleation and activation of the mitochondrial apoptotic pathway. Profiling of the proapoptotic signal transduction pathway induced by sagopilone with a panel of small interfering RNAs revealed that sagopilone acts similarly to paclitaxel. In HCT 116 colon carcinoma cells, sagopilone-induced apoptosis was partly antagonized by the knockdown of proapoptotic members of the Bcl-2 family, including Bax, Bak, and Puma, whereas knockdown of Bcl-2, Bcl-X(L), or Chk1 sensitized cells to sagopilone-induced cell death. Related to its improved subcellular pharmacokinetics, however, sagopilone is more cytotoxic than other epothilones in a large panel of human cancer cell lines in vitro and in vivo. In particular, sagopilone is highly effective in reducing the growth of paclitaxel-resistant cancer cells. These results underline the processes behind the therapeutic efficacy of sagopilone, which is now evaluated in a broad phase II program.

Acquired Cisplatin Resistance in the Head-neck Cancer Cell Line Cal27 is Associated with Decreased DKK1 Expression and Can Partially Be Reversed by Overexpression of DKK1

International Journal of Cancer. Journal International Du Cancer. Nov, 2008  |  Pubmed ID: 18688867

Head and neck cancers are treated by a combination of surgery, radiotherapy and/or chemotherapy. The clinical success of cisplatin-based chemotherapy, mostly in combination with 5-FU or a taxane, is however limited by multifactorial intrinsic or acquired resistance. So far, known genes involved in cisplatin resistance do not sufficiently allow the prediction of cancer chemosensitivity. Thus, the purpose of this study was to search for further genes involved in cisplatin resistance by differential gene expression analysis of the parental tongue cancer cell line Cal27 and its 10-fold more resistant sub-cell line Cal27cis, which was obtained by treating Cal27 with increasing concentrations of cisplatin. As found by the suppression subtractive hybridization, expression of DKK1, an inhibitor of canonical WNT signaling, was decreased in Cal27cis. Microarray analysis, qPCR and ELISA confirmed the approximately 2-fold difference in expression. Cisplatin treatment and serum starvation increased by 2-fold the secretion of DKK1 in Cal27 and Cal27cis, thus rendering DKK1-levels significantly different in both cell lines under basal and stress conditions. Recombinant overexpression of DKK1 in Cal27 and Cal27cis resulted in clonal cell lines, which were both 2.2- to 3-fold more sensitive toward cisplatin in cell viability (MTT) and in proliferation (BrdU) assays. In conclusion, acquired (10-fold) resistance of Cal27 against cisplatin is associated with decreased DKK1 expression and could partially be reversed by DKK1 overexpression, thus suggesting DKK1 and the WNT signaling pathway as a marker and target for cisplatin chemosensitivity.

Novel Insight in Structure-activity Relationship and Bioanalysis of P-glycoprotein Targeting Highly Potent Tetrakishydroxymethyl Substituted 3,9-diazatetraasteranes

Journal of Medicinal Chemistry. Sep, 2008  |  Pubmed ID: 18714979

Novel 3,9-diazatetraasteranes have been synthesized with varied aromatic substitution patterns and evaluated as P-glycoprotein (P-gp) inhibitors. Structure-activity relationships (SAR) are discussed in relation to determined physicochemical properties. The potential to induce P-gp expression has been evaluated in cancer cell lines. The bioanalytical results indicate favorable noninducing properties compared to P-gp inducing drug standard.

Estradiol, Tamoxifen and ICI 182,780 Alter Alpha3 and Beta1 Integrin Expression and Laminin-1 Adhesion in Oral Squamous Cell Carcinoma Cell Cultures

Oral Oncology. Jan, 2008  |  Pubmed ID: 17418616

Adhesion molecules such as integrins and extracellular matrix proteins like laminins have been identified to play an important role in cell proliferation, migration and invasion by regulating cell-extracellular matrix interaction in various cancers including oral squamous cell carcinoma (OSCC). In this study, the effect of estradiol (E2), and the E2 antagonists tamoxifen (TAM) and ICI 182,780 (ICI) on the expression of integrins and adhesion to laminin-1 in different OSCC in vitro models was analyzed. TAM and ICI inhibited growth in all OSCC cell lines. Dependent on estrogen receptor (ER) status E2 displayed a significant influence on growth after long-term administration. ICI reduced laminin-1 adhesion in all cell lines. beta1 Integrin transcription is reduced with TAM and E2 and alpha3 cell surface expression with TAM. This study shows that OSCC is estrogen and SERM sensitive and that these compounds can modulate cell-matrix interaction in part by modulating integrin expression and translation. The investigation also confirms that growth is significantly influenced by these adjuvant therapeutics. These data suggest that a greater understanding of basic biology and mechanisms of the ER and its ligands in oral squamous cells is needed to elucidate the use of specific pharmacological agents as therapeutics of anti-tumorigenic pathways.

Positive Correlation Between Cyclooxygenase 2 and the Expression of ABC Transporters in Non-small Cell Lung Cancer

Anticancer Research. Sep-Oct, 2008  |  Pubmed ID: 19031941

The primary method of treatment of non-small cell lung cancer (NSCLC) in stage IIIB and IV is chemotherapy. Previous data suggested a correlation between cyclooxygenase-2 (COX-2) expression and the multidrug-resistant phenotype of cancer cells.

Topoisomerase IIalpha MRNA and Protein Expression in Ovarian Carcinoma: Correlation with Clinicopathological Factors and Prognosis

Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc. Apr, 2009  |  Pubmed ID: 19270648

Topoisomerase IIalpha (Top IIalpha) is a nuclear enzyme that plays a central role in DNA metabolism, and is a molecular target for a variety of chemotherapeutic agents. Top IIalpha has recently gained attention as a biomarker for therapy response and patient survival. In this study, we attempted to assess the feasibility of measuring Top IIalpha gene expression in RNA, isolated from archival formalin-fixed paraffin-embedded tissue specimens, which are used routinely in pathology laboratories. We have employed a new technique on the basis of magnetic particles' separation and purification of nucleic acids, and evaluated both protein and mRNA expressions from the same routinely processed tissue blocks. We investigated the expression of Top IIalpha mRNA and protein by real-time reverse transcription polymerase chain reaction and immunohistochemistry, in a cohort of 133 primary ovarian carcinomas, and evaluated the association between Top IIalpha expression and clincopathological variables as well as patient outcome. Elevated Top IIalpha mRNA expression was observed in high-grade tumors (P=0.003) and advanced stage disease (P=0.011). In univariate Kaplan-Meier analysis, patients with higher expression of Top IIalpha nuclear protein had a significantly decreased overall survival (P=0.045). Interestingly, we detected cytoplasmic protein expression of Top IIalpha in a subset of samples. Cytoplasmic expression of Top IIalpha was associated with the expression of chromosomal region maintenance/exportin 1 (CRM1)-a nuclear export protein (P=0.008). Our study suggests that Top IIalpha overexpression is involved in the progression of ovarian cancer in a subset of the patients. Our results encourage the further evaluation of the prognostic and predictive values of Top IIalpha expression in ovarian carcinoma, which might help to assess the patients' risk profile, and the planning of an individualized therapy.

Therapeutic Potential of RNA Interference in Drug-resistant Cancers

Future Oncology (London, England). Mar, 2009  |  Pubmed ID: 19284376

Resistance including multidrug resistance to chemotherapy is a common clinical problem in patients suffering from cancer. Multidrug resistance is often mediated by overexpression of transmembrane xenobiotic transport molecules belonging to the superfamily of ATP-binding cassette (ABC)-transporters. Inhibition of ABC-transporters by low-molecular weight compounds in cancer patients has been extensively investigated in clinical trials, but the results have been disappointing. Thus, alternative experimental therapeutic strategies for overcoming multidrug resistance are under investigation. These include the application of RNA interference (RNAi) technology. Various RNAi strategies were applied to reverse multidrug resistance in different tumor models in vitro and in vivo. Results and conclusions of these RNAi studies as well as their potential impact for the development of potential RNAi therapeutics will be discussed.

A Prognostic Gene Expression Index in Ovarian Cancer - Validation Across Different Independent Data Sets

The Journal of Pathology. Jun, 2009  |  Pubmed ID: 19294737

Ovarian carcinoma has the highest mortality rate among gynaecological malignancies. In this project, we investigated the hypothesis that molecular markers are able to predict outcome of ovarian cancer independently of classical clinical predictors, and that these molecular markers can be validated using independent data sets. We applied a semi-supervised method for prediction of patient survival. Microarrays from a cohort of 80 ovarian carcinomas (TOC cohort) were used for the development of a predictive model, which was then evaluated in an entirely independent cohort of 118 carcinomas (Duke cohort). A 300-gene ovarian prognostic index (OPI) was generated and validated in a leave-one-out approach in the TOC cohort (Kaplan-Meier analysis, p = 0.0087). In a second validation step, the prognostic power of the OPI was confirmed in an independent data set (Duke cohort, p = 0.0063). In multivariate analysis, the OPI was independent of the post-operative residual tumour, the main clinico-pathological prognostic parameter with an adjusted hazard ratio of 6.4 (TOC cohort, CI 1.8-23.5, p = 0.0049) and 1.9 (Duke cohort, CI 1.2-3.0, p = 0.0068). We constructed a combined score of molecular data (OPI) and clinical parameters (residual tumour), which was able to define patient groups with highly significant differences in survival. The integrated analysis of gene expression data as well as residual tumour can be used for optimized assessment of the prognosis of platinum-taxol-treated ovarian cancer. As traditional treatment options are limited, this analysis may be able to optimize clinical management and to identify those patients who would be candidates for new therapeutic strategies.

Interleukin-1 Beta and Tumor Necrosis Factor-alpha Increase ABCG2 Expression in MCF-7 Breast Carcinoma Cell Line and Its Mitoxantrone-resistant Derivative, MCF-7/MX

Inflammation Research : Official Journal of the European Histamine Research Society ... [et Al.]. Oct, 2009  |  Pubmed ID: 19333723

In this study, we aimed to evaluate the influence of proinflammatory cytokines on ABCG2 expression and function in human MCF-7 breast cancer cell line and its mitoxantrone-resistant derivative MCF-7/MX.

Comparision of the Cytotoxic Effects of Birch Bark Extract, Betulin and Betulinic Acid Towards Human Gastric Carcinoma and Pancreatic Carcinoma Drug-sensitive and Drug-resistant Cell Lines

Molecules (Basel, Switzerland). 2009  |  Pubmed ID: 19396022

Betulin and betulinic acid are naturally occurring pentacyclic triterpenes showing cytotoxicity towards a number of cancer cell lines. These compounds can be found in the bark of the many plants. In this report we have compared the cytotoxic activity of crude birch bark extract and purified betulin and betulinic acid towards human gastric carcinoma (EPG85-257) and human pancreatic carcinoma (EPP85-181) drug-sensitive and drug-resistant (daunorubicin and mitoxantrone) cell lines. Our results show significant differences in sensitivity between cell lines depending on the compound used, and suggest that both betulin and betulinic acid can be considered as a promising leads in the treatment of cancer.

Expression of Multidrug Resistance-associated Protein 1 in Invasive Ovarian Carcinoma: Implication for Prognosis

Histopathology. May, 2009  |  Pubmed ID: 19438741

Multidrug resistance is a major impediment in chemotherapeutic treatment of ovarian carcinoma patients. The aim of this study was to investigate the expression of multidrug resistance-associated protein 1 (MRP1) and to assess the possible associations with clinicopathological variables and patient outcome in primary ovarian carcinoma.

Influx and Efflux Transport As Determinants of Melphalan Cytotoxicity: Resistance to Melphalan in MDR1 Overexpressing Tumor Cell Lines

Biochemical Pharmacology. Jul, 2009  |  Pubmed ID: 19447222

There is a considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines (r(2)=0.47, P=0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan (r(2)=0.56, P=0.03 and r(2)=0.62, P=0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1 overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non-MDR1 expressing one. Again, the resistance was reversed by cyclosporine and by MDR1-specific shRNA. LAT1 was the major influx transporter in tumor cell lines with 4000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.

Evaluation of Microarray Preprocessing Algorithms Based on Concordance with RT-PCR in Clinical Samples

PloS One. 2009  |  Pubmed ID: 19461970

Several preprocessing algorithms for Affymetrix gene expression microarrays have been developed, and their performance on spike-in data sets has been evaluated previously. However, a comprehensive comparison of preprocessing algorithms on samples taken under research conditions has not been performed.

Delivery of Short Hairpin RNAs by Transkingdom RNA Interference Modulates the Classical ABCB1-mediated Multidrug-resistant Phenotype of Cancer Cells

Cell Cycle (Georgetown, Tex.). Oct, 2009  |  Pubmed ID: 19770582

Delivery of RNA interference (RNAi)-mediating agents to target cells is one of the major obstacles for the development of RNAi-based therapies. One strategy to overcome this barrier is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. In this study, the tkRNAi approach was used for modulation of the "classical" ABCB1-mediated multidrug resistance (MDR) in human cancer cells. Subsequent to treatment with anti-ABCB1 shRNA expression vector bearing E. coli, MDR cancer cells (EPG85 257RDB) showed 45% less ABCB1 mRNA expression. ABCB1 protein expression levels were reduced to a point at which merely a weak band could be detected. Drug accumulation was enhanced 11-fold, to an extent that it reached 45% of the levels in non-resistant cells and resistance to daunorubicin was decreased by 40%. The data provide the proof-of-concept that tkRNAi is suitable for modulation of "classical" MDR in human cancer cells. Overall, the prototype tkRNAi system tested here did not yet attain the levels of gene silencing seen with conventional siRNAs nor virally delivered shRNAs; but the tkRNAi system for gene-silencing of ABCB1 is still being optimized, and may become a powerful tool for delivery of RNAi effectors for the reversal of cancer MDR in future.

Positive Correlation Between Cyclooxygenase-2 and ABC-transporter Expression in Non-Hodgkin's Lymphomas

Oncology Reports. Dec, 2009  |  Pubmed ID: 19885582

One of the leading causes of chemotherapy failure in non-Hodgkin's lymphomas (NHLs) is multidrug resistance (MDR). MDR can be associated with expression of members of the family of ABC-transporters. Since a correlation between expression of cyclooxygenase-2 (COX-2) and MDR in various cancer cells was described, the expression of COX-2 and the ABC-transporters MDR1/P-glycoprotein (P-gp), MRP1, MRP2 and BCRP was examined in 56 previously non-treated patients by immunohistochemistry. The data show that: i) P-gp is not expressed in non-treated NHLs; ii) MRP2 can be localized in the nuclear membranes of NHL cells; iii) expression of MRP2 in the cytoplasm membrane correlates with clinical response; iv) elevated expression of BCRP is typical for the patients, who did not respond to primary chemotherapy and for cases with shorter progression-free survival time in a 30 months follow-up; and v) there is a strong correlation between COX-2 and MRP1, MRP2 and BCRP. It can be concluded that: i) BCRP may be a crucial factor involved in primary resistance of NHLs, thus it may be useful for prediction of chemotherapeutic treatment and risk of relapse; and ii) since there is strong correlation between COX-2 expression and MDR in NHLs, the application of COX-2 inhibitors may be considered for chemosensitization.

Impact of Novel MDR Modulators on Human Cancer Cells: Reversal Activities and Induction Studies

Pharmaceutical Research. Jan, 2009  |  Pubmed ID: 18972190

Novel multidrug resistance (mdr) modulators have been proved as inhibitors of P-glycoprotein (P-gp). We first investigated the in vitro effects of selected compounds in human cancer cells on multidrug resistance reversal effects compared to drug standards and on P-gp induction to characterize the potential of the compounds as clinical candidates.

Proteomic Approaches for Investigation of Therapy Resistance in Cancer

Proteomics. Clinical Applications. Aug, 2009  |  Pubmed ID: 21136994

Resistance to anticancer therapy is a major obstacle for successful management of patients in oncology. Although in the past, various biological mechanisms involved in therapy resistance, in particular multidrug resistance, have been identified, cancer patients did not really benefit. The mechanisms include the enhanced activity of drug extrusion pumps, modulation of cellular death pathways, alteration and repair of target molecules and various other mechanisms. Together they build a complex network mediating an individual therapy-resistant phenotype. The improved description of this multifactorial network should be useful for prediction of treatment response and would allow to design an individual-tailored therapy regiment. Proteome analyzing technologies appear as powerful tools for identifying new factors and protein expression profiles associated with anticancer therapy resistance. In the last years, the application of proteomic techniques identified multiple new factors or protein expression signatures in drug-resistant cell models and cancerous tissues. However, the functional role and the clinical impact of these findings are not yet clarified. So far, none of the proteomic data were useful for the development of improved diagnostic tests, for prediction of individual therapy response or for development of updated chemosensitizers. Here, the previous therapy resistance-related proteome data and future perspectives will be discussed.

Betulinic Acid Exhibits Stronger Cytotoxic Activity on the Normal Melanocyte NHEM-neo Cell Line Than on Drug-resistant and Drug-sensitive MeWo Melanoma Cell Lines

Molecular Medicine Reports. Jul-Aug, 2009  |  Pubmed ID: 21475863

Betulinic acid is a triterpene isolated from the bark of many plants that exhibits cytotoxicity in several cancer cell lines and is capable of inducing apoptosis. In this study, we examined the cytotoxic activity and apoptotic ability of betulinic acid in the drug-sensitive (MeWo) and drug-resistant melanoma MeWo CIS (cisplatin), MeWo ETO (etoposide), MeWo VIN (vinblastin) and MeWo FOTE (fotemusine) cell lines, as well as in the normal melanocyte NHEM-neo cell line. The results show that betulinic acid exhibited significant cytotoxicity on all the cell lines. However, a sulphorhodamine B cell proliferation assay and immunocytochemical analysis of Ki67 expression revealed the strongest cytotoxicity on the normal melanocyte cell line, NHEM-neo. Flow cytometry and immunocytochemical analysis of caspase 3 expression was used to confirm cell death by apoptosis. In conclusion, betulinic acid is a potential candidate for anticancer research, and may also have an application in the cosmetics industry.

Overcoming Multidrug Resistance by RNA Interference

Methods in Molecular Biology (Clifton, N.J.). 2010  |  Pubmed ID: 19949936

The ATP-binding cassette (ABC)-transporter P-glycoprotein (Pgp, also known as ABCB1) is the best characterized factor involved in multidrug resistance (MDR) of cancer cells. Pgp, which is encoded by the MDR1 gene, acts as a membrane-embedded drug extrusion pump for multiple structurally unrelated cytotoxic drugs. Inhibition of the pump activity of Pgp by low-molecular weight pharmacologically active compounds as a method to reverse MDR in cancer patients has been studied extensively, but so far clinical trials have generally been disappointing. Thus, experimental strategies for overcoming MDR are under investigation. These approaches include the application of the RNA interference (RNAi) technology. RNAi is a physiological mechanism triggered by small double-stranded RNA molecules resulting in a sequence-specific gene-silencing. Besides its potential for development of novel therapeutics, RNAi also offers the possibility for specific inhibition of cellular targets in functional investigations. For specific inhibition of Pgp by triggering the RNAi pathway, transient gene-silencing by application of small interfering RNA (siRNA), and stable inhibition by transfection of MDR cancer cells with short hairpin RNA (shRNA) encoding expression cassettes encoded on plasmid DNA are described. Efficacy of RNAi on MDR1 mRNA expression level is determined by quantitative real-time RT-PCR and Northern blot. The consequences of RNAi on protein expression level are measured by Western blot and immunohistochemistry. The effects on the drug extrusion activity are measured by a drug accumulation assay based on flow cytometry, and reversal of the drug-resistant phenotype by assessment of drug-specific IC(50)-values by a cell proliferation assay based on colorimetry.

Phenolic Compounds As Selective Antineoplasic Agents Against Multidrug-resistant Human Cancer Cells

Planta Medica. Jul, 2010  |  Pubmed ID: 20157880

Twelve phenolic compounds, including three stilbenes, two flavonoids, two coumarins, one neolignan, and four lignans, isolated from Euphorbia and Pycnanthus species or obtained by derivatization, were assayed for their potential antineoplastic efficacy in three human cancer cell lines: gastric (EPG85-257), pancreatic (EPP85-181), and colon (HT-29) carcinomas as well as derived multidrug-resistant sublines. In each case, two different multidrug-resistant variants, i.e., cell lines with classical and atypical MDR phenotype, were used. The majority of the MDR cancer sublines showed increased sensitivities to the studied compounds when compared to the parental sublines. The most active compound was the flavonoid naringenin, found to be 15-fold more effective against the atypical MDR subline of gastric carcinoma than in parental drug-sensitive cells. Furthermore, the stilbene trans-3,5,3',4'-tetramethoxypiceatannol and the lignans 4'-hydroxy-3,3',4-trimethoxylignan and heliobuphthalmin also exhibited high antineoplasic activities against the classical MDR subline derived from gastric carcinoma. The results of this study suggest that some phenolic compounds might be valuable for the treatment of multidrug-resistant cancer cells.

Effect of YB-1 on the Regulation of Micro RNA Expression in Drug-sensitive and Drug-resistant Gastric Carcinoma Cells

Anticancer Research. Feb, 2010  |  Pubmed ID: 20332481

The multifunctional Y-Box protein 1 (YB-1) exerts positive and negative regulatory effects on gene expression by different mechanisms. Since transcription can be controlled by micro RNAs (miRNAs), YB-1 could also cause effects on gene expression by regulation of cellular miRNAs. To test this hypothesis, a previously established and well-characterized cell model derived from drug-sensitive (EPG85-257P/tetR/YB-1) and multidrug-resistant (EPG85-257RDB/tetR/YB-1) gastric carcinoma cells, in which the expression of YB-1 can be inhibited by tetracycline-dependent triggering of the RNA interference (RNAi) pathway, was investigated concerning their miRNA expression profiles in the presence and absence of YB-1. Microarray hybridizations demonstrated that six miRNAs (miR-96*, miR-210, miR-503, miR-623, miR-1275, miR-1290) were up-regulated more than 1.5-fold in drug-sensitive cells following YB-1 inhibition, but no differences in miRNA expression could be detected in multidrug-resistant cells. Independent validation of these findings by quantitative real-time reverse transcriptase polymerase chain reaction did not confirm these effects. Likewise, an in silico analysis of potential regulatory effects of the miRNAs on their target genes did not support the potential miRNA regulatory effects of YB-1. In conclusion, the data provide evidence that YB-1 has no direct influence on global miRNA expression pattern in different variants of gastric carcinoma cells and, therewith, does not control gene expression by regulation of miRNAs.

Regulation of MRNA Expression in Drug-sensitive and Drug-resistant Gastric Carcinoma Cells is Independent of YB-1 Expression

Anticancer Research. Feb, 2010  |  Pubmed ID: 20332492

Y-Box protein 1 (YB-1) is a multifunctional cellular protein expressed in a range of mammalian cells, including human cancer cells. It is involved in the regulation of various genes including cancer-associated genes, but the full range of target genes and regulatory mechanisms have not been fully elucidated. To identify global mRNA expression patterns that are potentially regulated by YB-1, a previously established and well-characterized cell model derived from drug-sensitive (EPG85-257P/tetR/YB-1) and multidrug-resistant (EPG85-257RDB/tetR/YB-1) gastric carcinoma cells in which the expression of YB-1 can be inhibited by tetracycline-dependent activation of the RNA interference (RNAi) pathway, was analyzed by microarray technology. By this approach, various potentially regulated genes encoding members of important cellular pathways such as the Jak/STAT, VEGF and the MAP-kinase signaling pathways were identified. Independent validation of these findings by quantitative real-time reverse transcriptase polymerase chain reaction and Western blot did not confirm these regulatory effects. In conclusion, the findings suggest that YB-1 is not directly involved in the regulation of mRNA expression in drug-sensitive or drug-resistant gastric carcinoma cells.

Jet-injection of Short Hairpin RNA-encoding Vectors into Tumor Cells

Methods in Molecular Biology (Clifton, N.J.). 2010  |  Pubmed ID: 20387147

The use of the RNA interference (RNAi) through the expression of small hairpin RNA (shRNA) is a promising approach for efficient gene silencing for therapeutic applications. In this chapter, we describe the in vivo reversal of the classical MDR1/P-glycoprotein (MDR1/P-gp)-mediated multidrug resistance (MDR) phenotype by shRNA. For local intratumoral delivery of naked shRNA-encoding vector constructs, the nonviral jet-injection was used. This jet-injector system uses compressed air to inject small volumes (5-10 muL) of naked nucleic acid solutions into tumor tissues. Furthermore, the design of the jet-injector allows multiple injections. Under our experimental design, the delivery of plasmid DNA encoding anti-MDR shRNA by jet-injection into human MDR1/P-gp overexpressing MaTu/ADR breast cancer xenografts resulted in a decrease of MDR1 mRNA expression level to more than 90%. Accordingly, the corresponding MDR1/P-gp protein is no longer detectable in the tumors after anti-MDR1 shRNA vector injection. Furthermore, combination of two intratumoral jet-injections of anti-MDR1 shRNA vectors with two intravenous administrations of doxorubicin is sufficient for a complete reversal of the MDR phenotype in association with tumor growth inhibition.

Influence of Tamoxifen on Cisplatin-sensitivity and Estrogen Receptors Expression in Ovarian Carcinoma Cell Lines

Ginekologia Polska. Mar, 2010  |  Pubmed ID: 20486538

Tamoxifen, used in breast cancer treatment, competitively inhibits estrogen receptor (ER) and also demonstrates direct antiproliferative effect on cancer cells even in ER lacking cancer tissue. However its molecular mechanism of action is still unclear

Novel Structure-activity Relationships and Selectivity Profiling of Cage Dimeric 1,4-dihydropyridines As Multidrug Resistance (MDR) Modulators

Bioorganic & Medicinal Chemistry. Jul, 2010  |  Pubmed ID: 20598550

Synthesized series of cage dimeric 1,4-dihydropyridines have been systematically evaluated as MDR modulators in in vitro assays to investigate structure-dependent selectivity properties of inhibiting most cancer-relevant efflux pump proteins. Structure-activity relationships of each P-glycoprotein (P-gp) and multidrug resistance associated protein (MRP) 1 and MRP2 inhibition are discussed and prove to be mainly determined by certain aromatic substitution patterns. The characterization of breast cancer resistance protein (BCRP) inhibition results in the discovery of benzyloxy substituted derivatives as selective P-gp inhibitors.

Multidrug Resistance Reversal Properties and Cytotoxic Evaluation of Representatives of a Novel Class of HIV-1 Protease Inhibitors

The Journal of Pharmacy and Pharmacology. Dec, 2010  |  Pubmed ID: 21054396

P-Glycoprotein (P-gp) plays a central role in the development of resistance against cytostatics in anticancer therapy and against human immunodeficiency virus (HIV) therapeutics of the HIV-1 protease inhibitor type. An approach to reverse the so-called multidrug resistance (MDR) phenomenon by the use of P-gp inhibiting agents is a challenge in the therapy of cancer and AIDS. Effective in-vitro inhibitors have P-gp substrate properties so that the expected in-vivo effects have been disappointing so far. Consequent higher dosages cause toxic effects.

YB-1 Dependent Virotherapy in Combination with Temozolomide As a Multimodal Therapy Approach to Eradicate Malignant Glioma

International Journal of Cancer. Journal International Du Cancer. Nov, 2010  |  Pubmed ID: 21086421

The human Y-box binding protein 1 (YB-1) is known to be a promising target for cancer therapy. We have demonstrated that YB-1 plays an important role in the adenoviral life cycle by regulating the adenoviral E2-gene expression. Thus, we studied the oncolytic effect of the recombinant adenovirus Ad-Delo3-RGD, in which the transactivation domain CR3 of the E1A protein is ablated to enable viral replication only in YB-1 positive cancer cells. In vitro Southern Blot analysis and cytopathic effect assays demonstrate high anti-glioma potency, which was significantly increased in combination with temozolomide (TMZ), daunorubicin and cisplatin. Since vascular endothelial growth factor (VEGF) is thought to promote the hypervascular phenotype of primary, malignant brain tumors, we also tested Ad-Delo3-RGD in regard to the inhibition of VEGF expression. Indeed, we found that Ad-Delo3-RGD induced VEGF down regulation, which was even amplified under hypoxic conditions. Tumor-bearing nudemice treated with the YB-1 dependent oncolytic adenovirus showed significantly smaller tumors than untreated controls. Furthermore, combination therapy with TMZ led to a regression in all treated animals with complete tumor regression in 33 % of analyzed mice, which was verified by bioluminescence imaging and histological studies. In addition, histopathological evaluation revealed enhanced apoptosis and a reduction in tumor vessel formation, indicating that Ad-Delo3-RGD has an anti-angiogenic effect in addition to its oncolytic capacity in vivo. Hence, our results demonstrate that the combination therapy of YB-1 dependent virotherapy and TMZ is effective in a xenograft glioma mouse model and might be useful in a YB-1 based clinical setting.

Healing of Ungrafted and Grafted Extraction Sockets After 12 Weeks: a Prospective Clinical Study

The International Journal of Oral & Maxillofacial Implants. Mar-Apr, 2011  |  Pubmed ID: 21483892

In this prospective study, bone formation in human extraction sockets augmented with Bio-Oss Collagen after a 12-week healing period was quantified and compared to bone formation in unaugmented extraction sockets.

CD46 Expression is an Unfavorable Prognostic Factor in Breast Cancer Cases

Applied Immunohistochemistry & Molecular Morphology : AIMM / Official Publication of the Society for Applied Immunohistochemistry. Dec, 2011  |  Pubmed ID: 21617523

The membrane cofactor protein, CD46 represents a complement inhibitor, which protects autologous cells from complement-mediated cytotoxicity. On tumor cells, CD46 may exhibit the potential to protect them from immune responses of the host. The present study aimed at evaluation of prognostic significance of CD46 expression in breast cancers. The analyses were performed on 70 samples of breast cancer. Immunohistochemical reactions were performed on paraffin sections of studied tumors using monoclonal antibodies directed against CD46. Results of the immunohistochemical reactions and of clinical observations were subjected to statistical analysis. Multivariate analysis showed that expression of CD46 and involvement of lymph nodes represent independent risk factors for disease-free survival and overall survival. Kaplan-Meier analysis showed that patients with tumors negative for CD46 have an increased progression-free time and overall survival time as compared with patients with the CD46-positive tumors. The study demonstrates that breast cancers manifest CD46 expression and that it is linked to a less favorable prognosis.

YB-1 Dependent Virotherapy in Combination with Temozolomide As a Multimodal Therapy Approach to Eradicate Malignant Glioma

International Journal of Cancer. Journal International Du Cancer. Sep, 2011  |  Pubmed ID: 21710499

The human Y-box binding protein 1 (YB-1) is known to be a promising target for cancer therapy. We have demonstrated that YB-1 plays an important role in the adenoviral life cycle by regulating the adenoviral E2-gene expression. Thus, we studied the oncolytic effect of the recombinant adenovirus Ad-Delo3-RGD, in which the transactivation domain CR3 of the E1A protein is ablated to enable viral replication only in YB-1 positive cancer cells. In vitro Southern Blot analysis and cytopathic effect assays demonstrate high anti-glioma potency, which was significantly increased in combination with temozolomide (TMZ), daunorubicin and cisplatin. Since vascular endothelial growth factor (VEGF) is thought to promote the hypervascular phenotype of primary, malignant brain tumors, we also tested Ad-Delo3-RGD in regard to the inhibition of VEGF expression. Indeed, we found that Ad-Delo3-RGD induced VEGF down regulation, which was even amplified under hypoxic conditions. Tumor-bearing nudemice treated with the YB-1 dependent oncolytic adenovirus showed significantly smaller tumors than untreated controls. Furthermore, combination therapy with TMZ led to a regression in all treated animals with complete tumor regression in 33 % of analyzed mice, which was verified by bioluminescence imaging and histological studies. In addition, histopathological evaluation revealed enhanced apoptosis and a reduction in tumor vessel formation, indicating that Ad-Delo3-RGD has an anti-angiogenic effect in addition to its oncolytic capacity in vivo. Hence, our results demonstrate that the combination therapy of YB-1 dependent virotherapy and TMZ is effective in a xenograft glioma mouse model and might be useful in a YB-1 based clinical setting.

Elevated YB-1 Expression is a New Unfavorable Prognostic Factor in Non-Hodgkin's Lymphomas

Anticancer Research. Sep, 2011  |  Pubmed ID: 21868545

Y-Box-binding protein-1 (YB-1) acts as a transcription factor for multiple genes and is linked to DNA replication and repair, cell proliferation and resistance to cytostatic drugs.

ABCC2 (MRP2, CMOAT) Localized in the Nuclear Envelope of Breast Carcinoma Cells Correlates with Poor Clinical Outcome

Pathology Oncology Research : POR. Oct, 2011  |  Pubmed ID: 21986666

Nuclear expression of ABCC2 can be specific for lower differentiated cells and stem cells. The study aimed at examination of ABCC2 expression in breast cancers. The immunohistochemical analyses were performed on 70 samples of breast cancer. We have also studied prognostic value of the ABCC2 mRNA expression using the KM plotter which assessed the effect of 22,277 genes on survival in 1809 breast cancer patients. Immunohistochemical studies demonstrated that ABCC2 expression may be manifested in nuclear envelope of neoplastic cells (ABCC2n) as well as in their cell membrane and cytoplasm (ABCC2c). The univariate and multivariate analyses showed that higher expression of ABCC2n and ABCC2c was typical for cases of a shorter overall survival time. Higher ABBC2n expression was also typical for cases of a shorter disease-free survival and a shorter progression-free time. The KM plotter analysis of the prognostic value of ABCC2 mRNA expression showed that elevated ABCC2 expression was specific for cases of a shorter relapse-free survival only in the estrogen receptor-negative subgroup. The study demonstrated hat breast cancers manifest ABCC2 expression and that it is linked to a less favourable prognosis. Our results suggested that immunohistochemical tests represent a reliable way to detect prognostic value of ABCC2 expression, allowing to demonstrate differences related to subcellular localization of the protein. Cases with nuclear expression of ABCC2 manifested a more aggressive clinical course, which might reflect a less advanced differentiation of neplastic cells, resistance to the applied cytostatic drugs and tamoxifen.

Estrogen Receptor Alpha Expression in Ovarian Cancer Predicts Longer Overall Survival

Pathology Oncology Research : POR. Sep, 2011  |  Pubmed ID: 21207255

Estrogen as a potential factor of ovarian carcinogenesis, acts via two nuclear receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), but the cellular signal pathways involved are not completely clear so far. In this study we have described the expression of ERα, detected by immunocytochemistry in 11 ovarian carcinoma cell lines and by immunohistochemistry in 43 Federation Internationale des Gyneacologistes et Obstetristes stage III ovarian carcinoma specimens prepared before and after treatment with cisplatin-based schemes. For cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma, analysis of cisplatin sensitivity in 11 ovarian carcinoma cell line was also performed. The strong nuclear ERα expression was only shown in the single A2780P cell line. Expression of ERα in tissue specimens did not reveal any correlations between histopathological parameters (histologic type and grading). We demonstrated a significant association with ERα expression in specimens from primary laparotomies (PL) and cause-specific survival. In the cases terminated by death of the patient, overall immunoreactivity score of ERα expression at PL was significantly lower than in surviving patients. In addition, Kaplan-Meier analysis revealed significantly shorter overall survival time and progression-free time in cases with lower immunoreactivity score of ERα expression at PL. Our findings support the hypothesis that aberrant hormone activity, by way of altered receptor expression, might be an important factor in the malignant transformation of ovarian cancer.

Loss of Estrogen Receptor Beta Expression Correlates with Shorter Overall Survival and Lack of Clinical Response to Chemotherapy in Ovarian Cancer Patients

Anticancer Research. Feb, 2011  |  Pubmed ID: 21378361

Estrogen receptor beta (ERβ) belongs to a large family of nuclear receptors. Recent studies have suggested that ERβ in contrast to ERα might act as a tumour suppressor in ovarian cancer (OVCA).

Osteogenic Potential of Mesenchymal Cells Embedded in the Provisional Matrix After a 6-week Healing Period in Augmented and Non-augmented Extraction Sockets: an Immunohistochemical Prospective Pilot Study in Humans

Clinical Oral Implants Research. Jan, 2012  |  Pubmed ID: 21435013

The aim of the present clinical study was the evaluation of the osteogenic potential of mesenchymal cells embedded in the provisional matrix of non-augmented and with Bio-Oss collagen-augmented human extraction sockets after 6 weeks of healing time.

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