Submit
Browse All

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

Recommend to Librarian

In JoVE (1)

Other Publications (65)

Articles by Martin R. Berger in JoVE

 JoVE Clinical and Translational Medicine

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound


JoVE 4178 8/14/2012

1, 1, 2, 1

1Department of Medical Physics in Radiology, German Cancer Research Center, Heidelberg, Germany, 2Unit of Chemotherapy and Toxicology, German Cancer Research Center, Heidelberg, Germany

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Other articles by Martin R. Berger on PubMed

Combination Effects of Alkylphosphocholines and Gemcitabine in Malignant and Normal Hematopoietic Cells

Cancer Letters. Aug, 2002  |  Pubmed ID: 12048162

Cytosine arabinoside (ara-C) and 2',2'-difluorodeoxycytidine (Gem) were compared in leukemia cells, with Gem being more potent than ara-C. Gem was combined with hexadecylphosphocholine (HPC) or erucylphospho-N,N,N-trimethylpropanolamine (ErPC(3)) in resistant CML cells. Supra-additive effects were seen in K-562 cells after concomitant and sequential exposure of Gem followed by HPC. The reverse sequence resulted in antagonism. Both effects were more significant when HPC was exchanged for ErPC(3). Gem or HPC failed to induce DNA laddering in K-562 cells, but apoptotic signals were transferred by the Gem-exposed SKW-3 cytosolic fraction to K-562 nuclei. HPC did not increase the clastogenicity of Gem and counteracted its mitotic inhibition in murine bone marrow. Thus, the combination of Gem and an alkylphosphocholine is advantageous in terms of their complementary mode of action, resulting in increased cytotoxicity and lowered myelotoxicity.

Combination with an Antisense Oligonucleotide Synergistically Improves the Antileukemic Efficacy of Erucylphospho-N,N,N-trimethylpropylammonium in Chronic Myeloid Leukemia Cell Lines

Molecular Cancer Therapeutics. Aug, 2002  |  Pubmed ID: 12492121

The aim of this study was to enhance the antileukemic efficacy of the alkylphosphocholine erucylphospho-N,N,N-trimethylpropylammonium (ErPC3) in chronic myeloid leukemia (CML)-derived cell lines by a bcr-directed antisense oligonucleotide (ASO-bcr). The mechanism was substantiated by Western blotting of the BCR-ABL expression level of CML cells, and the efficacy was substantiated by inhibition of colony formation compared with normal hematopoietic cells. The clonogenicity of K-562 cells expressing high levels of p210(BCR-ABL) was inhibited significantly by the ASO-bcr (T/C%, 30; P < 0.05) but not by ErPC3 (T/C%, 70). Combined sequential exposure to ErPC3 and the ASO-bcr, however, inhibited synergistically colony growth (T/C%, 3; P < 0.01). The colony growth of BV-173 cells expressing lower levels of p210(BCR-ABL) than K562 cells was inhibited to a greater extent by the ASO-bcr (T/C%, 15; P < 0.01). AR-230 cells that express high levels of p230(BCR-ABL) showed an intermediate decrease in colony formation in response to the ASO-bcr (T/C%, 20; P < 0.05). BCR-ABL levels of BV-173, CML-T1, and LAMA-84 cells were reduced in response to the ASO-bcr, as evidenced by Western blot. However, K-562 and AR-230 cells showed reduced BCR-ABL expression only after repeated treatment. ErPC3 and the ASO-bcr did not reduce colony formation (CFU-GM) of normal mouse bone marrow cells from long-term bone marrow cell cultures; instead, ErPC3 stimulated colony formation (P < 0.05) and did not induce chromosomal aberrations in mouse bone marrow. In conclusion, the combination of ErPC3 with a suitable antisense oligonucleotide inhibited synergistically colony formation of CML cell lines without damaging normal cells and thus might have a bearing on the purging of autologous hematopoietic transplants in CML patients.

Hepatic Disseminated Tumor Cells in Colorectal Cancer UICC Stage 4 Patients: Prognostic Implications

International Journal of Oncology. Sep, 2003  |  Pubmed ID: 12888919

Approximately 30-50% of all colorectal cancer patients with a resectable primary tumor will subsequently develop metastatic disease due to tumor cell dissemination. In the case of limited solid hepatic metastasis, resection of the primary tumor and the respective hepatic metastasis can be curative. Our aim was to evaluate the incidence of hepatic DTC in patients with solid liver metastasis and to describe their prognostic impact. Therefore, we applied a sensitive PCR-RFLP assay detecting one K-ras mutant among one million wild-type cells. Tumor tissue and liver biopsies from 32 colorectal cancer patients staged UICC 4 undergoing palliative surgery could be obtained intra-operatively and were thereupon screened for the presence of hepatic DTC. The primary tumor of 16 patients (50%) harbored a K-ras mutation and survival of K-ras positive patients was reduced as compared to K-ras negatives (P=0.039). In 8 of the patients (50%) with a K-ras mutated primary, no hepatic DTC were detected, whereas the liver of the remaining 8 patients (50%), showed a coincidence of solid liver metastasis and hepatic DTC. Median survival of patients with solid liver metastasis and hepatic DTC was decreased as compared with patients without any hepatic DTC burden (median survival 165 vs. 240 days; log-rank P=0.951). A subgroup analysis revealed that survival was significantly decreased in the case of bilobal DTC affection as compared with monolobal hepatic DTC affection (median survival 68 vs. 355 days; log-rank P=0.002). We conclude that the detection of hepatic DTC is a powerful prognostic factor. Furthermore, bilobal hepatic DTC involvement might be a contraindication for the resection of solid liver metastasis.

Superiority of Combined Chemo-embolization and Portal Infusion with 5-fluorouracil over Locoregional Infusion Concepts in Novikoff Hepatoma-bearing Rats

Journal of Cancer Research and Clinical Oncology. Nov, 2003  |  Pubmed ID: 14513367

The aim of this study was to investigate experimentally whether there is a superior effect of the combination of hepatic artery chemo-embolization with portal vein infusion over either of the two treatment modalities alone. Novikoff hepatoma cells transplanted under the liver capsule of Sprague Dawley rats were used as a model. Tumor growth was assessed at 7 and 21 days after tumor inoculation. The prolamine solution Ethibloc was employed for embolization, and 5-fluorouracil was used as a chemotherapeutic agent for both infusion and chemo-embolization. All arterial treatment modalities were administered in a super-selective manner. There was no intolerable toxicity after dosages of 55 to 125 mg 5-fluorouracil/kg body weight. With regard to therapeutic efficacy the results show that embolization is an effective therapeutic means for inducing tumor necrosis in selected liver areas. As a consequence, the ranking of all treatment modalities was based on the combined evaluation of tumor size and extent of tumor necrosis. According to this evaluation, hepatic artery chemo-embolization was superior to the respective type of infusion (P<0.01). In addition, the combination of both modalities in the form of hepatic artery chemo-embolization and portal vein infusion was effective in destroying more than 97% of vital tumor tissue (P<0.01). These results suggest the need for a comparative clinical study.

Cytokeratin 20 and Guanylyl Cyclase C MRNA is Largely Present in Lymph Node and Liver Specimens of Colorectal Cancer Patients

International Journal of Cancer. Journal International Du Cancer. Nov, 2003  |  Pubmed ID: 14520701

The aim of our prospective study was to detect circulating epithelial cells (CEC) indicating the presence of disseminated tumor cells (DTC) in tissues affected by lymphatic and hematogenic colorectal cancer metastasis. DTC were tracked in lymph node, liver or bone marrow samples of 245 colorectal cancer patients using 2 independent RT-PCR assays for cytokeratin 20 (CK20) and guanylylcyclase C (GCC) that demonstrated a sensitivity of 1 colorectal cancer cell in 10(6) nucleated hematopoietic cells. CK20 mRNA was detected in 79% of lymph nodes, 35% of both liver lobes and 11% of bone marrow samples. GCC mRNA was found in 68% of lymph nodes, 60% of both liver lobes and 6% of bone marrow specimens. Both markers were recorded in 63% of lymph nodes, 45% of at least 1 liver lobe and 1% of bone marrow samples. There was no significant difference when comparing lymph node samples tested positive for both markers in patients with (N1/2; 65%) and without (N0; 56%) nodal involvement. The same was true when comparing the percentages of patients with and without clinically overt distant metastasis who were positive for both markers in at least 1 liver lobe (62% vs. 41%) or in bone marrow (4% vs. 0%). A score denoting the cumulative sum of tests indicating presence of CK20 and GCC mRNA in the liver was significantly related with UICC classification (p = 0.039). However, addition of lymph node results to this score decreased the correlation. The high incidence of clinically inconspicuous lymph node and liver samples tested positive for both markers emphasizes the function of these organs as primary filters for epithelial cells possibly shed from colorectal carcinomas. The potential prognostic significance of these findings warrants verification, especially regarding the importance of CEC or DTC resident in the liver of colorectal cancer patients.

Induction of Apoptosis by Erucylphospho-N,N,N-trimethylammonium is Associated with Changes in Signal Molecule Expressionand Location

Annals of the New York Academy of Sciences. Dec, 2003  |  Pubmed ID: 15033740

At concentrations effecting apoptosis, the alkylphosphocholine ErPC3 induced increased expression of the Rb protein in breast cancer (MCF-7) and leukemia (SKW-3, AR-230) cell lines as well as hypophosphorylation (K-562, CMLT-1, DOHH-2) and fragmentation of Rb (BV-173, SKW-3) in leukemia cell lines. ErPC3 exerts at least part of its antineoplastic activity by apoptosis, and this chain of events comprises early changes in the lipid raft fraction of the cellular membrane as well as modulation of different signal molecules, such as Abl, Bcr-Abl (fusion protein), and Rb.

Decreased Levels of Osteopontin and Bone Sialoprotein II Are Correlated with Reduced Proliferation, Colony Formation, and Migration of GFP-MDA-MB-231 Cells

International Journal of Oncology. May, 2004  |  Pubmed ID: 15067347

MDA-MB-231 human breast cancer cells transfected with GFP were used as model to determine the reduction in proliferation, colony formation, and migration in response to agents with anti-metastatic properties. These agents consisted of antisense oligonucleotides (ASOs) directed against osteopontin (OPN), bone sialoprotein II (BSP II), and osteonectin (ON), as well as an antibody directed against BSP II. A bisphosphonate derivative (ibandronate) and an alkylphosphocholine (erucylphospho-NNN-trimethylpropanolamine; ErPC3) were used as positive controls. The ASOs directed against OPN, BSP II and ON suppressed the expression of their respective target proteins by 81%, 74% and 69%, respectively. They were barely but significantly active in inhibiting the proliferation, but intermediately to highly active in inhibiting the colony formation and migration of GFP-MDA-MB-231 breast cancer cells. The antibody against human BSP II was significantly more active than all ASOs used and was equally active or even surpassed the activity of ibandronate and ErPC3 in all three assays. The results obtained suggest a specific anti-metastatic activity of this antibody as well as of the ASOs found effective in decreasing OPN and BSP II expression.

Chemoembolization of Rat Liver Metastasis with Microspheres and Gemcitabine Followed by Evaluation of Tumor Cell Load by Chemiluminescence

Oncology Reports. May, 2004  |  Pubmed ID: 15069554

An orthotopic, isogenic rat model was used to determine the potential of chemoembolization (CHE) for reducing the tumor cell load of a diffusely metastatic liver. Seven days after injecting CC531-lac-Z cells intraportally to male WAG/Rij rats, tumor positive animals were treated by injection into the hepatic artery with solvent (n=17), degradable starch microspheres (DSM, 30 mg/kg; n=16), DSM plus 5-fluorouracil (5-FU, dosages: 90, 60, and 40 mg/kg) or DSM plus gemcitabine (Gem, dosages: 100, 80, 50, and 10 mg/kg). After 3 more weeks the experiment was terminated, the livers were weighted and the number of CC531-lac-Z cells per liver was determined. Injection of DSM reduced the tumor cell load by 21% (T/C%=79), the combination with 5-FU caused a stimulation of growth at 40 mg/kg (T/C%=291; n=10), but effected dose-dependent reductions in tumor cell number at 60 mg/kg (T/C%=86; n=16), and 90 mg/kg (T/C=19; n=17). None of these effects was significantly different from controls. The combination of DSM plus Gem was toxic at the highest dose (100 mg/kg), but well tolerated and highly effective at 80 mg/kg (T/C%= 16; n=12), 50 mg/kg (T/C%=9; n=12), and 10 mg/kg (T/C%=26; n=14). These results were significantly different from controls (p<0.05), respectively. In summary, the comparison of CHE with 5-FU or Gem shows that the efficacy of Gem in reducing the hepatic tumor cell load was significantly higher and its therapeutic ratio was greater than that of 5-FU.

Fluoropyrimidine Chemotherapy in a Rat Model: Comparison of Fluorouracil Metabolite Profiles Determined by High-field 19F-NMR Spectroscopy of Tissues Ex Vivo with Therapy Response and Toxicity for Locoregional Vs Systemic Infusion Protocols

NMR in Biomedicine. May, 2004  |  Pubmed ID: 15137037

A Sprague-Dawley rat model with DS sarcoma transplanted in the thigh was used to compare transcatheter locoregional i.a. and systemic i.v. administration of 5-fluorouracil (FU) at 12 dose-rate schedules: 25, 50 and 100 mg/kg; bolus, 1, 5 and 24 h infusions. In experiment A tumor (62/67 animals) as well as liver and kidney (56/67 animals) were excised 1 h after a single bolus or 1 h infusion or at the end of 5 and 24 h infusions. (19)F-NMR spectroscopy at 11.7 T was used to quantitate FU and its metabolites in ca. 1 g of tissue at 4 degrees C. In experiment B analogous FU treatments were repeated for 5 days (rats 80+11 controls). Tumor volumes vs time, various blood parameters and survival times were recorded, and a log growth rate parameter log GR, a response index RI, and a toxicity index TI were calculated. The i.a. vs i.v. ratios for tumor concentrations of FU and total anabolites (F-Nucl) were >1 for nearly all treatments and increased with infusion time at the higher doses. F-Nucl in tumor correlated linearly with total fluorine concentration (Tot. F range 30-1100 nmol/g) over all treatments (r=0.92, slope=0.45, p<0.0001). For non-bolus i.v. treatments [FU+F-Nucl] decreased linearly with decreasing FU dose rate (r(2)=0.74, zero intercept), while i.a. treatments showed non-linear behavior. For non-bolus treatments the mean log GR per treatment group showed a negative correlation (r=-0.87) with log[F-Nucl]. The most effective non-toxic treatments were 25 mg/kg over 5 or 24 h; the i.a. route was superior to i.v. on the basis of [FU+F-Nucl], RI, the reduction in log GR, and Kaplan-Meier survival statistics. For liver and kidney Tot. F (>83% FU catabolites) reached ca. 3-4 and 6-7 micromol/g, respectively, at the highest dose rates for either route; F-Nucl were detected only for Tot. F>500 nmol/g and increased exponentially as Tot. F increased (toxic treatments). The concentrations of the main catabolite (alpha-fluoro-beta-alanine, FBAL) in tumor did not correlate with Tot. F but rather with FBAL levels in kidney (r=0.90, all treatments), indicating that uptake of liver-derived FBAL from the circulation is the major source of FBAL in tumor.

K-ras Codon 12 and 13 Mutations Are Correlated with Differential Patterns of Tumor Cell Dissemination in Colorectal Cancer Patients

International Journal of Oncology. Jun, 2004  |  Pubmed ID: 15138598

The aim of this prospective study was to relate the incidences of cytokeratin 20 (CK20) and guanylylcyclase C (GCC) in lymph node, liver, and bone marrow specimens of 245 colorectal cancer (CRC) patients with the K-ras oncogene status of the corresponding primary tumor. Qualitative RT-PCR detection of CK20 and GCC mRNA was used as marker of circulating epithelial cells (CEC). Samples were considered positive for CEC only when both markers were detected concomitantly. For the detection of K-ras mutations, a PCR-RFLP assay was used. In the group with K-ras mutated primary carcinomas (n=92), CEC were detected in 62% of lymph node-, 43% of liver-, and 2% of bone marrow samples. No statistical significance was found when comparing these results with those from patients with K-ras wild-type carcinoma (59%, 46%, and 0%, respectively). In contrast to this combined evaluation, separate analysis of K-ras codons 12 (n=75, 82%) and 13 (n=17, 18%) revealed significantly differing CEC incidences. Lymph node specimens from corresponding K-ras codon 13 mutated carcinomas showed a significantly higher CEC incidence (82%) than the groups with codon 12 mutation (57%, p<0.05) or K-ras wild-type sequence (59%, p<0.05). Unlike these findings in lymph nodes, liver biopsies from corresponding carcinomas with K-ras codon 12 mutation or wild-type sequence were significantly more often positive for CEC (31% and 29%) than specimens from K-ras codon 13 mutated primary CRC (12%, p<0.04, respectively). In conclusion, colorectal carcinomas with K-ras codon 12 mutation showed the same pattern of tumor cell dissemination as their K-ras wild-type counterparts. Since K-ras codon 12 mutations prevailed 4-fold over codon 13 mutations, combined analysis of the two codons showed the same result. However, sub-analysis of patients with K-ras codon 13 mutation revealed that the respective CEC incidence was significantly increased in lymph nodes, but decreased in liver biopsies.

Prognostic Value of Disseminated Colorectal Tumor Cells in the Liver: Results of Follow-up Examinations

International Journal of Colorectal Disease. Jul, 2004  |  Pubmed ID: 14634776

Dissemination of tumor cells is an initial step in metastatic disease. Detection of disseminated tumor cells (DTC) in blood, bone marrow and lymph nodes has been associated with reduced disease-free survival, but to date there are no data for hepatic DTC. We investigated the prognostic relevance of hepatic DTC that are present in patients with colorectal cancer (CRC) at the time of surgery.

Downregulation of Osteopontin and Bone Sialoprotein II is Related to Reduced Colony Formation and Metastasis Formation of MDA-MB-231 Human Breast Cancer Cells

Cancer Gene Therapy. Feb, 2004  |  Pubmed ID: 14647232

Osteopontin (OPN), bone sialoprotein (BSPII), and osteonectin (ON) belong to a family of glycoproteins, which have been linked to cancer metastasis and progression. Here, we report on the selection of antisense oligonucleotides (ASOs), which are effective in reducing their protein levels. In human MDA-MB-231 breast cancer cells, the maximum inhibition of protein expression ranged from 84% (OPN) to 75% (BSPII) and 70% (ON). Erucylphospho-NNN-trimethylpropanolamine (ErPC3) was used as positive control and combination partner. Exposure to ErPC3 inhibited colony formation of MDA-MB-231 cells by 11% (10 microM), 45% (14 microM) and 78% (20 microM). The clonogenicity of breast cancer cells was reduced by 15%, 11%, 8% (5 microM), 39%, 19%, 14% (10 microM) and 46%, 39%, 21% (20 microM) in response to ASO-OPN-04, ASO-BSPII-06 and ASO-ON-03, respectively. Combination of ErPC3 with the ASOs caused additive combination effects. Pre-exposure to the ASOs, but not to the NSO, inhibited formation of osteolytic metastasis in three of four (ASO-OPN-04, P<0.03) and two of four (ASO-BSPII-06) nude rats, and reduced metastasis lesions significantly (T/C%=4.3 and 9.1, P=0.05, respectively). We conclude that downregulation of OPN and BSPII reduces colony formation of MDA-MB-231 cells and formation of osteolytic metastasis in nude rats.

Chemoembolization of Rat Liver Metastasis with Irinotecan and Quantification of Tumor Cell Reduction

Journal of Cancer Research and Clinical Oncology. Apr, 2004  |  Pubmed ID: 14740207

Hematogenic metastasis of patients with colorectal cancer most frequently effects the liver; the prognosis of affected patients is dramatically worsened by the presence of this lesion. The aim of this study was to evaluate the effect of hepatic arterial chemoembolization (HACE) with irinotecan versus 5-fluorouracil as a standard agent in a rat liver metastasis model.

Detection of Isolated Tumor Cells by Polymerase Chain Reaction-restriction Fragment Length Polymorphism for K-ras Mutations in Tissue Samples of 199 Colorectal Cancer Patients

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Jan, 2004  |  Pubmed ID: 14760087

The aim of this study was to identify K-ras mutations as marker for isolated tumor cells in liver, lymph node, and bone marrow specimens of colorectal cancer patients. To detect these, a PCR-RFLP assay was used with a sensitivity exceeding that of routine histopathology by at least 1 order of magnitude. In addition, the ratio of mutated versus wild-type alleles was determined by an internal standard. Of 199 patients, 74 (37.5%) were found to bear a K-ras-positive tumor. Of these, 60 (81%) were mutated in codon 12 and 14 (19%) in codon 13 (P < 0.001). In addition, 14 organs were found K-ras positive, 13 of which were from 12 patients with a K-ras-positive tumor (16%) and 1 from a patient with a K-ras-negative tumor (0.8%). Eight patients exhibited liver involvement and 6 showed lymph node involvement. Remarkably, no bone marrow specimen was found K-ras positive (P < 0.017 versus liver involvement). Sequence analysis of tumor DNA revealed that GGT (Gly) was replaced by GAT (Asp; 35%), GTT (Val; 32%), AGT (Ser; 13%), GCT (Ala; 10%), TGT (Cys; 8%), and CGT (Arg; 2%) for codon 12, and by GAC (Asp) as the only type of mutation for codon 13. In colorectal carcinomas the ratio of K-ras mutated versus wild-type alleles ranged over 4 orders of magnitude (10(0)-10(-4), median: 10(-2)) and was correlated with both, residual tumor load (R1/2; P = 0.028) and distant metastasis (M1; P = 0.057). These results show that detection of K-ras mutated alleles by PCR-RFLP in patients with colorectal carcinoma may aid in the identification of isolated tumor cells. High ratios of K-ras alleles were correlated with certain negative prognostic parameters (R,M). In accord with its function as a primary filter for colorectal carcinoma cells, the liver was more often contaminated with K-ras-positive cells than bone marrow.

Delivery and Expression of PDNA Embedded in Collagen Matrices

Journal of Controlled Release : Official Journal of the Controlled Release Society. Mar, 2004  |  Pubmed ID: 14980779

Collagen matrices can be used as non-viral biocompatible gene carriers for localized implantable gene therapy. Collagen matrices embedding pDNA with enhanced binding through condensing agent linkage to the matrix or to the pDNA have been formulated, and characterized in various systems. pDNA and condensed pDNA were released intact from the matrices within 1-2 days. In vitro transfection with collagen matrices containing pDNA (luciferase encoding), pDNA in liposome (LIP), and pDNA with polyethylenimine (PEI) resulted in significantly higher expression levels in comparison to naked pDNA. pDNA-LIP matrices exhibited a dose response transfection of NIH 3T3, 293, MDA-MB-231 and smooth muscle cells (SMCs) in cell cultures. Subdermal implantations of collagen-polylysine-pDNA matrices in rats resulted in significantly higher gene expression levels in comparison to non-condensed pDNA matrices. Perivascular treatment with pDNA matrix and of naked pDNA solution in balloon-injured rat carotid arteries resulted in significant expression. In conclusion, a facile method for embedding cationic formulations of pDNA in collagen matrices was developed. These bioactive matrices seem to be suitable for tissue engineering and local gene therapy strategies.

Molecular Detection of Clinical Colorectal Cancer Metastasis: How Should Multiple Markers Be Put to Use?

International Journal of Colorectal Disease. Mar, 2005  |  Pubmed ID: 15459772

Up to 45% of colorectal cancer (CRC) patients will develop local recurrence or metastasis following curative resection. The latter is due to cells shed from the primary carcinoma prior to or during surgery. The aim of this study was to contribute toward a "rational"-approach for detecting these disseminated tumor cells (DTC) using a combination of independent markers and detection methods.

Lifelong Exposure to Di-(2-ethylhexyl)-phthalate Induces Tumors in Liver and Testes of Sprague-Dawley Rats

Toxicology. Jan, 2005  |  Pubmed ID: 15588926

The plasticizer di-(2-ethylhexyl)-phthalate (DEHP) is the most important phthalate with respect to its production, use and occurrence in the environment. In standard carcinogenicity experiments with F344 rats and B6C3F1 mice, DEHP has been shown to induce hepatocellular tumors. Moreover, DEHP is strongly suspected to be a developmental and reproductive toxicant. The present study aimed at determining the long-term toxic effects of lifetime exposure to low concentrations of DEHP in Sprague-Dawley rat strain. Seven hundred and thirty male rats, stratified into four groups, received DEHP with the diet, resulting in dosages of 300, 95, 30 and 0 mg/kg per day for up to 159 weeks and were only sacrificed when moribund. All organs of the dead and sacrificed animals were histopathologically examined. Significantly increased tumor incidences after exposure to 300 mg/kg per day DEHP (P = 0.04 for testes and 0.05 for liver) and a significant dose-related trend (P(Trend) = 0.02 for testes and 0.03 for liver) were detected in both organs liver and testes. Time to tumor analysis revealed that DEHP-induced testicular tumors developed earlier in lifetime than hepatocellular neoplasias, and their multiplicity increased with time. In addition, animals exposed to the highest DEHP dose showed a significantly increased rate of testicular tubular atrophy (P < 0.01). In conclusion, this study shows for the first time that the rat testes are a target organ of DEHP carcinogenicity in Sprague-Dawley rats upon lifetime exposure. This new finding indicates the importance of evaluating the effects of lifetime exposure in assessing the potential human health risks of DEHP. In addition, the carcinogenicity should be evaluated in rat strains with low spontaneous tumor incidence in the organs known as target of DEHP toxicity.

Combination Treatment of CC531-lac-Z Rat Liver Metastases by Chemoembolization with Pemetrexed Disodium and Gemcitabine

Journal of Cancer Research and Clinical Oncology. May, 2005  |  Pubmed ID: 15657768

The aim of this study was to evaluate the combination effect of pemetrexed disodium (MTA; Alimta; LY 231514) and gemcitabine (GEM) administered by hepatic artery and portal vein chemoembolization (HACE and PVCE) in a colorectal cancer rat liver metastasis model.

Characterization of a Rat Model with Site-specific Bone Metastasis Induced by MDA-MB-231 Breast Cancer Cells and Its Application to the Effects of an Antibody Against Bone Sialoprotein

International Journal of Cancer. Journal International Du Cancer. Jun, 2005  |  Pubmed ID: 15688393

Metastasis into the skeleton is a serious complication of certain neoplastic diseases such as breast, prostate and lung cancer, but the reasons for this osteotropism are poorly understood. Our aim was to establish a physiologically relevant animal model that is characterized by osteolytic lesions confined to the hind leg of nude rats. For this purpose, we injected 1x10(5) MDA-MB-231 human breast cancer cells transfected with GFP into the superficial epigastric artery, which is an anastomosing vessel between the femoral and iliac arteries. As assessed with the aid of X-rays, computed tomography and immunohistochemisty, osteolytic lesions occurred exclusively in the femur, tibia and fibula of the animals. The tumor take rate was 93% in a series of 96 rats and the increase in lesion size was observed up to 110 days after tumor cell inoculation. When applying this animal model to the effects of an antibody against bone sialoprotein (BSP), a significantly reduced osteolytic lesion size was observed after preincubation of cells (2 hr, 600 microg/ml anti-BSP) prior to intra-arterial tumor cell injection resulting in 19 T/C% at day 60 after tumor implantation (p < 0.05). In addition, the osteolytic lesion size was also significantly reduced after s.c. treatment of the animals with the antibody (20 mg/kg anti-BSPx3 within 5 days after tumor implantation), resulting in 30 T/C% at day 60 after tumor cell implantation (p < 0.05). In conclusion, the novel rat model for site-specific osteolytic lesions provides in vivo evidence that preincubation of MDA-MB-231GFP cells and treatment of rats after tumor implantation with an antibody against BSP significantly reduces the size of lytic lesions in bone.

Reduced Expression of Hugl-1, the Human Homologue of Drosophila Tumour Suppressor Gene Lgl, Contributes to Progression of Colorectal Cancer

Oncogene. Apr, 2005  |  Pubmed ID: 15735678

The human gene, human giant larvae (Hugl-1/Llg1/Lgl1) has significant homology to the Drosophila tumour suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that binds Myosin II and is involved in maintaining cell polarity and epithelial integrity. The human protein, Hugl-1 contains several conserved functional domains found in Lgl, suggesting that these proteins may have closely related functions. Whether loss of Hugl expression plays a role in human tumorigenesis has so far not been extensively investigated. Thus, we evaluated tumour tissues from 94 patients undergoing surgery for colorectal cancer (CRC) for loss of Hugl-1 transcription and compared our findings with the clinical data from each of these patients. We found that Hugl-1 was lost in 75% of tumour samples and these losses were associated with advanced stage and particularly with lymph node metastases. Reduced Hugl-1 expression during the adenoma-carcinoma sequence occurring as early as in colorectal adenomas was detected by both immunohistochemical and reverse transcription-polymerase chain reaction analysis. Functional assays with ecdysone-inducible cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Our studies thus indicate that downregulation of Hugl-1 contributes to CRC progression.

Effect of Chemokine Receptors CXCR4 and CCR7 on the Metastatic Behavior of Human Colorectal Cancer

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Mar, 2005  |  Pubmed ID: 15755995

The expression of chemokine receptors CXCR4 and CCR7 has been associated with tumor dissemination and poor prognosis in a limited number of tumor entities. However, no data are currently available on the impact of chemokine receptor expression on disease progression and prognosis in human colorectal cancer.

Osteonectin Influences Growth and Invasion of Pancreatic Cancer Cells

Annals of Surgery. Aug, 2005  |  Pubmed ID: 16041213

We sought to examine the expression and functional role of osteonectin in primary and metastatic pancreatic ductal adenocarcinoma (PDAC).

Identification of Potent Anticancer Activity in Ximenia Americana Aqueous Extracts Used by African Traditional Medicine

Toxicology and Applied Pharmacology. Mar, 2006  |  Pubmed ID: 16005923

The antineoplastic activity of a plant powder used in African traditional medicine for treating cancer was investigated by analyzing the activity of various extracts in vitro. The most active, aqueous extract was subsequently subjected to a detailed investigation in a panel of 17 tumor cell lines, showing an average IC50 of 49 mg raw powder/ml medium. The sensitivity of the cell lines varied by two orders of magnitude, from 1.7 mg/ml in MCF7 breast cancer cells to 170 mg/ml in AR230 chronic-myeloid leukemia cells. Immortalized, non-tumorigenic cell lines showed a marginal sensitivity. In addition, kinetic and recovery experiments performed in MCF7 and U87-MG cells and a comparison with the antineoplastic activity of miltefosine, gemcitabine, and cisplatinum in MCF7, U87-MG, HEp2, and SAOS2 cells revealed no obvious similarity between the sensitivity profiles of the extract and the three standard agents, suggesting a different mechanism of cytotoxicity. The in vivo antitumor activity was determined in the CC531 colorectal cancer rat model. Significant anticancer activity was found following administration of equitoxic doses of 100 (perorally) and 5 (intraperitoneally) mg raw powder/kg, indicating a 95% reduced activity following intestinal absorption. By sequencing the mitochondrial gene for the large subunit of the ribulose bis-phosphate carboxylase (rbcL) in DNA from the plant material, the source plant was identified as Ximenia americana. A physicochemical characterization showed that the active antineoplastic component(s) of the plant material are proteins with galactose affinity. Moreover, by mass spectrometry, one of these proteins was shown to contain a stretch of 11 amino acids identical to a tryptic peptide from the ribosome-inactivating protein ricin.

Treatment of Bone Metastasis Induced by MDA-MB-231 Breast Cancer Cells with an Antibody Against Bone Sialoprotein

International Journal of Oncology. Mar, 2006  |  Pubmed ID: 16465361

The extracellular bone matrix protein bone sialoprotein (BSP) is considered to play an important role in the pathogenesis of lytic skeletal lesions which are associated with severe morbidity in breast, prostate or lung cancer patients. In addition to in vitro studies, nude rats were implanted with 10(5) MDA-MB-231 cells transfected with GFP into a small branch of the femoral artery. Osteolytic lesions of the respective hind leg were detected by X-ray and CT analysis as well as by immunohistochemistry. Exposure of MDA-MB-231GFP cells in vitro to an antibody against BSP (0-400 microg/ml) decreased proliferation, colony formation and migration of these cells by up to 95, 83 and 89 T/C%, respectively. In nude rats, pre-incubation of MDA-MB-231GFP cells prior to inoculation (25-100 microg/ml) reduced the mean osteolytic lesion size to 22 T/C% after 90 days of observation (p<0.05). Treatment of overt lytic metastasis with the anti-BSP antibody (10 mg/kg) resulted in a significantly smaller mean lesion size of 57 T/C% at the end of the observation period (p<0.05) as well as in new bone formation. Immunohistochemical analysis revealed the presence of BSP in MDA-MB-231GFP cells and in vessel endothelium cells during processes such as migration and invasion. In conclusion, an anti-BSP antibody decreased proliferation, colony formation and migration of MDA-MB-231GFP cells in vitro and reduced osteolysis besides inducing bone formation in a nude rat model.

Identification and Characterization of Riproximin, a New Type II Ribosome-inactivating Protein with Antineoplastic Activity from Ximenia Americana

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Jun, 2006  |  Pubmed ID: 16641197

The aim of this study was to identify and characterize the active component(s) of Ximenia americana plant material used to treat cancer in African traditional medicine. By a combination of preextraction, extraction, ion exchange and affinity chromatography, a mixture of two cytotoxic proteins was isolated. Using degenerated primers designed on the de novo sequence of two tryptic peptides from one of these proteins, a DNA fragment was amplified and the sequence obtained was used to determine the complete cDNA sequence by the RACE method. Sequence analysis and molecular modeling showed that the new protein, riproximin, belongs to the family of type II ribosome inactivating proteins. These results are in good agreement with the ability of riproximin to inhibit protein synthesis in a cell-free system, as well as with the cytotoxicity of riproximin, as demonstrated by its IC50 value of 0.5 pM in MCF7, 1.1 pM in HELA and 0.6 pM in CC531-lacZ cells. To assess the antineoplastic efficacy of the purified riproximin in vivo, the CC531-lacZ colorectal cancer rat metastasis model was used. Significant anticancer activity was found after administration of total dosages of 100 (perorally) and 10 (intraperitoneally) pmol riproximin/kg. These results suggest that riproximin has distinct potential for cancer treatment.

The Balanced Induction of K-ras Codon 12 and 13 Mutations in Mucosa Differs from Their Ratio in Neoplastic Tissues

International Journal of Oncology. Oct, 2006  |  Pubmed ID: 16964391

The aim of this study was to compare the ratio of K-ras codon 12 and 13 mutations in various tissues of colorectal cancer patients. Multiple samples of inconspicuous mucosa and a sample of carcinoma tissue were taken from 36 colorectal cancer patients (group I) and these results were compared with those from polyp and carcinoma tissues of another 48 colorectal cancer patients (group II). A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect the respective point mutations. The results of this assay were complemented by sequencing the K-ras mutations. In mucosa tissue, the ratio of codon 12 and 13 mutations was nearly equal (0.9:1) whereas the respective ratio in tumour tissue showed a strong preponderance of K-ras codon 12 mutations (14:1, p=0.004). In polyp tissue of patients from group II, the ratio was 2.7:1 and that in carcinomas was 19:1 (p=0.053). The prevalence of both types of mutation was 14.6% in all mucosa samples, corresponding to 30.6% of group I patients. The K-ras mutation rate in carcinoma tissue of the same patients was 38.9%. Similarly, 33.4% of all polyp and 41.7% of all carcinoma samples from group II harboured K-ras codon 12 and/or 13 mutations. Sequencing confirmed 59 of 60 K-ras codon 12 mutations, but due to the detection limit for sequencing (1:10(4)) only 10 of 20 K-ras codon 13 mutations were confirmed. It is concluded that after balanced induction K-ras codon 12 mutations increase in frequency relative to K-ras codon 13 mutations during tumour progression.

Strong Expression of Chemokine Receptor CXCR4 by Pancreatic Cancer Correlates with Advanced Disease

Oncology Reports. Dec, 2006  |  Pubmed ID: 17089032

Certain chemokines have been proposed to distinctly contribute to tumor growth, dissemination and local immune escape. Expression of the chemokine receptor CXCR4 has been linked to tumor progression in diverse tumor entities. The aim of this study was to evaluate if the expression of CXCR4 influences progression of human pancreatic cancer. CXCR4 expression of pancreatic cancer was retrospectively assessed by immunohistochemistry in 103 patients with pancreatic cancer. Intensity of CXCR4 expression was correlated with both tumor and patient characteristics. Human pancreatic cancer revealed variable intensities of CXCR4 expression. Strong CXCR4 expression was significantly associated with advanced UICC stages (P=0.03) and revealed a trend for hematogenous metastasis (P=0.09) and progressed local tumor stages (P=0.15). In summary, strong expression of CXCR4 was significantly associated with advanced pancreatic cancer.

Antineoplastic and Anticlastogenic Properties of Curcumin

Annals of the New York Academy of Sciences. Jan, 2007  |  Pubmed ID: 17404048

Curcumin is the pigment of turmeric and has been reported as a signal transduction modulator and inhibitor of transcription factors, for example, NF-kappaB. In our article we found a concentration-dependent cytotoxic activity of curcumin in a panel of eight leukemic cell lines (SKW-3, CEM, U-937, HL-60, HL-60/Dox, K-562, LAMA-84, and AR-230). Additive to synergistic interactions was recorded for combinations with bendamustine and idarubicine in SKW-3 and LAMA-84 cells. Noteworthy, in multiple myeloma cells (RPMI-8226 and U-266) a potentiation of the efficacy of bendamustine by curcumin application was found. Moreover, curcumin increased the bendamustine cytotoxicity in cultures of cells isolated from the bone marrow of a patient with non-Hodgkin's lymphoma (NHL). The increased bendamustine efficacy could be explained by NF-kappaB inhibition, because this factor is activated in many cancers, especially leukemia and multiple myeloma. Curcumin is characterized by low toxicity and was described to have a chemoprotective activity. Therefore, the level of reduced glutathione (GSH) was measured and a concentration-dependent increase of GSH levels was recorded in AR-230 and SKW-3 cells (concentration range 5-25 muM). Experiments with mice showed significant protection against cisplatin-induced chromosomal aberrations (clastogenic effect) and inhibition of mitoses in bone marrow cells. Curcumin alone caused reduction of the mitotic index. In combination with cisplatin, however, this parameter was increased when compared to cisplatin alone. Our data indicate that curcumin has pleiotropic effects on signal transduction by inhibiting transcription thus exerting antitumor activity. In addition, curcumin has protective and anticlastogenic activity by enhancing the scavenging of free radicals.

The Expression Level of the Tumor Suppressor Retinoblastoma Protein (Rb) Influences the Antileukemic Efficacy of Erucylphospho-N,N,N-trimethylpropylammonium (ErPC3)

Cancer Biology & Therapy. Jun, 2007  |  Pubmed ID: 17495525

The alkylphosphocholine erucylphospho-N,N,N-trimethylpropylammonium (ErPC3) is a promising new drug for treating various types of cancer. Its mechanism of action is not yet fully understood but is related to the Rb tumor suppressor protein. In the present study, we investigated the role of decreased Rb expression levels for the antileukemic efficacy of ErPC3 in BV-173 and K-562 CML-derived cell lines. We used antisense technique to knock down Rb levels in the two cell lines in addition to ErPC3 treatment. Cells with reduced Rb expression showed a diminished sensitivity to ErPC3 exposure, as determined by MTT (BV-173 and K-562) and clonogenicity assays (K-562 only), if concentrations below the IC50 were used. The feasibility of Rb knockdown varied between BV-173 and K-562 cells, with the former being distinctly more sensitive than the latter. We conclude that sufficient Rb levels are important for the cytotoxic and anticlonogenic effects of ErPC3 at levels below the IC(50), but that higher concentrations of ErPC3 are less dependent on Rb status.

The T-box Transcription Factor Eomesodermin Controls CD8 T Cell Activity and Lymph Node Metastasis in Human Colorectal Cancer

Gut. Nov, 2007  |  Pubmed ID: 17566017

BACKGROUND/AIMS: An efficient cytolytic T cell function is essential for immune mediated rejection of colorectal cancer. However, the molecular mechanisms driving T cell mediated cancer rejection are still poorly understood. Here, we assessed the relevance of the T-box transcription factor eomesodermin in colorectal cancer. METHODS/ RESULTS: By analysing tissue probes from 88 different colorectal tumours, a significant (p<0.02) inverse correlation between eomesodermin expression in colorectal cancers and the presence of lymph node metastases could be shown, whereas no such correlation was noted for the master transcription factor of regulatory T cells, FoxP3 and CD8 alpha expression. To evaluate whether this effect might be due to effects of eomesodermin on tumour infiltrating CD8 T cells, we subsequently analysed the regulated expression and function of this transcription factor in human T cells. Whereas overexpression of this factor induced perforin but not granzyme expression, siRNA mediated suppression of eomesodermin expression led to significantly reduced IFN-gamma production, perforin levels and cytolytic activity of CD8 T cells. Furthermore, TGF-beta and IL4 could be identified as important inducer of eomesodermin expression. CONCLUSION: These data define for the first time a regulatory role of eomesodermin for CD8 T cell activity in humans. Our findings are consistent with a model in which eomesodermin expression in tumour infiltrating T cells regulates cytolytic functions of CD8 T cells via perforin expression. These data provide novel insights into control mechanisms governing the functional activity of human CD8 T lymphocytes via T-box transcription factors in cancer.

Effect of Zoledronic Acid and an Antibody Against Bone Sialoprotein II on MDA-MB-231(GFP) Breast Cancer Cells in Vitro and on Osteolytic Lesions Induced in Vivo by This Cell Line in Nude Rats

Clinical & Experimental Metastasis. 2007  |  Pubmed ID: 17636409

The aim of this study was to investigate the combined effect of zoledronic acid and an antibody against bone sialoprotein II (BSPII) on proliferation and osteolytic activity of MDA-MB-231(GFP) breast cancer cells. For this purpose, the cells were exposed to zoledronic acid (10-20 microg/ml [25-50 microgM]) and an anti-BSPII IgY (10-100 microg/ml) for up to 5 days alone or in combination. The combined treatment showed synergistic antiproliferative effects at the higher dose of zoledronic acid. Following inoculation of 1 x 10(5) MDA-MB-231 (GFP) breast cancer cells into a branch of the femoral artery of nude rats, lytic lesions developed in the tibia, femur or fibula of the injected hind leg after approximately 30 days. The appearance and development of these lesions were monitored radiographically. Rats with lytic lesions were treated with zoledronic acid (60 microg/kg/week sc x 8; n = 10), zoledronic acid and an anti-BSPII IgY antibody (60 microg/kg/week sc x 8 + 10 mg/kg/week sc x 8; n = 10), or left untreated (n = 20). In addition, rats were treated for 4 weeks (n = 10) with both regimens starting right after tumor cell inoculation. Finally, ten rats were treated with zoledronic acid for 2 weeks before tumor cell inoculation (60 microg/kg/week sc x 2). The antiosteolytic effect of zoledronic acid was high as shown by inhibition of osteolytic growth. Addition of the anti-BSPII IgY further decreased the incidence of femoral osteolytic lesions (40% reduction), indicating remineralization, and reduced periosteal defects of cortical bone (20% reduction). These observations favor using the IgY-antibody in addition to zoledronic acid in order to stimulate osteoblast-induced remineralization.

Coexpression of Receptor-tyrosine-kinases in Gastric Adenocarcinoma--a Rationale for a Molecular Targeting Strategy?

World Journal of Gastroenterology : WJG. Jul, 2007  |  Pubmed ID: 17659711

To define the (co-)expression pattern of target receptor-tyrosine-kinases (RTK) in human gastric adenocarcinoma.

Effects of Bone Sialoprotein on Pancreatic Cancer Cell Growth, Invasion and Metastasis

Cancer Letters. Jan, 2007  |  Pubmed ID: 16488077

Bone sialoprotein (BSP) is an acidic glycoprotein that plays an important role in cancer cell growth, migration and invasion. The expression, localization and possible function of BSP in chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC) were analyzed by QRT-PCR, laser capture microdissection, DNA microarray analysis, immunoblotting, radioimmunoassays and immunohistochemistry as well as cell growth, invasion, scattering, and adhesion assays. BSP mRNA was detected in 40.7% of normal, in 80% of CP and in 86.4% of PDAC samples. The median BSP mRNA levels were 6.1 and 0.9copies/microl cDNA in PDAC and CP tissues, respectively, and zero copies/microl cDNA in normal pancreatic tissues. BSP was weakly present in the cytoplasm of islet cells and ductal cells in 20% of normal pancreatic tissues. BSP was localized in the tubular complexes of both CP and PDAC, as well as in pancreatic cancer cells. Five out of 8 pancreatic cancer cell lines expressed BSP mRNA. Recombinant BSP (rBSP) inhibited Capan-1 and SU8686 pancreatic cancer cell growth, with a maximal effect of -46.4+/-12.0% in Capan-1 cells and -45.7+/-14.5% in SU8686 cells. rBSP decreased the invasion of SU8686 cells by -59.1+/-11.2% and of Capan-1 cells by -13.3+/-3.8% (P<0.05), whereas it did not affect scattering or adhesion of both cell lines. In conclusion, endogenous BSP expression levels in pancreatic cancer cells and low to absent BSP expression in the surrounding stromal tissue elements may indirectly act to enhance the proliferation and invasion of pancreatic cancer cells.

Chemoembolisation of Rat Colorectal Liver Metastases with Drug Eluting Beads Loaded with Irinotecan or Doxorubicin

Clinical & Experimental Metastasis. 2008  |  Pubmed ID: 18259882

Systemic chemotherapy has limited success in treating liver metastasis of colorectal cancer. Alternative approaches such as hepatic arterial infusion or trans arterial chemoembolisation aim to deliver the chemotherapy locally to address the predominant liver disease. Chemoembolisation with drug eluting beads (DEB) designed to deliver drug at the target over a protracted period of time is a new strategy to reduce the tumor burden of liver metastases. To test this hypothesis, DEB possessing anionic groups capable of ionically complexing with cationic drugs were synthesised by a suspension polymerisation method and were fractionated to produce an average size of 75 microm. The DEB were loaded with the desired concentration of either doxorubicin hydrochloride or irinotecan hydrochloride prior to administration by immersion in the drug solution, yielding essentially 100% loading efficiency. To determine their effect in vivo, a transplantable orthotopic and isogenic rat liver metastasis model was used which is based on intraportal injection of 4 x 10(6) beta-galactosidase transfected CC531 rat colorectal cancer cells into male WAG/Rij rats. By MTT assay, the cells were shown to be sensitive to both drugs in vitro with the IC(50) being by two orders of magnitude lower for doxorubicin (110 nM after 72 h) compared to irinotecan (25 microM after 72 h). For the in vivo phase, a differential expression of the ERK MAP kinase between tumor cells cultured in vitro and those inoculated in vivo was noted using Western blotting techniques. This was considered to be indicative of passage-induced cell senescence that reduced the sensitivity of the tumor cells to DEB chemoembolisation. This notwithstanding, administration of DEB loaded with irinotecan or doxorubicin by single injection into the hepatic artery showed significant anticancer activity, as measured by a reduction in the tumor burden of the liver and a corresponding reduction in liver weight. Comparing the two agents, irinotecan appears more advantageous because of its significant activity and excellent tolerability following administration at two dosages of either 20 or 30 mg/kg. Doxorubicin showed a narrower window of activity, being effective at 4 mg/kg but ineffective at the lower dose of 2 mg/kg. We conclude that chemoembolisation with DEB with either agent may have potential for treating patients with colorectal liver metastasis, although irinotecan DEB appeared to have a more favourable safety profile.

PDGFRalpha/beta Expression Correlates with the Metastatic Behavior of Human Colorectal Cancer: a Possible Rationale for a Molecular Targeting Strategy

Oncology Reports. Mar, 2008  |  Pubmed ID: 18288404

As new multi-target tyrosine kinase inhibitors are emerging in the therapy of various malignancies, our aim was to define the co-expression pattern of receptor-tyrosine-kinase platelet-derived growth factor receptors alpha and beta (PDGFRalpha/beta) in human colorectal cancer. The co-expression pattern of PDGFRalpha/beta was analyzed by RT-PCR in 99 histologically confirmed human colorectal carcinomas and five colorectal cancer cell lines. In addition, immunohistochemical (IHC) staining was applied for confirmation of expression and analysis of receptor tyrosine kinase (RTK) localisation. The colorectal cancer cell lines that were analysed revealed varying expression intensities of PDGFRalpha and PDGFRbeta. The majority of human colorectal cancer specimens revealed a PDGFRalpha (83%) or PDGFRbeta (60%) expression. While PDGFRalpha showed a predominantly cytoplasmic staining in tumor cells as well as in stromal pericytes, PDGFRbeta was restricted to stromal pericytes only. Furthermore, PDGFRalpha expression significantly correlated with lymph node metastasis (P=0.0082) and advanced UICC stages III/IV (P=0.018) in older patients (P=0.043). PDGFRbeta expression only revealed a trend towards lymphatic dissemination (P=0.099). Co-expression of PDGFRalpha/beta occurred in 57% of the colorectal cancer samples, whereas another 29% of the samples depicted mono-expression of PDGFRalpha or PDGFRbeta. Notably, PDGFRalpha/beta expression significantly correlated with lymphatic metastasis (P=0.007) and advanced UICC stages III/IV (P=0.017) in older patients (P=0.03). In summary, our results revealed that PDGFRalpha/beta expression significantly correlates with lymphatic dissemination and therefore encourages application of PDGFRalpha/beta RTK-inhibitors within a combination therapy.

Bevacizumab Inhibits Breast Cancer-induced Osteolysis, Surrounding Soft Tissue Metastasis, and Angiogenesis in Rats As Visualized by VCT and MRI

Neoplasia (New York, N.Y.). May, 2008  |  Pubmed ID: 18472968

The aim of this study was to evaluate the effect of an antiangiogenic treatment with the vascular endothelial growth factor antibody bevacizumab in an experimental model of breast cancer bone metastasis and to monitor osteolysis, soft tissue tumor, and angiogenesis in bone metastasis noninvasively by volumetric computed tomography (VCT) and magnetic resonance imaging (MRI). After inoculation of MDA-MB-231 human breast cancer cells into nude rats, bone metastasis was monitored with contrast-enhanced VCT and MRI from day 30 to day 70 after tumor cell inoculation, respectively. Thereby, animals of the treatment group (10 mg/kg bevacizumab IV weekly, n = 15) were compared with sham-treated animals (n = 17). Treatment with bevacizumab resulted in a significant difference versus control in osteolytic as well as soft tissue lesion sizes (days 50 to 70 and 40 to 70 after tumor cell inoculation, respectively; P < .05). This observation was paralleled with significantly reduced vascularization in the treatment group as shown by reduced increase in relative signal intensity in dynamic contrast-enhanced MRI from days 40 to 70 (P < .05). Contrast-enhanced VCT and histology confirmed decreased angiogenesis as well as new bone formation after application of bevacizumab. In conclusion, bevacizumab significantly inhibited osteolysis, surrounding soft tissue tumor growth, and angiogenesis in an experimental model of breast cancer bone metastasis as visualized by VCT and MRI.

Analysis of the K-ras/B-raf/Erk Signal Cascade, P53 and CMAP As Markers for Tumor Progression in Colorectal Cancer Patients

Oncology Reports. Jul, 2008  |  Pubmed ID: 18575712

Colorectal cancer patients may succumb to their disease because of local recurrence or formation of metastasis. To develop a prognostic tool for these fatal types of progression, 23 patients with colorectal carcinoma were included in this study for the detection at the time of surgery of the incidence of K-ras, B-raf and p53 mutations, the phosphorylation status of Erk and the expression of cystatin-like metastasis-associated protein (CMAP) in tumor, mucosa and liver samples. Polymerase chain reaction-restriction fragment length polymorphism and PCR-SSCP were used to detect the respective mutations. The results of these assays were complemented by sequencing the K-ras, B-raf and p53 mutations. A multiplex RT-PCR assay was used to detect the CMAP mRNA levels and the phosphorylation status of Erk in tumor samples was assessed by Western blot using a phosphospecific Erk antibody. The carcinomas were classified as stages T4 (70%), T3 (17%), T2 (9%) and T1 (4%) and thus represent a group of advanced colorectal carcinomas. The carcinomas (8 out of 23, 39.1%) were mutated in K-ras codons 12 or 13 and two patients had a B-raf (V599) mutation in their tumor. Of 22 tumors, 11 (50%) were positive for pErk, indicating the activation of the RAS/RAF/ERK signaling pathway. Of the 23 tumors, 13 (65.5%) showed an increased CMAP RNA level. Notably, 10 of these 13 patients have already died and two developed liver metastasis. Mutations in p53 were found in only 6 patients (26%), with 6 being detected in carcinoma, 1 in mucosa and 1 in liver tissue. These alterations were classified as non-sense (n=1), mis-sense (n=2) and frame-shift mutations (n=1) as well as intron polymorphisms (n=5). There was a significant correlation between Erk activation and K-ras codon 12 mutation (p=0.016), but not between K-ras codon 13 or B-raf mutations and Erk activation. Furthermore, there was a significant correlation of each positive marker with tumor stage (p=0.001).

Bcl-x(L) and Myeloid Cell Leukaemia-1 Contribute to Apoptosis Resistance of Colorectal Cancer Cells

World Journal of Gastroenterology : WJG. Jun, 2008  |  Pubmed ID: 18609706

To explore the role of Bcl-x(L) and Myeloid cell leukaemia (Mcl)-1 for the apoptosis resistance of colorectal carcinoma (CRC) cells towards current treatment modalities.

Strong Expression of Chemokine Receptor CXCR4 by Renal Cell Carcinoma Correlates with Advanced Disease

Journal of Oncology. 2008  |  Pubmed ID: 19266088

Diverse chemokines and their receptors have been associated with tumor growth, tumor dissemination, and local immune escape. In different tumor entities, the level of chemokine receptor CXCR4 expression has been linked with tumor progression and decreased survival. The aim of this study was to evaluate the influence of CXCR4 expression on the progression of human renal cell carcinoma. CXCR4 expression of renal cell carcinoma was assessed by immunohistochemistry in 113 patients. Intensity of CXCR4 expression was correlated with both tumor and patient characteristics. Human renal cell carcinoma revealed variable intensities of CXCR4 expression. Strong CXCR4 expression of renal cell carcinoma was significantly associated with advanced T-status (P = .039), tumor dedifferentiation (P = .0005), and low hemoglobin (P = .039). In summary, strong CXCR4 expression was significantly associated with advanced dedifferentiated renal cell carcinoma.

Cytotoxic Activity of Platinum(II) and Palladium(II) Complexes of N-3-pyridinylmethanesulfonamide: the Influence of Electroporation

Zeitschrift Für Naturforschung. C, Journal of Biosciences. Mar-Apr, 2009  |  Pubmed ID: 19526709

The series of complexes: cis-[Pd(PMSA)2X2], cis-[Pt(PMSA)2X2], trans-[Pt(PMSA)2I2] and [Pt(PMSA)4]Cl2 (PMSA = N-3-pyridinylmethanesulfonamide; X = Cl, Br, I), previously synthesized and characterized by us, as well as the free ligand PMSA, were tested for their cytotoxic activity without electroporation -- against murine leukemia F4N and human SKW-3 and MDA-MB-231 tumour cell lines -- and with electroporation -- against the latter two cell lines. The majority of the complexes exhibited cytotoxic effects (IC50 < 100 micromol/l) under the conditions of electroporation. Both cis- and trans-[Pt(PMSA)2I2] had pronounced cytotoxic effects (29-61 micromol/l against MDA-MB-231 cells).

Erucylphospho-N,N,N-trimethylpropylammonium Shows Substantial Cytotoxicity in Multiple Myeloma Cells

Annals of the New York Academy of Sciences. Aug, 2009  |  Pubmed ID: 19723075

Multiple myeloma (MM) is a frequent hematological malignancy that is incurable despite recent developments, such as proteasome and angiogenic inhibitors. Erucylphospho-N,N,N-trimethylpropylammonium (erufosine) is an i.v. injectable alkylphosphocholine with antineoplastic activity based on an unusual mode of action and is currently undergoing clinical trials in leukemia patients. The aim of this investigation was to evaluate the efficacy of erufosine in MM cells and to study the modulation of cell-death pathways. The cytotoxicity of erufosine against three MM cell lines (RPMI-8226, U-266, and OPM-2) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-dye reduction assay. All MM cell lines responded to erufosine, RPMI-8226 cells being most and U-266 being least sensitive. The respective IC(50) values were 3.2 and 16.2 micromol/L. Various cell-death characteristics were studied in response to erufosine, such as morphological changes, oligonucleosomal DNA fragmentation, caspase activation, and poly (ADP)-ribose polymerase cleavage. Erufosine was found to cause cell shrinkage, chromatin condensation, and caspase-8 and -3 activation. Taken together, our data indicate that erufosine is a potential antimyeloma drug eliciting specific features of apoptotic cell death in vitro.

Sustained Delivery and Efficacy of Polymeric Nanoparticles Containing Osteopontin and Bone Sialoprotein Antisenses in Rats with Breast Cancer Bone Metastasis

International Journal of Cancer. Journal International Du Cancer. Apr, 2010  |  Pubmed ID: 19739076

Poor prognosis in mammary carcinoma is associated with a certain expression profile of a defined set of genes including osteopontin and bone sialoprotein. Efficient and specific delivery of antisenses (AS) and a protection of the sequences from degradation are the crucial conditions for AS therapeutic efficiency. We hypothesized that effective and safe AS delivery direceted against these genes could be achieved by polymeric nanoparticles (NP) fabricated from a biocompatible polymer. Due to their nano-size range and small negative charge, AS-NP can overcome the absorption barrier offering increased resistance to nuclease degradation, sustained duration of AS administration, and consequently, prolonged antisense action. The ASs designed against OPN and BSP-II were successfully encapsulated in NP composed of the biodegradable and biocompatible polylactide-co-glycolide polymer (PLGA), exhibiting sustained release and stability of the ASs. The therapeutic efficacy of the AS-NP delivery system was examined in vitro, and in a breast cancer bone metastasis animal model of MDA-MB-231 human breast cancer cells in nude rats. Treatment with OPN-AS or BSP-AS loaded NP in comparison with osmotic mini-pumps (locoregional injection and SC implants, respectively) resulted in a significant decrease in both, tumor bone metastasis incidence and in the size of the lesions in rats with metastases. Despite its smaller dose, AS-NP exhibited a better therapeutic efficacy than osmotic mini-pumps in terms of lesion ratio at later time periods (8-12 weeks). It may be concluded that AS delivery by NP is a promising therapeutic modality providing stability of the encapsulated AS and a sustained release.

K-ras Mutation Status Correlates with the Expression of VEGFR1, VEGFR2, and PDGFRalpha in Colorectal Cancer

International Journal of Colorectal Disease. Feb, 2010  |  Pubmed ID: 19936766

We initiated this study in order to analyze whether the expression level of targeted receptor tyrosine kinases (RTK) is associated with the K-ras mutation status.

Low Expression of Chemokine Receptor CCR5 in Human Colorectal Cancer Correlates with Lymphatic Dissemination and Reduced CD8+ T-cell Infiltration

International Journal of Colorectal Disease. Apr, 2010  |  Pubmed ID: 20054600

Chemokines and their receptors have been proposed to distinctly contribute to tumor growth, dissemination, and local immune escape. The aim of this study was to evaluate the relevance of the chemokine receptor CCR5 expression for the progression of human colorectal cancer.

Does Expression of Receptor Tyrosine Kinases in Gastric Adenocarcinoma Correlate with Clinicopathological Parameters?

Hepato-gastroenterology. Mar-Apr, 2010  |  Pubmed ID: 20583450

This study was initiated in order to define the (co-)expression patterns of target receptor tyrosine kinases (RTKs) in human gastric adenocarcinoma and to correlate them with clinicopathological parameters.

Regulation of Osteopontin and Related Proteins in Rat CC531 Colorectal Cancer Cells

International Journal of Oncology. Aug, 2010  |  Pubmed ID: 20596651

Colorectal cancer accounts for 11% of all cancers and is the second most frequent cause of cancer-related death, with the majority of deaths attributable to hepatic metastasis. The main aim of the study was to investigate changes which occur in CC531 rat colon adenocarcinoma cells and are instrumental to the metastatic phenotype after homing to the liver. RT-PCR and Western blotting were used to detect the expression of certain proteins, transcription factors and enzymes, which are intimately linked to colorectal metastasis. These included osteopontin (OPN), bone sialoprotein ll (BSP ll), Runx2, Hoxc8, matrix metalloprotease-7 (MMP-7) and matrix-metalloprotease-9 (MMP-9). Subsequently, in order to detect the role of the hepatocytes in these changes seen in tumor cells, two models of co-culturing hepatocytes with CC531 cells were established. OPN, Runx2 and MMP-7 were found to be highly expressed in CC531 metastases explanted from the liver, but showed subsequent down-regulation and/or disappearance in cell culture. The inverse regulation of Hoxc8, OPN and Runx2 suggests that these genes may be regulated in a feed-back loop manner. MMP-9 mRNA and active MMP-7 protein were expressed in tumor cells themselves. The presence of hepatocytes was insufficient to induce induction of OPN and Runx2 in tumor cells in vitro, so was the addition of OPN or TGF-beta1. Whereas TGF-beta1 induced over-expression of OPN and Runx2 in hepatocytes, it did not exert the same effect on hepatocytes co-cultured with CC531 cells, indicating that this response was abrogated by CC531 cells.

Quantitative Analysis of Conditional Gene Inactivation Using Rationally Designed, Tetracycline-controlled MiRNAs

Nucleic Acids Research. Sep, 2010  |  Pubmed ID: 20639530

The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo.

Electrodelivery of Drugs into Cancer Cells in the Presence of Poloxamer 188

Journal of Biomedicine & Biotechnology. 2010  |  Pubmed ID: 20706647

In the present study it is shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation, decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can pass/diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Tumors were implanted subcutaneously in both flanks of nude mice using HeLa cells, transfected with genes for red fluorescent protein and luciferase. The volume of tumors stopped to grow after electrochemotherapy and the use of poloxamer 188 reduced the edema near the electrode and around the subcutaneously growing tumors.

Biodistribution of Antisense Nanoparticles in Mammary Carcinoma Rat Model

Drug Delivery. Aug, 2010  |  Pubmed ID: 20429847

Efficient and specific delivery of antisenses (ASs) and protection of the sequences from degradation are critical factors for effective therapy. Sustained release nanoparticles (NP) offer increased resistance to nuclease degradation, increased amounts of AS uptake, and the possibility of control in dosing and sustained duration of AS administration. The biodegradable and biocompatible poly(D,L-lactic-co-glycolic acid) copolymer (PLGA) was utilized to encapsulate AS directed against osteopontin (OPN), which is a promising therapeutic target in mammary carcinoma. Whole body biodistribution of OPN AS NP was evaluated in comparison to naked AS, in intact and mammary carcinoma metastasis model bearing rats. Naked and NP encapsulated AS exhibited different biodistribution profiles. AS NP, in contrast to naked AS, tended to accumulate mostly in the spleen, liver, and at the tumor inoculation site. Drug levels in intact organs were negligible. The elimination of naked AS was faster, due to rapid degradation of the unprotected sequence. It is concluded that AS NP protect the AS from degradation, provide efficient AS delivery to the tumor tissue, and minimize AS accumulation in intact organs due to the AS sustained release profile as well as the favorable NP physicochemical properties.

Osteopontin but Not Osteonectin Favors the Metastatic Growth of Pancreatic Cancer Cell Lines

Cancer Biology & Therapy. Jul, 2010  |  Pubmed ID: 20495387

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in western countries and among the malignancies with the worst prognosis. Osteonectin and osteopontin, two proteins of the extracellular matrix, have been found to be upregulated in PDAC. In the present study the expression of osteopontin mRNA as determined in a panel of 14 human pancreatic cancer cell lines was significantly related to the growth of these cell lines in the liver of nude rats (p = 0.001); whereas osteonectin showed a trend of being negatively related to pancreatic cancer cell growth in vivo (p = 0.10). In an in vitro co-culture model of human Suit2-007 and rat AsML PDAC cells with rat hepatocytes, a clearly increased expression of OPN mRNA was found in the tumor cells. In addition, both downregulation of osteopontin with specific antisense oligonucleotides and treatment with exogenous rh-osteonectin were associated with reduced cell proliferation. In accordance with the latter finding downregulation of osteonectin was coupled with increased proliferation. This evidence supports a protumorigenic role of osteopontin and points to an antitumorigenic role of osteonectin in PDAC.

Disseminated Colorectal Tumor Cells in Organs Prone to Metastasis Detected by New Double Enriched Nested-PCR in Comparison with Recognized Assays

Oncology Reports. May, 2011  |  Pubmed ID: 21369707

Hypothetically, K-ras mutations can be used as a marker of disseminated tumor cells (DTCs) in patients with K-ras mutated primary carcinoma. This study focused on the development of a useful assay for detecting low numbers of DTCs in potential target tissues of metastatic K-ras codon 12 mutated colorectal cancer. Tumor, liver, lymph node and bone marrow tissues from 46 colorectal carcinoma patients were examined for K-ras codon 12 mutations with a new double enriched nested (DEN)-PCR and the incidence of mutations was compared to those obtained from three established assays. DEN-PCR followed by sequencing found one mutated cell within 107 K-ras codon 12 wild-type cells and was more sensitive than other methods (1:106-1:102). Colon carcinomas (26/46) and adenomas (1/3) harbored mutations in K-ras codon 12. Sixteen of these 27 mutated tumors were found with all assays, two with three methods and one with the two most sensitive assays. In 8 cases, only DEN-PCR identified the K-ras mutation, and thus prevailed over the other methods used (p<0.002). Eight of 26 patients with K-ras mutated colorectal carcinoma also harbored K-ras mutated DTCs in liver and lymph nodes, respectively, and 4 in bone marrow. For liver and lymph node samples, DTC-mutations were identical to those in the primary carcinoma but those in bone marrow differed from the respective mutation in the primary carcinoma. In conclusion, DEN-PCR is a highly sensitive method for detecting K-ras mutations as marker of early and late tumor cell dissemination in tissues potentially harboring colorectal carcinoma metastases.

Expression of Chemokine Receptor CCR5 Correlates with the Presence of Hepatic Molecular Metastases in K-ras Positive Human Colorectal Cancer

Journal of Cancer Research and Clinical Oncology. Jul, 2011  |  Pubmed ID: 21468700

Molecular metastases are precursors of postoperative recurrence, detected by molecular-biological tools. Chemokines and their receptors contribute to dissemination and local immune recognition. A strong expression of the chemokine receptor CCR5 is associated with non-metastatic colorectal cancer and increased CD8+ T-cell infiltration. The aim of this study was to analyze whether CCR5 expression correlates with the presence of hepatic molecular metastases (MM).

The Insulin-like Growth Factor Binding Proteins 3 and 7 Are Associated with Colorectal Cancer and Liver Metastasis

Cancer Biology & Therapy. Jul, 2011  |  Pubmed ID: 21525788

Over the last few decades, a great deal of attention has been directed to the IGF system for its vital role in regulating cell and tissue survival, growth and differentiation. The insulin-like growth factor binding proteins (IGFBPs), a main constituent of this system, have been implicated in the tumorigenesis of colorectal cancer (CRC). In this study, we intended to shed more light on two essential members; IGFBP3 as representative for the six main IGFBPs and IGFBP7 to represent their related proteins (IGFBP-rps). Our experiments on silencing IGFBP3 or IGFBP7 in the two human CRC cell lines SW480, Caco2, and in the rat CRC cell line CC531 show reduced proliferation, colony formation, and for IGFBP3, also reduced migration. The expression of both genes in 68 human CRC samples was higher in UICC stages II and III than in stages I and IV. Additionally, IGFBP3 was negatively correlated with age (p = 0.05) and positively related to IGFBP7 expression (p = 0.0001). Further, in a liver metastasis experiment, the expression of both genes was drastically increased in response to early metastatic growth in vivo. Since these high levels returned gradually to normal thereafter, it could be assumed that the up-regulation of IGFBPs is vital during the process of homing into the liver and early metastatic dissemination. Our results indicate that IGFBP3 and 7 cannot be simply considered as tumor suppressors but have additional properties, which become evident only during cancer progression and metastasis formation.

Cilengitide Inhibits Metastatic Bone Colonization in a Nude Rat Model

Oncology Reports. Oct, 2011  |  Pubmed ID: 21725616

Integrins αvβ3 and αvβ5 are considered to play an important role in the pathogenesis of breast cancer bone metastases. This study investigates the effects of the αvβ3/αvβ5 integrin-specific inhibitor cilengitide during early metastatic bone colonization. The impact of cilengitide on the migration, invasion and proliferation of MDA-MB-231 human breast carcinoma cells as well as on bone resorption by osteoclasts was investigated in vitro. For in vivo experiments, nude rats were treated with cilengitide for 30 days starting one day after site-specific tumor cell inoculation in the hind leg, and the course of metastatic changes in bone was followed using flat-panel volumetric computed tomography (VCT) and magnetic resonance imaging (MRI). Vascular changes in bone metastases were investigated using dynamic contrast-enhanced (DCE-) MRI-derived parameters amplitude A and exchange rate coefficient kep. In vitro, cilengitide treatment resulted in a decrease in proliferation, migration and invasion of MDA-MB-231 cells, as well as of osteoclast activity. In vivo, the development of bone metastasis in the hind leg of rats was not prevented by adjuvant cilengitide treatment, but cilengitide reduced the volumes of osteolytic lesions and respective soft tissue tumors of developing bone metastases as assessed with VCT and MRI, respectively. DCE-MRI revealed significant changes in the A and kep parameters including decreased relative blood volume and increased vessel permeability after cilengitide treatment indicating vessel remodeling. In conclusion, during early pathogenic processes of bone colonization, cilengitide treatment exerted effects on tumor cells, osteoclasts and vasculature reducing the skeletal lesion size of experimental skeletal metastases.

Cilengitide Inhibits Progression of Experimental Breast Cancer Bone Metastases As Imaged Noninvasively Using VCT, MRI and DCE-MRI in a Longitudinal in Vivo Study

International Journal of Cancer. Journal International Du Cancer. May, 2011  |  Pubmed ID: 20648558

The aim of this study was to investigate the effect of inhibiting αvβ(3)/α(v) β(5) integrins by cilengitide in experimentally induced breast cancer bone metastases using noninvasive imaging techniques. For this purpose, nude rats bearing established breast cancer bone metastases were treated with cilengitide, a small molecule inhibitor of αvβ(3) and αvβ(5) integrins (75 mg/kg, five days per week; n = 12 rats) and compared to vehicle-treated control rats (n = 12). In a longitudinal study, conventional magnetic resonance imaging (MRI) and flat panel volumetric computed tomography were used to assess the volume of the soft tissue tumor and osteolysis, respectively, and dynamic contrast-enhanced (DCE-) MRI was performed to determine functional parameters of the tumor vasculature reflecting blood volume and blood vessel permeability. In rats treated with cilengitide, VCT and MRI showed that osteolytic lesions and the respective bone metastatic soft tissue tumors progressed more slowly than in vehicle-treated controls. DCE-MRI indicated a decrease in blood volume and an increase in vessel permeability and immunohistology revealed increased numbers of immature vessels in cilengitide-treated rats compared to vehicle controls. In conclusion, treatment of experimental breast cancer bone metastases with cilengitide resulted in pronounced antiresorptive and antitumor effects, suggesting that αvβ(3)/αvβ(5) inhibition may be a promising therapeutic approach for bone metastases.

Erucylphospho-N,N,N-trimethylpropylammonium (erufosine) is a Potential Antimyeloma Drug Devoid of Myelotoxicity

Cancer Chemotherapy and Pharmacology. Jan, 2011  |  Pubmed ID: 20177898

Erufosine is an i.v. injectable alkylphosphocholine which is active against various haematological malignancies in vitro. In the present study, its effects on multiple myeloma (MM) cell lines and on murine and human hematopoietic progenitor cells (HPCs) were investigated.

Sequential Biphasic Changes in Claudin1 and Claudin4 Expression Are Correlated to Colorectal Cancer Progression and Liver Metastasis

Journal of Cellular and Molecular Medicine. Feb, 2012  |  Pubmed ID: 21388515

Terminal progression of colorectal cancer (CRC) culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyse mRNA expression profiles of colorectal carcinoma (CC)531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag-Rij rats and re-isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8-fold initially down-regulated genes. The co-culture of tumour cells with isolated rat hepatocytes and Kupffer cells did not induce down-regulation of either claudin1 or 4. When the environment effective on circulating tumour cells was simulated by cell culture conditions favouring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by small interfering RNA caused significantly increased migration and decreased clonogenic growth of tumour cells (P < 0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human CRC samples in Union for International Cancer Control (UICC) stages I-III, as evaluated by real-time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log-rank test, P= 0.018). Further, significantly (P < 0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of rat CC531 CRC cells to the liver. This modulation is reflected by significant changes in claudin expression in human primary and metastatic CRC.

Purification and Characterization of Riproximin from Ximenia Americana Fruit Kernels

Protein Expression and Purification. Mar, 2012  |  Pubmed ID: 22178181

Highly pure riproximin was isolated from the fruit kernels of Ximenia americana, a defined, seasonally available and potentially unlimited herbal source. The newly established purification procedure included an initial aqueous extraction, removal of lipids with chloroform and subsequent chromatographic purification steps on a strong anion exchange resin and lactosyl-Sepharose. Consistent purity and stable biological properties were shown over several purification batches. The purified, kernel-derived riproximin was characterized in comparison to the African plant material riproximin and revealed highly similar biochemical and biological properties but differences in the electrophoresis pattern and mass spectrometry peptide profile. Our results suggest that although the purified fruit kernel riproximin consists of a mixture of closely related isoforms, it provides a reliable basis for further research and development of this type II ribosome inactivating protein (RIP).

Erufosine Simultaneously Induces Apoptosis and Autophagy by Modulating the Akt-mTOR Signaling Pathway in Oral Squamous Cell Carcinoma

Cancer Letters. Jun, 2012  |  Pubmed ID: 22202640

We investigated the anticancer activity of erufosine in oral squamous carcinoma cell lines in terms of cell proliferation, colony formation, induction of autophagy/apoptosis, cell cycle and mTOR signaling pathway. Erufosine showed dose-dependent cytotoxicity in all cell lines, it induced autophagy as well as apoptosis, G2 cell cycle arrest and modulation of cyclin D1 expression. Further erufosine downregulated the phosphorylation of major components of mTOR pathway, like p-Akt at Ser473 and Thr308 residues, p-Raptor, p-mTOR, p-PRAS40 and its downstream substrates p-p70S6K and p-4EBP1 in a dose-dependent manner. The pre-treatment of tumor cells with p-mTOR siRNA increased cytotoxic effects of erufosine comparable to cisplatin but higher than rapamycin.

Silencing of Skeletal Metastasis-associated Genes Impairs Migration of Breast Cancer Cells and Reduces Osteolytic Bone Lesions

Clinical & Experimental Metastasis. Jun, 2012  |  Pubmed ID: 22407340

Bone sialoprotein (BSP) and osteopontin (OPN) are important factors in the metastasis of breast cancer, which were examined as targets for antineoplastic therapy by siRNA. In addition, the effect of gene silencing on their transcription factor Runx2 and their interaction partners integrin β(3) and matrix metalloproteinase 2 was studied. The effect of siRNAs directed against these genes was assessed by monitoring expression levels followed by functional assays in cell culture as well as skeletal metastases caused by human MDA-MB-231(luc) breast cancer cells in nude rats. Upon silencing of the targets, cell migration was profoundly impaired (p < 0.001 for BSP-siRNA), but the impact on proliferation was low. Systemic administration by osmotic mini-pumps of BSP-siRNA but not OPN-siRNA decreased osteolytic lesions (p = 0.067). Extraosseous tumour growth was not affected. As an alternative approach, non-viral, polymeric based formulations of siRNAs in nanoparticles (NP) were developed. Locoregional administration of the two siRNAs targeting OPN and BSP encapsulated in these biodegradable NP reduced skeletal lesions even more efficiently (p = 0.03). Compared to systemic administration, this treatment caused not only a more pronounced anti-osteolytic effect at a 25-fold lower total siRNA dose, but also had a slight reducing effect on tumour incidence (p = 0.095). In conclusion, the siRNA treatment had a small effect on cellular proliferation but a significant efficacy against migration of and osteolysis induced by MDA-MB-231 cells. Our data underline that siRNA mediated knockdown is a powerful tool for identifying targets for pharmacological intervention. In addition, encapsulation of siRNA into biodegradable NP is a strategy, which promises well for using siRNA.

Interleukin-6, Osteopontin and Raf/MEK/ERK Signaling Modulate the Sensitivity of Human Myeloma Cells to Alkylphosphocholines

Leukemia Research. Jun, 2012  |  Pubmed ID: 22421411

Alkylphosphocholines are highly active against multiple myeloma (MM) cells in vitro and are devoid of myelotoxicity. Little is known about the determinants of MM cell responsiveness or resistance to these drugs. In this study we investigated the effects of disease-relevant cytokines, such as interleukin-6 (IL-6) and osteopontin (OPN), on the in vitro antimyeloma activity of erufosine and perifosine. The role of the Raf/MEK/ERK pathway was also studied. Exogenous IL-6 reduced the cytotoxicity of erufosine against OPM-2 cells and, to a smaller extent, against U-266 cells. This was accompanied by inhibition of apoptosis in OPM-2 cells. The efficacy of perifosine was similarly affected, but to a greater extent. IL-6 slightly enhanced the sensitivity of RPMI-8226 cells to erufosine, thus emphasizing the heterogeneity of MM. Induced overexpression of OPN isoforms made OPM-2 cells less sensitive to erufosine. In all cases of IL-6- or OPN-induced resistance, the effective concentrations of erufosine were still within the clinically achievable range. Like other alkylphosphocholines, erufosine enhanced Raf/MEK/ERK signaling in MM cells but in some cases this contributed to cytotoxicity.

Prognostic Value of Tumor Progression-related Gene Expression in Colorectal Cancer Patients

Journal of Cancer Research and Clinical Oncology. May, 2012  |  Pubmed ID: 22614156

PURPOSE: Colorectal cancer (CRC) is driven by genetic alterations causing its progression. Besides accepted tumor suppressor- and onco- genes, a series of genes have been identified, which contribute to transformation into a more malignant stage. We investigated whether the expression level of such genes, alone or in combination, could add to predict the prognosis of CRC patients. METHODS: Tumor samples from 118 CRC patients were screened in a retrospective analysis by qRT-PCR for expression of the four tumor progression-associated genes osteopontin (Opn), transforming growth factor β (Tgf-β), matrix metalloproteinase-2 (Mmp-2) and cyclooxigenase-2 (Cox-2). The resulting qRT-PCR values were related to those of housekeeping genes. All patients were clustered for similar expression levels between the four genes with R statistical software using the package pvclust, which provides bootstrap agglomerative hierarchical clustering. Clusters with similar expression of the four genes were analyzed for correlation with UICC stages and survival time. RESULTS: Expression of the four genes varied considerably within the cohort of patients. Cluster analysis of patients revealed a subgroup (n = 33) who in comparison with the other patients showed tenfold higher expression levels of all four genes (p < 0.001, respectively). However, there was no correlation between patients expressing high or low levels of these four genes and known parameters of clinical prognosis (UICC stages, survival time). CONCLUSIONS: In conclusion, tenfold increased expression levels of Opn, Tgf-β, Mmp-2 and Cox-2 in a subset of CRC patients did not predict for a clinical outcome that is different from that of the remaining patients.

A Particular Expression Pattern of CD13 Epitope 7H5 in Chronic Lymphocytic Leukaemia - a Possible New Therapeutic Target

Hematology (Amsterdam, Netherlands). May, 2012  |  Pubmed ID: 22664111

A total of 50 patients with chronic lymphocytic leukaemia (CLL), as well as the B-cell leukaemia cell lines MEC-1, JVM-3, and BV-173 were studied in order to assess the incidence of CD13/aminopeptidase N (APN) immunolabelling with a monoclonal antibody 7H5 compared to LeuM7 and to CD13 mRNA levels, and to correlate these data with the cytotoxic and apoptosis-induction activity of the natural phenolic APN inhibitor curcumin. CD13/APN was detected in a significant proportion of B-CLL patients (42/50, 84%), immunolabelled by 7H5 (42/50) ± LeuM7 (10/50). Molecular analysis for CD13 transcripts confirmed these data, resulting in a specific RT-PCR product in CD13 positive cases. Curcumin showed concentration-dependent cytoreductive efficacy and apoptosis-induction activity in all tested cell lines and primary cultures from CLL mononuclear cells. There was a clear tendency for a better response in CD13 positive cases. The incidence of CD13/APN in CLL suggests that the inhibition of APN/CD13 by curcumin may be an effective new molecular target for a more efficient therapy for these patients and warrants further investigations.

Erufosine Suppresses Breast Cancer in Vitro and in Vivo for Its Activity on PI3K, C-Raf and Akt Proteins

Journal of Cancer Research and Clinical Oncology. Jun, 2012  |  Pubmed ID: 22752602

PURPOSE: This study investigated the antineoplastic effect of the membrane active alkylphosphocholine erufosine in breast carcinoma models in vitro and in vivo and determined its influence on the PI3K/Akt and Ras/Raf/MAPK signaling pathways. METHODS: The antiproliferative effect of erufosine in vitro was determined by the MTT dye reduction assay, and the antineoplastic efficacy on tumor growth was investigated by relating the mean total tumor volumes of treated and control rats. Immunoblot analysis was used for detecting changes in the expression level of the signal molecules p-PI3K (p-p85), p-Akt at Thr 308 and p-cRaf. RESULTS: Based on their IC(50) (40 μM, respectively), the breast carcinoma cell lines MCF-7 and MDA-MB 231, which are estrogen receptor positive and negative, respectively, were equally sensitive to erufosine. In addition, erufosine caused dose-dependent decreases in the phosphorylation of PI3K (p85), Akt (PKB) at Thr 308 and cRaf in both cell lines. Moreover, administration of erufosine to rats bearing autochthonous methylnitrosourea-induced rat mammary carcinomas caused a significant dose-related tumor remission by more than 85 % (p < 0.05), which was well tolerated, as evidenced by a body weight loss of maximally 7 % and reduced tumor-related mortality (2 of 35 instead of 6 of 18 controls, p < 0.002). CONCLUSIONS: The results clearly indicate that erufosine possesses high antineoplastic activity not only in human breast cancer cell lines in vitro but also in rat mammary carcinoma in vivo. In addition, it can be derived that the mechanism of action of erufosine involves influence on both, PI3K/Akt and Ras/Raf/MAPK signaling pathways.

Evaluation of Riproximin Binding Properties Reveals a Novel Mechanism for Cellular Targeting

The Journal of Biological Chemistry. Aug, 2012  |  Pubmed ID: 22872642

Riproximin is a cytotoxic type II ribosome inactivating protein showing high selectivity for tumor cell lines. Its binding to cell surface glycans is crucial for subsequent internalization and cytotoxicity. In this paper, we describe a unique mechanism of interaction and discuss its implications for riproximin's cellular targeting and cytotoxicity. On a carbohydrate microarray, riproximin specifically bound to two types of asialo-glycans, namely to bi- and tri-antennary complex N-glycan structures (NA2/ NA3) and to repetitive N-acetyl-D-galactosa-mine (GalNAc), the so-called clustered Tn antigen, a cancer-specific O-glycan on mucins. Two glycoproteins showing high riproximin binding, the NA3-presenting asialofetuin (ASF) and the clustered Tn-rich asialo bovine submaxillary mucin (aBSM), were subsequently chosen as model to mimic riproximin's binding interactions with the two types of glycans. ELISA analyses were used to relate the two binding specificities of riproximin to its two sugar binding sites. Riproximin's ability to crosslink the two model proteins revealed that binding of the two types of glycoconjugates occurs within different binding sites. The biological implications of these binding properties were analyzed in cellular assays. The cytotoxicity of riproximin was found to depend on its specific and concomitant interaction with the two glycoconjugates, as well as on dynamic avidity effects typical for lectins binding to multivalent glycoproteins. The presence of definite, cancer related structures on the cells to be targeted determines the therapeutic potency of riproximin. Due to its crosslinking ability, riproximin is expected to show a high degree of specificity for cells exposing both NA2/NA3 and clustered Tn structures.

Waiting
simple hit counter