Recent studies have reported that hyper-methylation in the promoter region of miRNAs could silence the expression of tumor suppressive miRNAs and might play significant roles in the process of tumor development. However, the potential mechanisms regarding how methylation of miRNA CpG Island could regulate cancer cell chemo-resistance have not yet been studied. Using microarray and BSP (Bisulfate Sequencing PCR) assays, we found that compared with the parent SGC7901/VCR cells, expression of miR-129-5p was restored in SGC7901/VCR gastric cancer multi-drug resistant cell line treated by de-methylation reagent (5-AZA-dC). Using gain or loss of function assays, we found the over-expressed miR-129-5p reduced the chemo-resistance of SGC7901/VCR and SGC7901/ADR cells, while down-regulation of miR-129-5p had an opposite effect. Furthermore, three members of multi-drug resistance (MDR) related ABC transporters (ABCB1, ABCC5 and ABCG1) were found to be direct targets of miR-129-5p using bioinformatics analysis and report gene assays. The present study indicated that hyper-methylation of miR-129-5p CpG island might play important roles in the development of gastric cancer chemo-resistance by targeting MDR related ABC transporters and might be used as a potential therapeutic target in preventing the chemo-resistance of gastric cancer.
miRNA-16 (miR16) plays an important role in modulating the drug resistance of SGC7901 cell lines to adriamycin (ADR). A variety of viral carriers have been designed for miRNA delivery. However, the safety concerns are currently perceived as hampering the clinical application of viral vector-based therapy. Herein a type of magnetic nanoparticles (MNPs) was designed and synthesized using poly(ethylene glycol) (PEG)-coated Fe3O4 nanoparticles as a miRNA delivery system for the purpose of reducing drug resistance of gastric cancer cells by enforcing miR16 expression in SGC7901/ADR cells. The MNPs with good biocompatibility were synthesized by thermal decomposition, and then conjugated with miRNA via electrostatic interaction producing miR16/MNPs. After co-culture with miR16/MNPs, ADR-induced apoptosis of SGC7901/ADR was examined by MTT and TUNEL. miR16/MNPs treatment significantly increased cell apoptosis in vitro. SGC7901/ADR(fluc) tumor-bearing nude mice under ADR therapy were treated with miR16/MNPs by tail vein injection for in vivo study. After intraperitoneal injection of ADR, tumor volume measurement and fluorescence imaging were performed to for the death of SGC7901/ADR cells in vivo. Results showed that miR16/MNPs were able to significantly suppress SGC7901/ADR tumor growth, probably through increasing SGC7901/ADR cells' sensitivity to ADR. Our results suggest the efficient delivery of miR16 by MNPs as a novel therapeutic strategy for drug resistant tumor treatment.
By integrating the clinically used endoscope with the emerging Cerenkov luminescence imaging (CLI) technology, a new endoscopic Cerenkov luminescence imaging (ECLI) system was developed. The aim is to demonstrate the potential of translating CLI to clinical studies of gastrointestinal (GI) tract diseases. We systematically evaluated the feasibility and performance of the developed ECLI system with a series of in vitro and pseudotumor experiments. The ECLI system is comprised of an electron multiplying charge coupled device (EMCCD) camera coupled with a clinically used endoscope via an optical adapter. A 1951-USAF test board was used to measure the white-light lateral resolution, while a homemade test chart filled with (68)Ga was employed to measure the CL lateral resolution. Both in vitro and pseudotumor experiments were conducted to obtain the sensitivity of the ECLI system. The results were validated with that of CLI using EMCCD only, and the relative attenuation ratio of the ECLI system was calculated. Results showed that The white-light lateral resolution of the ECLI system was 198 µm, and the luminescent lateral resolution was better than 1 mm. Sensitivity experiments showed a theoretical sensitivity of [Formula: see text] ([Formula: see text]) and [Formula: see text] ([Formula: see text]) for the in vitro and pseudotumor studies, respectively. The relative attenuation ratio of ECLI to CLI was about 96%. The luminescent lateral resolution of the ECLI system was comparable with that of positron emission tomography (PET). The pseudotumor study illustrated the feasibility and applicability of the ECLI system in living organisms, indicating the potential for translating the CLI technology to the clinic.
Mesenchymal stem cells (MSCs) have been shown to integrate into the tumor stroma; however, the precise mechanisms of this process are still elusive. In this study, the EMT phenotype and the enhanced metastatic ability of tumor cells were observed using transwell and trans-endothelial migration assays, respectively, as well as by using electron and laser confocal microscopy. Critical genes were screened and validated using gene arrays and clinical samples, and the changes at the protein level were examined both in vitro and in vivo. Cancer cells acquired an "activated" carcinoma-associated fibroblasts (CAFs) phenotype after being in close contact with MSCs and enhancing tumor metastasis and growth in vivo. Paracrine signals also induced EMT and promoted transwell and trans-endothelial migration, the changes were dependent on ?-catenin, MMP-16, snail and twist. Notably, the higher expression levels of ?-catenin and MMP-16 were correlated with tumor invasion and distant organ and lymph node metastases in intestinal type gastric cancer. MSCs within the tumor niche significantly facilitated tumor growth and metastasis by paracrine cues and close physical connection. This occurred partly through snail, twist and its downstream targets, specifically ?-catenin/MMP-16. This article is protected by copyright. All rights reserved.
The expression of membrane-bound complement regulatory proteins (mCRPs) that inhibit the complement system in normal tissues is essential for self-protection against an autologous immune reaction. However, the expression patterns of mCRPs, including CD46, CD55, and CD59, are inconsistent in different types of cancer cells.
Multidrug resistance (MDR) is the most common cause of chemotherapy failure in gastric cancer (GC) treatment; however, the underlying molecular mechanisms remain elusive. Long noncoding RNAs (lncRNAs) can be involved in carcinogenesis, but the effects of lncRNAs on MDR are poorly understood. We show here that the lncRNA MRUL (MDR-related and upregulated lncRNA), located 400 kb downstream of ABCB1 (ATP-binding cassette, subfamily B, member 1), was significantly upregulated in two multidrug-resistant GC cell sublines, SGC7901/ADR and SGC7901/VCR. Furthermore, the relative expression levels of MRUL in GC tissues were negatively correlated with in vitro growth inhibition rates of GC specimens treated with chemotherapeutic drugs and indicated a poor prognosis for GC patients. MRUL knockdown in SGC7901/ADR and SGC7901/VCR cells led to increased rates of apoptosis, increased accumulation, and reduced doxorubicin (Adriamycin [ADR]) release in the presence of ADR or vincristine. Moreover, MRUL depletion reduced ABCB1 mRNA levels in a dose- and time-dependent manner. Heterologous luciferase reporter assays demonstrated that MRUL might positively affect ABCB1 expression in an orientation- and position-independent manner. Our findings indicate that MRUL promotes ABCB1 expression and is a potential target to reverse the MDR phenotype of GC MDR cell sublines.
Ran, a member of the RasGTPase family, has been showed to function in diverse cellular processes of cancer. In the present study, we examined the effects of Ran on the cell motility in pancreatic cancer cells and explored the possible mechanism of Ran's function in the metastasis of pancreatic cancer. We demonstrated that the expression of Ran was remarkably higher in lymph lode metastases than in primary pancreatic cancer tissues. In the functional studies, stable knockdown of Ran by shRNA could efficiently inhibit the migration and invasion of pancreatic cancer cells both in vitro and in vivo. By PCR array, we analyzed the differences in the expression levels of metastasis-associated genes before and after the downregulation of Ran, and it was showed that the regulation of pancreatic cancer metastasis by Ran was partially mediated by AR and CXCR4. We further confirmed that AR and CXCR4 were significantly decreased following knockdown of Ran. These data indicated that Ran could regulate the invasion and metastasis of pancreatic cancer cells through AR and CXCR4.
FOXJ1 is a member of the forkhead transcription factor family, which has been mostly studied for its role in the development of ciliated epithelium and immunology. However, the role of FOXJ1 in tumorigenesis remains largely unknown or even conflicting. We thus investigated FOXJ1 expression in gastric cancer and analyzed its correlations with tumor progression and prognosis.
The transcription factor CUTL1 (CCAAT displacement protein 1) has been reported to participate in the proliferation of diverse types of cancer. In the present study, we investigated the potential involvement of CUTL1 in the proliferation of malignant melanoma. We found that CUTL1 expression was upregulated in malignant melanoma tissues and cell lines, and CUTL1 expression was selected as a prognostic predictor for malignant melanoma patients by both univariate and multivariate analysis. Knockdown of CUTL1 by short hairpin RNA significantly reduced the colony-forming ability of malignant melanoma cells in vitro and reduced tumor growth in vivo, whereas forced overexpression of CUTL1 produced the opposite results. Consistently, cell cycle progression was impaired upon downregulation of CUTL1 and enhanced when CUTL1 was upregulated. Additional experiments suggested that CUTL1 may regulate the proliferation of malignant melanoma by modulating the expression of cell cycle-related proteins.
Ubiquitin specific protease 33 (USP33) is a multifunctional protein regulating diverse cellular processes. The expression and role of USP33 in lung cancer remain unexplored. In this study, we show that USP33 is down-regulated in multiple cohorts of lung cancer patients and that low expression of USP33 is associated with poor prognosis. USP33 mediates Slit-Robo signaling in lung cancer cell migration. Downregulation of USP33 reduces the protein stability of Robo1 in lung cancer cells, providing a previously unknown mechanism for USP33 function in mediating Slit activity in lung cancer cells. Taken together, USP33 is a new player in lung cancer that regulates Slit-Robo signaling. Our data suggest that USP33 may be a candidate tumor suppressor for lung cancer with potential as a prognostic marker.
The Drosophila Groucho protein and its mammalian orthologues the transducin-like enhancers of split (TLEs) are critical transcriptional corepressors that repress Wnt and other signaling pathways. Although it is known that Groucho/TLEs are recruited to target genes by pathway-specific transcription factors, molecular events after the corepressor recruitment are largely unclear. We report that association of TLEs with O-GlcNAc transferase, an enzyme that catalyzes posttranslational modification of proteins by O-linked N-acetylglucosamine, is essential for TLE-mediated transcriptional repression. Removal of O-GlcNAc from Wnt-responsive gene promoters is critical for gene activation from Wnt-responsive promoters. Thus, these studies identify a molecular mechanism by which Groucho/TLEs repress gene transcription and provide a model whereby O-GlcNAc may control distinct intracellular signaling pathways.
Chemoresistance remains the most significant obstacle to successful chemotherapy for leukemia, and its exact mechanism is still unknown. In this work, we used the cell-surface capturing method together with quantitative proteomics to investigate differences in the glycoproteomes of adriamycin-sensitive and adriamycin-resistant leukemia cells. Two quantitative methods, isotopic dimethyl labeling and SWATH, were used to quantify glycoproteins, and 35 glycoproteins were quantified by both methods. High correlation was observed between the glycoproteins quantified by the above two methods, and 15 glycoproteins displayed a consistent significant change trend in both sets of quantitative results. These 15 proteins included classical multidrug resistance-related glycoproteins such as ABCB1 as well as a set of novel glycoproteins that have not previously been reported to be associated with chemoresistance in leukemia cells. Further validation with quantitative real-time PCR and Western blotting confirmed the proteomic screening results. Subsequent functional experiments based on RNA interference technology showed that CTSD, FKBP10, and SLC2A1 are novel genes that participate in the acquisition and maintenance of the adriamycin-resistant phenotype in leukemia cells.
FOXO4, a member of the FOXO family of transcription factors, is currently the focus of intense study. Its role and function in gastric cancer have not been fully elucidated. The present study was aimed to investigate the expression profile of FOXO4 in gastric cancer and the effect of FOXO4 on cancer cell growth and metastasis.
Multi-drug resistance (MDR) has become the largest obstacle to the success of cancer chemotherapies. The mechanisms of MDR and the approaches to test MDR have been discovered, yet not fully understood. This review covers the in vivo and in vitro approaches for the detection of MDR in the laboratory and the mechanisms of MDR in cancers. This study also envisages the future developments toward the clinical and therapeutic applications of MDR in cancer treatment. Future therapeutics for cancer treatment will likely combine the existing therapies with drugs originated from MDR mechanisms such as anti-cancer stem cell drugs, anti-miRNA drugs or anti-epigenetic drugs. The challenges for the clinical detection of MDR will be to find new biomarkers and to determine new evaluation systems before the drug resistance emerges.
Understanding the mechanism underlying multidrug resistance and identifying effective targets that can overcome it is of critical importance. In this study, mRNA and miRNA expression profiling of the drug resistant sublines, SGC7901/VCR and SGC7901/ADR, and their parental gastric cancer cell line SGC7901 were performed. A significant number of genes and a limited subset of miRNAs were commonly dysregulated, which were further validated using qRT-PCR. GO and KEGG pathway analyses of the commonly dysregulated genes indicated that the MAPK signalling pathway may be involved in multidrug resistance, which was further validated using immunoblotting and MTT assay. Finally a primary multidrug resistance network in gastric cancer, consisting of the commonly dysregulated genes and miRNAs, was established and functional miRNA-mRNA pairs were identified. The commonly dysregulated genes and miRNAs identified in this study may represent good therapeutic targets and further study of these targets may increase our understanding of the mechanisms underlying multidrug resistance.
Ran, a member of the Ras GTPase family, has important roles in nucleocytoplasmic transport. Herein, we detected Ran expression in pancreatic cancer and explored its potential role on tumour progression. Overexpressed Ran in pancreatic cancer tissues was found highly correlated with the histological grade. Downregulation of Ran led to significant suppression of cell proliferation, cell cycle arrest at the G1/S phase and induction of apoptosis. In vivo studies also validated that result. Further studies revealed that those effects were at least partly mediated by the downregulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, CDK4, phospho-Rb and Survivin proteins and up regulation of cleaved Caspase-3.
GX1 is a tumor targeting peptide. In this study, we evaluated the antitumor efficacy of a GX1-derived fusion toxin, GX1-rmhTNF?, and investigated its targeting efficiency and pharmacokinetics in vivo using multimodality imaging. Flow cytometry revealed a greater level of cell apoptosis induced by GX1-rmhTNF? (27.1%) compared with rmhTNF? or a saline control (13.7% and 4.7%, respectively). SPECT (single-photon emission computed tomography) demonstrated high accumulation of GX1-rmhTNF? in tumor site. Biodistribution studies indicated GX1-rmhTNF? was cleared by the liver and kidney, and the drug may not cross the blood-brain barrier. In addition, bioluminescence imaging (BLI) showed that GX1-rmhTNF? caused a satisfactory delay in tumor growth in both subcutaneous and orthotopic cancer models. Contrast-enhanced ultrasound (CEUS) and CD31 staining revealed a loss in blood perfusion and vasculature. TUNEL and Ki67 staining validated the in vivo results. Biochemical analyses revealed limited renal and hepatic toxicity of GX1-rmhTNF?. This study demonstrated that GX1-rmhTNF? is a safe and potent anticancer agent that may have great potential for the targeted therapy of gastric cancer.
Gastric cancer is one of the most common cancers and accounts for a large proportion of cancer-related deaths in the world, while the pathogenesis of it is still not clear. Epigenetic changes have been found to participate in the development and progression of gastric cancer. Epigenetic changes involve methylation of cytosines in DNA, modifications of histone, chromatin remodeling, and alterations in the expression of microRNAs. MicroRNAs, a family of small non-coding RNAs, have been demonstrated to participate in many fundamental biological processes including the carcinogenesis of gastric cancer. Previous studies have shown that the downregulation of microRNAs are often caused by the methylation in the CpG islands of microRNA promoters. Here, we have summarized the functions and molecular mechanisms of gastric cancer related methylated microRNAs in gastric carcinogenesis. We further envisage the clinical application of microRNA methylation in the early diagnosis, treatment and prognosis assessment of gastric cancer.
The post-translational modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates essential cellular processes such as signal transduction, transcription, translation, and protein degradation. Misfolded, damaged, and unwanted proteins are tagged with a chain of ubiquitin moieties for degradation by the proteasome, which is critical for cellular homeostasis. In this review, we summarize the current knowledge of the interplay between O-GlcNAcylation and ubiquitination in the control of protein degradation. Understanding the mechanisms of action of O-GlcNAcylation in the ubiquitin-proteosome system shall facilitate the development of therapeutics for human diseases such as cancer, metabolic syndrome, and neurodegenerative diseases.
To facilitate the translation of cancer fluorescence imaging into clinical practice, the development of stable and highly specific and sensitive targeted fluorescence probes with low toxicity is desirable. GX1, a gastric tumor angiogenesis marker candidate, holds promise in the target-specific delivery of molecular imaging probes for early gastric cancer detection in vivo. In this study, we describe the design and synthesis of a series of novel penta-methine cyanine dyes using the symmetric synthesis method and further conjugated the dyes with GX1, allowing specific binding to the vasculature of gastric cancer. This efficient synthetic route can decrease the undesired byproducts, while increasing yield. Furthermore, in vivo fluorescence imaging revealed that this novel targeted probe accumulates selectively in the tumor site of SGC-7901 subcutaneous xenograft models. The combination of such novel vasculature-targeted molecular probes with fluorescence imaging technology may improve early detection, metastasis detection, and antitumor angiogenesis therapy for gastric cancer.
Targeted radiopharmaceutical is an effective treatment for solid tumors. By labeling with radionuclides, targeting peptide could achieve both noninvasive diagnosis and targeted radionuclide therapy. In order to evaluate the potential applicability of GEBP11 peptides in diagnosis and radiotherapy of gastric cancer, in this study, iodine 131 labeled GEBP11 peptides, including a novel bifid PEGlylated GEBP11 trimer and its corresponding monomer, were developed. The clinical potential of GEBP11 peptides, such as tumor binding affinity and antitumor efficacy was demonstrated and assessed with multimodality imaging methods. Cerenkov and SPECT imaging showed higher tumor uptake for (131)I-2PEG-(GEBP11)3 (P<0.05, day 1; P<0.01, day 2; vs. monomer). Biodistribution studies indicated higher tumor accumulation and better T/NT of (131)I-2PEG-(GEBP11)3. Bioluminescence imaging exhibited a significant tumor growth suppression in (131)I-2PEG-(GEBP11)3 treated group (P<0.001 vs. control; P<0.01 vs. monomer). After treatment with (131)I-2PEG-(GEBP11)3, the tumor volume and vasculature decreased significantly, and the survival time was prolonged to 75.5days. Meanwhile, no significant hepatic or renal toxicity was observed with (131)I-2PEG-(GEBP11)3 administered. In conclusion, (131)I-2PEG-(GEBP11)3 could be a promising candidate for peptide-based targeting therapy of gastric cancer. 2PEG-(GEBP11)3 might be a potential drug delivery vehicle for the antiangiogenic therapy of gastric cancer.
The interactions between cancer cells and the extracellular matrix (ECM) are important with respect to a number of cell behavoirs, yet remain unclear. In this study, self-assembled monolayers with different terminal chemical groups (hydroxyl (-OH), carboxyl (-COOH), animo (-NH2), mercapto (-SH), and methyl (-CH3)) were employed as substrates for the culture of MCF-7 cells to examine effects on cell behavior. Cell spreading was investigated by scanning electron microscopy, tallin expression by immunofluorescence, proliferation rate by counting cell numbers, cell cycle by flow cytometry, metabolism by high-performance liquid chromatography and cell migration by live cell imaging. Annexin V-FITC (fluorescein isothiocyanate) and JC-1 assays were performed to determine cell apoptosis and mitochondrial membrane potential, respectively. Our results demonstrate the varied behaviors of MCF-7 cells in response to different chemical groups. Specifically, NH2 and COOH terminal functional groups promote proliferation, the production of lactic acid and mobility of MCF-7 cells; SH and OH terminal groups enhance the expression and distribution of tallin but result in weak cell proliferation, metabolism, spreading and mobility. These results are meaningful for uncovering the interactions between the ECM and cancer cells; they are potentially useful for designing novel cancer treatment strategies.
The epithelial-mesenchymal transition (EMT) is a fundamental biological process that is involved in normal embryogenesis, would healing, and tissue repair, as well as numerous pathologies, including organ fibrosis, malignant transformation, and cancer progression. Both transcriptional and post-transcriptional regulatory mechanisms contribute to a complex and tightly controlled regulatory network during the EMT process, and a growing body of evidence now demonstrates that microRNAs (miRNAs) are crucial regulators of this network. miRNAs are a class of small non-coding RNAs that regulate gene expression through translational repression or mRNA degradation. A set of miRNAs have been discovered that have the potential to target multiple components of the signaling pathways and downstream effectors of the EMT. Our understanding of the roles that miRNAs play during the EMT process suggests that these miRNAs may eventually serve as novel biomarkers and therapeutic targets for various EMT-based pathological conditions. This review summarizes the current knowledge concerning how miRNAs mechanistically regulate the EMT and discusses the specific roles that miRNAs play in three EMT subtypes. We hope that a more comprehensive understanding of the functions of miRNAs in the EMT process will lead to the rapid development of novel diagnostic techniques and molecular-based strategies for controlling EMT.
Multidrug resistance (MDR) is the major cause of failure of gastric cancer chemotherapy. Members of the miR-17-92 cluster, including miR-19a/b, are considered oncomiRs and influence multiple aspects of the malignant phenotype of gastric cancer. However, the role of miR-19a/b in MDR in gastric cancer and its underlying mechanism remain unclear. In this study, we found that miR-19a/b were upregulated in MDR cell lines. Our results also showed that miR-19a/b upregulation decreased the sensitivity of gastric cancer cells to anticancer drugs. We further confirmed that miR-19a/b accelerated the ADR efflux of gastric cancer cells by increasing the levels of mdr1 and P-gp and that miR-19a/b suppressed drug-induced apoptosis by regulating Bcl-2 and Bax. Finally, we verified that PTEN, an inhibitor of AKT phosphorylation, is the functional target of miR-19a/b. Overall, these findings demonstrated that miR-19a/b promote MDR in gastric cancer cells by targeting PTEN.
Recurrence and metastasis remain the most common causes of lethal outcomes in hepatocellular carcinoma (HCC) after curative resection. Thus, it is critical to discover the mechanisms underlying HCC metastasis. Forkhead box C1 (FoxC1), a member of the Fox family of transcription factors, induces epithelial-mesenchymal transition (EMT) and promotes epithelial cell migration. However, the role of FoxC1 in the progression of HCC remains unknown. Here, we report that FoxC1 plays a critical role in HCC metastasis. FoxC1 expression was markedly higher in HCC tissues than in adjacent noncancerous tissues. HCC patients with positive FoxC1 expression had shorter overall survival times and higher recurrence rates than those with negative FoxC1 expression. FoxC1 expression was an independent, significant risk factor for recurrence and survival after curative resection. FoxC1 overexpression induced changes characteristic of EMT and an increase in HCC cell invasion and lung metastasis. However, FoxC1 knockdown inhibited these processes. FoxC1 transactivated Snai1 expression by directly binding to the Snai1 promoter, thereby leading to the inhibition of E-cadherin transcription. Knockdown of Snai1 expression significantly attenuated FoxC1-enhanced invasion and lung metastasis. FoxC1 expression was positively correlated with Snai1 expression, but inversely correlated with E-cadherin expression in human HCC tissues. Additionally, a complementary DNA microarray, serial deletion, site-directed mutagenesis, and a chromatin immunoprecipitation assay confirmed that neural precursor cell expressed, developmentally down-regulated 9 (NEDD9), which promotes the metastasis of HCC cells, is a direct transcriptional target of FoxC1 and is involved in FoxC1-mediated HCC invasion and metastasis.
Multidrug resistance (MDR) is a major clinical obstacle in treatment of gastric cancer (GC) and it accounts for the majority of cancer-related mortalities. Shugoshin1 (SGO1) is an important player in appropriate chromosome segregation and is involved in tumorigenesis. In this study, we found endogenous SGO1 overexpression in the multidrug-resistant GC cell lines SGC7901/VCR and SGC7901/ADR compared with their parental cell line SGC7901. By enhancing expression of SGO1, sensitivity of SGC7901 cells to vincristine (VCR), adriamycin, 5-fluorouracil (5-FU), and cisplatin (CDDP) was significantly diminished. Silencing its expression resulted in enhanced sensitivity of SGC7901/VCR and SGC7901/ADR cells to these antitumor drugs. Additionally, we confirmed that SGO1 increased capacity of cells to enable adriamycin (ADR) efflux and inhibit drug-induced apoptosis by regulating MRP 1, Bcl-2, and Bax genes so as to confer a MDR phenotype to GC cells. In brief, these findings suggest that SGO1 promotes MDR of GC cells and may be useful as a novel therapeutic target for preventing or reversing MDR.
Krüppel-like factor 8 (KLF8), a downstream transcription factor of transforming growth factor-?1 (TGF-?1), has a role in tumorigenesis, tumor progress and epithelial-to-mesenchymal transition (EMT) induction. Recent studies mainly focused on its role in breast cancer and hepatocellular carcinoma; however, little is studied in gastric cancer. Here, we aim to explore whether KLF8 is involved in TGF-?1-induced EMT in gastric cancer cells.
Multidrug resistance (MDR) remains a significant challenge to the clinical treatment of gastric cancer (GC). In the present study, using a phage display approach combined with MTT assays, we screened a specific peptide GMBP1 (Gastric cancer MDR cell-specific binding peptide), ETAPLSTMLSPY, which could bind to the surface of GC MDR cells specifically and reverse their MDR phenotypes. Immunocytochemical staining showed that the potential receptor of GMBP1 was located at the membrane and cytoplasm of MDR cells. In vitro and in vivo drug sensitivity assays, FACS analysis and Western blotting confirmed that GMBP1 was able to re-sensitize MDR cells to chemical drugs. Western blotting and proteomic approaches were used to screen the receptor of GMBP1, and GRP78, a MDR-related protein, was identified as a receptor of GMBP1. This result was further supported by immunofluoresence microscopy and Western blot. Additionally, Western blotting demonstrated that pre-incubation of GMBP1 in MDR cells greatly diminished MDR1, Bcl-2 and GRP78 expression but increased the expression of Bax, whereas downregulation of GRP78, function as a receptor and directly target for GMBP1, only inhibited MDR1 expression. Our findings suggest that GMBP1 could re-sensitize GC MDR cells to a variety of chemotherapeutic agents and this role might be mediated partly through down-regulating GRP78 expression and then inhibiting MDR1 expression. These findings indicate that peptide GMBP1 likely recognizes a novel GRP78 receptor and mediates cellular activities associated with the MDR phenotype, which provides new insight into research on the management of MDR in gastric cancer cells.
Our previous work identified thioredoxin-like protein 2 (Txl-2) as the target of the monoclonal antibody MC3 associated with colon cancer, but its underlying mechanisms remain poorly understood. Txl-2, a novel thioredoxin (Trx) and nucleoside diphosphate kinase family member, is alternatively spliced and gives rise to three different Txl-2 isoforms. In this study, Txl-2 expression in colon cancer, differential functions for Txl-2 isoforms in cell invasion and metastasis, and the downstream signaling were investigated.
Human gastric cancer is a big public health problem. Multidrug resistance is a main obstacle to successful chemotherapeutic treatment in gastric cancers and the underlying mechanism is not clear. Glycosylation, one of the most important post translational modifications of proteins, plays a vital role in diverse aspects of tumor progression. In the present study, we applied two multidrug resistance cell lines and their parental drug sensitive gastric cancer cell line to a modified cell surface capturing strategy with triplex labeling to characterize MDR related cell surface glycoproteome. Finally, 56 cell membrane glycoproteins were successfully identified via combination of identification by glycopeptides and quantitation by non-glycopeptides, and 11 of them were found to be differentially expressed with the same trend in both drug resistant cell lines compared with that in sensitive cell line. The further analysis by western blot and in vitro drug sensitivity assay demonstrated that our approach is reliable and accurate and suggested that these glycoproteins may represent as biomarkers for multidrug resistance in gastric cancer.
Apogossypolone (ApoG2), a potent small molecular inhibitor of Bcl-2 family proteins, is reported to have a significant anti-cancer effect in several types of cancers, but it has not been investigated in gastric cancer. In this study, we demonstrate in vitro and in vivo that ApoG2 inhibits human gastric cancer. Gastric carcinoma cell growth and proliferation was significantly hampered in vitro, as measured by MTT and colony formation assays. Real-time bioluminescence imaging indicated that ApoG2 causes tumor growth delay in a murine xenograft model. Further studies revealed that the ApoG2 induced apoptosis in gastric cancer cells was associated with the endoplasmic reticulum stress-induced apoptosis pathway. Conclusively, our results indicate that ApoG2 may be a promising agent for gastric cancer therapy.
Hepatocellular carcinoma (HCC) is a global health burden that is associated with limited treatment options and poor patient prognoses. Silybin (SIL), an antioxidant derived from the milk thistle plant (Silybum marianum), has been reported to exert hepatoprotective and antitumorigenic effects both in vitro and in vivo. While SIL has been shown to have potent antitumor activity against various types of cancer, including HCC, the molecular mechanisms underlying the effects of SIL remain largely unknown. The Notch signaling pathway plays crucial roles in tumorigenesis and immune development. In the present study, we assessed the antitumor activity of SIL in human HCC HepG2 cells in vitro and in vivo and explored the roles of the Notch pathway and of the apoptosis-related signaling pathway on the activity of SIL. SIL treatment resulted in a dose- and time-dependent inhibition of HCC cell viability. Additionally, SIL exhibited strong antitumor activity, as evidenced not only by reductions in tumor cell adhesion, migration, intracellular glutathione (GSH) levels and total antioxidant capability (T-AOC) but also by increases in the apoptotic index, caspase3 activity, and reactive oxygen species (ROS). Furthermore, SIL treatment decreased the expression of the Notch1 intracellular domain (NICD), RBP-J?, and Hes1 proteins, upregulated the apoptosis pathway-related protein Bax, and downregulated Bcl2, survivin, and cyclin D1. Notch1 siRNA (in vitro) or DAPT (a known Notch1 inhibitor, in vivo) further enhanced the antitumor activity of SIL, and recombinant Jagged1 protein (a known Notch ligand in vitro) attenuated the antitumor activity of SIL. Taken together, these data indicate that SIL is a potent inhibitor of HCC cell growth that targets the Notch signaling pathway and suggest that the inhibition of Notch signaling may be a novel therapeutic intervention for HCC.
IL-23 is a newly discovered proinflammatory cytokine that contributes to the maintenance and expansion of Th17 cells. IL-23 has recently been identified as playing a critical role in a number of chronic inflammatory diseases. However, the regulatory mechanism of IL-23 in chronic hepatitis B (CHB) remains largely unknown. The aims of this study were to detect the expression of IL-23 in CHB patients and to explore the molecular mechanism of hepatitis B virus (HBV)-induced IL-23 expression. Serum levels and hepatic expression of IL-23 were significantly upregulated in CHB patients. A positive correlation was found between IL-23 expression and the histological activity index score, HBV DNA load, and serum alanine aminotransferase and aspartate aminotransferase levels. HBx protein increased IL-23 expression in a dose-dependent manner. It also aided in the nuclear translocation of NF-?B, which directly bound to the promoters of IL-23 subunits p19 and p40 to facilitate their transcription. NF-?B inhibitors blocked the effect of HBx on IL-23 induction, and NF-?B subunits p65 and p50 increased the augmented IL-23 expression. Inhibition of ERK1/2 activation and transfection with ERK dominant-negative plasmid significantly blocked the HBx-induced IL-23 expression. Furthermore, PI3K and Ras-MEK-MAPK inhibitors significantly decreased the ERK1/2 activation and IL-23 expression. Thus, we report a new molecular mechanism for HBV-induced IL-23 expression, which involves the activation of the ERK/NF-?B pathway by HBx, leading to the transactivation of the IL-23 p19 and p40 promoters.
Epithelial-mesenchymal transition (EMT) is a key process that drives cancer invasion. Recently, hypoxia has been reported to induce EMT, accompanied by cytoskeleton remodeling. As RhoE is a key regulator in cytoskeleton formation, we hypothesized that RhoE may play a role in hypoxia-induced EMT. For the first time, we report that RhoE protein levels increase in gastric cancer cells under hypoxic conditions. Rigorous analysis revealed that RhoE up-regulation is at the transcriptional levels and requires hypoxia-inducible factor (HIF)-1? induction, and that HIF-1? binds a hypoxia-responsive element (HRE) on the RhoE promoter. Additionally, we discovered that hypoxia or overexpression of RhoE in normoxia up-regulates the mesenchymal marker Vimentin, down-regulates the epithelial marker E-cadherin, and significantly increases cell invasion in vitro. Silencing of HIF-1? or RhoE by specific siRNAs rescued these hypoxia-induced effects. Ectopic expression of RhoE also induced up-regulation of MMP2/MMP-9 in gastric cancer cells. This study identifies RhoE as a direct target for HIF-1 in gastric cancer cells. In addition, RhoE up-regulation represents a pivotal cellular adaptive response to hypoxia with implications in gastric cancer cell EMT and invasion. We propose that RhoE-targeted therapy might inhibit the high invasive potential of gastric cancer cells in hypoxic regions.
MicroRNAs (miRNAs) regulate tumor progression and invasion via direct interaction with target messenger RNAs (mRNAs). We defined miRNAs involved in cancer metastasis (metastamirs) using an established in vitro colorectal cancer (CRC) model of minimally metastatic cells (SW480 line) from a colon adenocarcinoma primary lesion and highly metastatic cells (SW620 line) from a metastatic lymph node from the same patient 1 year later. We used microarray analysis to identify miRNAs differentially expressed in SW480 and SW620 cells, focusing on miR-499-5p as a novel candidate prometastatic miRNA whose functions in cancer had not been studied. We confirmed increased miR-499-5p levels in highly invasive CRC cell lines and lymph node-positive CRC specimens. Furthermore, enhancing the expression of miR-499-5p promoted CRC cell migration and invasion in vitro and lung and liver metastasis in vivo, while silencing its expression resulted in reduced migration and invasion. Additionally, we identified FOXO4 and PDCD4 as direct and functional targets of miR-499-5p. Collectively, these findings suggested that miR-499-5p promoted metastasis of CRC cells and may be useful as a new potential therapeutic target for CRC.
The significance of carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7) expression in gastric carcinoma and precancerous lesions and its correlation with CEA expression has rarely been previously investigated.
Our previous study revealed that human ribosomal protein L6 (RPL6) was up-regulated in multidrug-resistant gastric cancer cells and over-expression of RPL6 could protect gastric cancer from drug-induced apoptosis. It was further demonstrated that up-regulation of RPL6 accelerated growth and enhanced in vitro colony forming ability of GES cells while down-regulation of RPL6 exhibited the opposite results. The present study was designed to investigate the potential role of RPL6 in therapy of gastric cancer for clinic. The expression of RPL6 and cyclin E in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemisty. It was found that RPL6 and cyclin E were expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa and the two were correlative in gastric cancer. Survival time of postoperative patients was analyzed by Kaplan- Meier analysis and it was found that patients with RPL6 positive expression showed shorter survival time than patients that with RPL6 negative expression. RPL6 was then genetically down-regulated in gastric cancer SGC7901 and AGS cell lines by siRNA. It was demonstrated that down-regulation of RPL6 reduced colony forming ability of gastric cancer cells in vitro and reduced cell growth in vivo. Moreover, down-regulation of RPL6 could suppress G1 to S phase transition in these cells. Further, we evidenced that RPL6 siRNA down-regulated cyclin E expression in SGC7901 and AGS cells. Taken together, these data suggested that RPL6 was over-expressed in human gastric tissues and caused poor prognosis. Down-regulation of RPL6 could suppress cell growth and cell cycle progression at least through down-regulating cyclin E and which might be used as a novel approach to gastric cancer therapy.
MGb2, a mouse-derived monoclonal antibody specific to gastric carcinoma, was developed in our laboratory. Nevertheless, the potential role of MGb2-antigen/TRAK1 (MGb2-Ag/TRAK1) in colorectal cancer (CRC) is unclear. The aim of this study was to investigate the relationship between MGb2-Ag/TRAK1 expression and the clinicopathological characteristics of CRC. The potential utility of MGb2-Ag/TRAK1 expression as a prognostic indicator was also evaluated.
MicroRNAs (miRNAs) are important regulators that play key roles in tumorigenesis and tumor progression. A previous report has shown that let-7 family members can act as tumor suppressors in many cancers. Through miRNA array, we found that let-7f was downregulated in the highly metastatic potential gastric cancer cell lines GC9811-P and SGC7901-M, when compared with their parental cell lines, GC9811 and SGC7901-NM; however, the mechanism was not clear. In this study, we investigate whether let-7f acts as a tumor suppressor to inhibit invasion and metastasis in gastric cancers.
Cancer stem cells are nowadays considered to be the origin of cancer. Also, stem cell associated genes are emerging as predictors of cancer malignancy. We investigated the association of several stemness genes (c-Myc, PTEN, p57 and p21) with clinic pathological parameters and survival in stomach cancer by performing immunohistochemistry on paraffin sections of gastric cancer patients who underwent surgical staging with following-up statistics. We discovered that expression of c-Myc was significantly related to distant metastasis, the combined expression of PTEN and p21 correlated positively to overall survival, while p57 was less useful in overall survival prediction in gastric cancer. Additionally, there is a positive correlation between expressions of p57 and p21. In conclusion, our present study indicated that expression of stemness genes (c-Myc, PTEN, p57 and p21) performed different predictive potential in the evaluation of clinical malignancy levels in gastric cancer.
Hepatic encephalopathy (HE) is a very common complication in patients after transjugular intrahepatic portosystemic shunt (TIPS). The purpose of this study is to determine the most robust predictors of post-TIPS HE by performing a systematic review of studies that identified the risk factors for patients with post-TIPS HE.
Sirt1 has been associated with various effects of calorie restriction, including an increase in lifespan. Here we show in mice that a central regulatory component in energy metabolism, the hypothalamic melanocortin system, is affected by Sirt1, which promotes the activity and connectivity of this system resulting in negative energy balance. In adult mice, the pharmacological inhibition of brain Sirt1 activity decreased Agrp neuronal activity and the inhibitory tone on the anorexigenic POMC neurons, as measured by the number of synaptic inputs to these neurons. When a Sirt1 inhibitor (EX-527) was injected either peripherally (i.p., 10 mg/kg) or directly into the brain (i.c.v., 1.5 nmol/mouse), it decreased both food intake during the dark cycle and ghrelin-induced food intake. This effect on feeding is mediated by upstream melanocortin receptors, because the MC4R antagonist, SHU9119, reversed Sirt1s effect on food intake. This action of Sirt1 required an appropriate shift in the mitochondrial redox state: in the absence of such an adaptation enabled by the mitochondrial protein, UCP2, Sirt1-induced cellular and behavioral responses were impaired. In accordance with the pharmacological results, the selective knock-out of Sirt1 in hypothalamic Agrp neurons through the use of Cre-Lox technology decreased electric responses of Agrp neurons to ghrelin and decreased food intake, leading to decreased lean mass, fat mass, and body weight. The present data indicate that Sirt1 has a central mode of action by acting on the NPY/Agrp neurons to affect body metabolism.
Muscle atrophy remains a significant concern in multiple inflammatory conditions, including injury, sepsis, cachexia, and HIV-associated wasting. Herein, we show that inflammatory stressors, including TNF-alpha, IFN-gamma, or lipopolysaccharide, potently induced the novel expression of the RNA editor ADAR1, an observation not previously described in muscle cells. We also observed that cytokine stimulation suppressed muscle-associated microRNAs, an observation also not previously demonstrated. To map potential effects of ADAR1 induction in the muscle program, we conducted knockdown and overexpression studies in the mouse C2C12 muscle precursor cell (MPC) line and in primary human MPCs. We show that knockdown of stress-induced ADAR1 increased inflammation-mediated declines in the muscle differentiation markers Myogenin and myosin heavy chain, and knockdown reduced levels of active phosphorylated Akt (phospho-Akt), but had no effect on microRNA transcript levels, suggesting a role for ADAR1 in buffering inflammatory stress effects on myogenic transcription and protein synthesis pathways. In addition, overexpression of recombinant ADAR1 suppressed active phosphorylated double-stranded RNA (dsRNA)-dependent protein kinase (phospho-PKR), consistent with a role for ADAR1 in limiting inflammation-driven catabolic atrophy pathways. Collectively, these data identify a novel regulatory role for ADAR1 activation under inflammatory stress to both promote muscle protein synthesis pathways and limit atrophy pathways.
Hepatocellular carcinoma (HCC) is the second most common malignancy in Asia, with a 5-year survival rate of less than 5% due to high recurrence after surgery and resistance to chemotherapy. A variety of therapeutic interventions to treat HCC, particularly gene therapy, have recently been investigated in tumor model systems to provide a more complete understanding of hepatocarcinogenesis and effectively design therapeutic strategies to treat this disease. In our study, we constructed an adenoviral vector expressing small interfering RNA (siRNA) targeting a newly discovered gene named upregulated gene 11 (URG11). We introduced this vector into HCC cells to investigate the role of URG11 in HCC carcinogenesis. We observed that upon URG11 knockdown, HCC cell proliferation was inhibited through downregulation of several G1-S phase related molecules including cyclin D1 and apoptosis was induced as a result of Bcl-2 downregulation. Besides decreased expression of cyclin D1, CDK4, pRb and Bcl-2, URG11 also suppressed several other proteins including CAPN9, which was identified by cDNA microarray and 2D gel electrophoresis. Moreover, Ad-URG11-siRNA significantly suppressed HCC tumor growth in nude mice. In conclusion, Ad-URG11-siRNA can significantly suppress HCC tumor growth in vitro and in vivo by silencing the URG11 gene, and the use of this vector for gene therapy may represent a novel strategy to treat human HCC.
Several monoclonal antibodies (McAbs) have been developed that show high sensitivity and specificity to gastric cancer and colorectal cancer. However, few of the antigens recognized by these antibodies have been identified. The authors now report the selection of anti-idiotype (anti-id) antibodies of MGb1 McAb against gastric cancer and MC5 McAb against colorectal cancer using phage-displayed single-chain variable fragment (ScFv) libraries. After purification, the anti-id antibodies were approximately 30 kd and could be recognized by MGb1/MC5 McAb. Anti-id antibodies significantly blocked the binding of MGb1 and MC5 to gastric cancer/colorectal cancer cells, respectively, suggesting that the antibodies were specific to MGb1 and MC5. Antibodies against gastric and colorectal cancer could be detected in mice at 6 weeks after immunization with the anti-id antibodies. At week 8, antibody titers reached 1:400. The anti-id antibodies may be useful as novel reagents for developing vaccines against gastric cancer and colorectal cancer.
MicroRNAs play key roles in tumor metastasis. Here, we describe the regulation and function of miR-218 in gastric cancer (GC) metastasis. miR-218 expression is decreased along with the expression of one of its host genes, Slit3 in metastatic GC. However, Robo1, one of several Slit receptors, is negatively regulated by miR-218, thus establishing a negative feedback loop. Decreased miR-218 levels eliminate Robo1 repression, which activates the Slit-Robo1 pathway through the interaction between Robo1 and Slit2, thus triggering tumor metastasis. The restoration of miR-218 suppresses Robo1 expression and inhibits tumor cell invasion and metastasis in vitro and in vivo. Taken together, our results describe a Slit-miR-218-Robo1 regulatory circuit whose disruption may contribute to GC metastasis. Targeting miR-218 may provide a strategy for blocking tumor metastasis.
Our previous study revealed that human ribosomal protein L6 (RPL6) was upregulated in multidrug-resistant gastric cancer cells and over-expression of RPL6 could protect gastric cancer cells from drug-induced apoptosis. The present study was designed to explore the role of RPL6 in tumorigenesis and development of gastric cancer. The expression of RPL6 in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemical staining. It was found RPL6 was expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa. RPL6 was then genetically overexpressed or knocked down in human immortalized gastric mucosa epithelial GES cells. It was demonstrated that upregulation of RPL6 accelerated the growth and enhanced in vitro colony forming ability of GES cells whereas downregulation of RPL6 showed adverse effects. Moreover, over-expression of RPL6 could promote G1 to S phase transition of GES cells. It was further evidenced that upregulation of RPL6 resulted in elevated cyclin E expression while downregulation of RPL6 caused decreased cyclin E expression in GES cells. Taken together, these data indicated that RPL6 was overexpressed in human gastric cancer and its over-expression could promote cell growth and cell cycle progression at least through upregulating cyclin E expression.
Previous studies in our laboratory have suggested that gankyrin expression is correlated with a malignant phenotype in colorectal cancer. Here, we investigated the possible role of gankyrin in pancreatic carcinogenesis. Gankyrin expression was significantly increased in pancreatic cancer compared to non-cancerous tissues. This expression significantly enhanced cancer cell proliferation and growth in vitro and in vivo. Suppression of gankyrin downregulated cyclin A, cyclin D1, cyclin E, CDK2, CDK4, PCNA and p-Rb but upregulated p27, Rb and p53. However, gankyrin overexpression led to opposite results. Thus, gankyrin could enhance pancreatic cancer cell proliferation by promoting cell cycle progression and p53 degradation.
Hepatic gluconeogenesis is a major contributing factor to hyperglycemia in the fasting and postprandial states in type 2 diabetes mellitus (T2DM). Because Sirtuin 1 (SirT1) induces hepatic gluconeogenesis during fasting through the induction of phosphoenolpyruvate carboxylase kinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase) gene transcription, we hypothesized that reducing SirT1, by using an antisense oligonucleotide (ASO), would decrease fasting hyperglycemia in a rat model of T2DM. SirT1 ASO lowered both fasting glucose concentration and hepatic glucose production in the T2DM rat model. Whole body insulin sensitivity was also increased in the SirT1 ASO treated rats as reflected by a 25% increase in the glucose infusion rate required to maintain euglycemia during the hyperinsulinemic-euglycemic clamp and could entirely be attributed to increased suppression of hepatic glucose production by insulin. The reduction in basal and clamped rates of glucose production could in turn be attributed to decreased expression of PEPCK, FBPase, and G6Pase due to increased acetylation of signal transducer and activator of transcription 3 (STAT3), forkhead box O1 (FOXO1), and peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), known substrates of SirT1. In addition to the effects on glucose metabolism, SirT1 ASO decreased plasma total cholesterol, which was attributed to increased cholesterol uptake and export from the liver. These results indicate that inhibition of hepatic SirT1 may be an attractive approach for treatment of T2DM.
Epithelial-to-mesenchymal transition (EMT) induced by chronic hypoxia is one of the critical causes of renal fibrosis. Twist, a basic helix-loop-helix transcription factor, is believed to be important in promoting EMT. We found that the expression of Twist was increased in human tubule cell lines (HK-2 and HKC) grown under hypoxic conditions. This was accompanied by reduced expression of the epithelial markers E-cadherin and ZO-1 and enhanced expression of the mesenchymal markers vimentin and alpha-smooth muscle actin. When Twist was overexpressed in these cells it induced a mesenchymal phenotype, whereas its knockdown by short interfering RNA (siRNA) effectively reversed hypoxia-induced EMT. We showed that transfection with siRNA to hypoxia-inducible factor-1alpha (HIF-1alpha), another basic helix-loop-helix transcription factor, reduced Twist expression. Twist promoters contain HIF1-alpha-binding sites and transfection of reporter constructs using the promoter showed increased transcription in cells subjected to hypoxia. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the presence of a functional HIF-1alpha-binding site within the proximal Twist gene promoter. In an in vivo assay using the rat remnant kidney we found that both Twist and HIF-1alpha were overexpressed in tubular epithelial cells showing EMT. These studies suggest that HIF-1alpha induces Twist expression in hypoxic tubular cells and that this plays a role in EMT during renal fibrogenesis.
Gastric cancer is the top lethal cancer in Asia. As the majority of cases present with advanced disease, conventional therapies (surgery, chemotherapy, and radiotherapy) have limited efficacy to reduce mortality. Emerging modalities provide promise to combat this malignancy. Target-protein-based cancer therapy has become available in clinical practice. Numerous molecules have been shown potential to target specific pathways for tumor cell growth. Cyclooxygenase-2 (COX-2) is overexpressed in and correlated with gastric cancer, and knockdown of COX-2 or administration of COX-2 inhibitors suppresses tumor formation in models of gastric cancer. Induction of apoptosis, reduction of angiogenesis, and blocking of potassium ion channels may present new mechanisms of COX-2 inhibition. Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene whose deficiency is causally related to gastric cancer. RUNX3 is downregulated in metastatic gastric cancer. RUNX3 activation inhibits angiogenesis in xenograft tumors in nude mice. Tumor microenvironment modulation also provides a powerful tool to inhibit cancer development and progress; details of the potential roles of angiopoietins are discussed in this review. Osteopontin is a secreted protein involved in stress response, inflammation, wound healing, and immune response. Inhibition of osteopontin by RNA interfering technique suppressed tumorigenesis as well as angiogenesis in gastric cancer. Immunotherapy remains another important choice of adjuvant therapy for cancer. A tumor-specific antigen MG7-Ag has been identified with great potential for inducing immune response in gastric cancer. Using HLA-A-matched allogeneic gastric cancer cells to induce tumor-specific cytotoxic T lymphocytes appeared to be an alternative option of immunotherapy for gastric cancer.
The present study aimed to describe the characterization of an antibody MGb2 that reacts with an epitope on gastric cancer cells, and identification of MGb2 antigen (MGb2-Ag). Immunostaining revealed its distribution in human tissues and demonstrated that the positive rate of MGb2-Ag was 81.48% in gastric cancer, 100% in gastric signet-ring cell carcinoma and mucinous adenocarcinoma, 13.16% in precancerous conditions, and 0% in chronic superficial gastritis. Using Western blotting, immunoprecipitation and MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry), MGb2-Ag was identified as TRAK1 (Trafficking protein, kinesin-binding 1), a new molecular gained limited recognition. Both MGb2 and commercial anti-TRAK1 Ab recognized prokaryotic expressed TRAK1. Immunostaining characteristics of TRAK1 were identical with MGb2-Ag in continuous sections of paraffin-embedded tissues of gastric tissues. This is the first report that TRAK1/MGb2-Ag is a promising diagnostic marker for gastric cancer and may help to detect signet-ring cell carcinoma and mucinous adenocarcinoma.
The fasting-activated longevity protein sirtuin 1 (SirT1, ref. 1) promotes gluconeogenesis in part, by increasing transcription of the key gluconeogenic genes pepck1 and g6pase, through deacetylating PGC-1alpha and FOXO1 (ref. 4). In contrast, signal transducer and activator of transcription 3 (STAT3) inhibits glucose production by suppressing expression of these genes. It is not known whether the inhibition of gluconeogenesis by STAT3 is controlled by metabolic regulation. Here we show that STAT3 phosphorylation and function in the liver were tightly regulated by the nutritional status of an animal, through SirT1-mediated deacetylation of key STAT3 lysine sites. The importance of the SirT1-STAT3 pathway in the regulation of gluconeogenesis was verified in STAT3-deficient mice in which the dynamic regulation of gluconeogenic genes by nutritional status was disrupted. Our results reveal a new nutrient sensing pathway through which SirT1 suppresses the inhibitory effect of STAT3, while activating the stimulatory effect of PGC-1alpha and FOXO1 on gluconeogenesis, thus ensuring maximal activation of gluconeogenic gene transcription. The connection between acetylation and phosphorylation of STAT3 implies that STAT3 may have an important role in other cellular processes that involve SirT1.
An increasing body of evidence indicates that miR-149 can both suppress and promote tumor growth depending on the tumor type. However, the role of miR-149 in the progression of gastric cancer (GC) remains unknown. Here we report that miR-149 is a tumor suppressor in human gastric cancer. miR-149 expression is decreased in GC cell lines and clinical specimens in comparison to normal gastric epithelial cell and tissues, respectively. The expression levels of miR-149 also correlate with the differentiation degree of GC cells and tissues. Moreover, ectopic expression of miR-149 in gastric cancer cells inhibits proliferation and cell cycle progression by down-regulating ZBTB2, a potent repressor of the ARF-HDM2-p53-p21 pathway, with a potential binding site for miR-149 in its mRNAs 3UTR. It is also found that ZBTB2 expression increases in GC cells and tissues compared to normal gastric epithelial cell and tissues, respectively. Silencing of ZBTB2 leads to suppression of cell growth and cell cycle arrest in G0/G1 phase, indicating that ZBTB2 may act as an oncogene in GC. Furthermore, transfection of miR-149 mimics into gastric cancer cells induces down-regulation of ZBTB2 and HDM2, and up-regulation of ARF, p53, and p21 compared to the controls. In summary, our data suggest that miR-149 functions as a tumor suppressor in human gastric cancer by, at least partially through, targeting ZBTB2.
Coronins are a family of highly evolutionary conserved proteins reportedly involved in the regulation of actin cytoskeletal dynamics, although only coronin 3 has been shown to be related to cancer cell migration. In glioblastoma cells, the knockdown of coronin 3 inhibits cell proliferation and invasion. Coronin 3 is also associated with the aggression and metastasis of hepatocellular carcinoma. In this paper, we analyze the migration, invasion and metastasis abilities of gastric cancer cells after up- or down-regulation of coronin 3, and explore the mechanism of coronin 3 in the process of gastric cancer metastasis.
RhoE, an atypical Rho protein, is differently deregulated in some solid tumors, and there are conflicting data describing the role of RhoE in tumor cell migration and invasion. This study aimed to investigate RhoE expression in human colorectal cancer and its relationship with clinicopathological features and prognosis.
The association between hepatocellular carcinoma (HCC) and the 61A>G polymorphism in the epidermal growth factor (EGF) gene has been analyzed in several studies, but results have been inconsistent. The aim of this study was to integrate previous findings and explore whether this polymorphism is associated with susceptibility to HCC. A meta-analysis was performed by searching PubMed, Web of Science, and Cochrane Library databases. Data were extracted using predefined form and pooled odds ratios (OR) with 95% confidence intervals (CI) and were calculated to evaluate the strength of this association. Five studies involving 690 cases, 514 healthy controls, and 1419 controls with cancer-free liver diseases were identified. On the basis of healthy controls, the significant main effects on HCC risk were observed in a heterozygote comparison (OR=1.76, 95% CI 1.07-2.90, p=0.02) and a dominant genetic model (OR=1.65, 95% CI 1.03-2.66, p=0.04). On the basis of the controls with cancer-free liver diseases, a significantly increased risk of HCC was found in all the genetic models. Subgroup analyses stratified by ethnicity and etiology of HCC also showed positive associations. The EGF 61G allele is a risk factor for developing HCC without the influence of ethnic and etiological diversity.
The proliferation-specific transcription factor Forkhead box M1 (FoxM1) acts as a master regulator of cancer cell growth and survival and plays an important role in the development of hepatocellular carcinoma. However, the molecular mechanisms that regulate FoxM1 expression remain largely unknown. In the current study, we demonstrated that tumor necrosis factor (TNF)-?? induced FoxM1 expression and transactivated its promoter activity in hepatoma cells. Serial 5" deletion and site-directed mutagenesis revealed that the induction of FoxM1 expression by TNF-? was dependent upon the hypoxia-inducible factor 1 (HIF1)-1 and HIF1-3/4 binding sites within the FoxM1 promoter. Furthermore, at the transcriptional level, the stabilization of HIF-1? via reactive oxygen species generation led to the binding of HIF-1? to the FoxM1 promoter and resulted in increased FoxM1 expression. The inhibition of both HIF-1? expression and reactive oxygen species generation significantly decreased TNF-?-induced FoxM1 overexpression. Consequently, the upregulation of FoxM1 promoted the proliferation of hepatoma cells and enhanced their resistance to TNF-?-induced apoptosis. Consistently, there was a positive correlation between HIF-1? and FoxM1 expression in 406 human hepatocellular carcinoma tissues, and the combination of these two parameters was a powerful predictor of poor prognosis in hepatocellular carcinoma patients after curative resection. Here, we report a new molecular mechanism by which FoxM1 expression is regulated by the TNF-?/reactive oxygen species/HIF-1 pathway, and this mechanism results in the proliferation of hepatoma cells and their resistance to apoptosis.
Liver fibrosis is characterized by accumulation of extracellular matrix. Our previous study found that osteopontin (OPN) increased in plasma of cirrhotic patients and indicative of cirrhosis staging. The present study was designed to investigate the expression of OPN in liver tissues and plasma of cirrhotic patients and further explore the role of OPN in human hepatic stellate cell (HSC) activation.
Forkhead box M1 (FoxM1) is a master regulator of tumor metastasis that plays an important role in the development of hepatocellular carcinoma (HCC). However, whether or not FoxM1 contributes to the progression of HBV-associated HCC (HBV-HCC) remains unknown. Therefore, we aimed at investigating the clinicopathologic significance of FoxM1 in HBV-HCC and the potential role of FoxM1 in hepatitis B virus X (HBx)-mediated invasiveness and metastasis.
Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.
Gastric cancer is the second leading cause of cancer mortality worldwide. Understanding the multistep process of carcinogenesis of gastric cancer is pivotal to develop novel therapeutic strategies. Molecular imaging in preclinical cancer models bridges the gap of laboratory-based experiment and clinical translation. To this end, the human gastric cancer cell line SGC-7901 was established to stably express luciferase and GFP by lentiviral transduction (SGC7901-Luc-GFP). Preclinical models were developed by orthotopic transplantation of SGC-7901-Luc-GFP into the sub-serosal layer of the stomach of immunocompromised mice. Tumor progression and therapeutic responses were dynamically tracked by bioluminescence imaging (BLI). Bioluminescence tomography (BLT) was used to monitor stereoscopic morphological and signal changes during tumor progression. Good correlation between cell number and bio-luminescence/fluorescence intensity was observed (R(2)=0.9983/r(2)=0.9974) in vitro. Tumor progression and therapeutic response could be successfully followed directly by BLI. Importantly, BLT provided a more accurate spatial location and tomographic quantification of the internal lesion. In conclusion, our novel bioluminescence-based preclinical gastric cancer models enable superior, noninvasive monitoring gastric cancer progression and their drug responses. The BLT technique in particular, may have great potential for future oncological studies.
Multidrug resistance (MDR) in gastric cancer remains a major challenge to clinical treatment. Activating transcription factor 4 (ATF4) is a stress response gene involved in homeostasis and cellular protection. However, the expression and function of ATF4 in gastric cancer MDR remains unknown. In this study, we investigate whether ATF4 play a role in gastric cancer MDR and its potential mechanisms.
Forkhead box Q1 (FoxQ1) is a master regulator of tumor metastasis. However, the molecular mechanism of FoxQ1 in regulating hepatocellular carcinoma (HCC) metastasis remains unknown. Here, we report a novel function for FoxQ1 in modifying the tumor microenvironment to promote HCC metastasis. FoxQ1 expression was an independent and significant risk factor for the recurrence and survival in two independent cohorts of 1002 HCC patients. FoxQ1 induced EMT through the trans-activation of ZEB2 expression by directly binding to the ZEB2 promoter. Knockdown of ZEB2 decreased FoxQ1-enhanced HCC metastasis, whereas up-regulation of ZEB2 rescued the decreased metastasis induced by FoxQ1 knocking down. Additionally, serial deletion, site-directed mutagenesis, and a chromatin immunoprecipitation assays showed that VersicanV1, which promoted HCC metastasis and macrophage attraction, was a direct transcriptional target of FoxQ1. FoxQ1-induced VersicanV1 expression promoted the secretion of CCL2 from HCC cells. Chemotaxis assay showed that the culture media from FoxQ1-overexpressing HCC cells increased the migratory activity of the macrophages. Inhibition of VersicanV1 and CCL2 expression significantly inhibited FoxQ1-mediated macrophage migration. In animal studies, the up-regulation of FoxQ1 in HCC cells promoted HCC metastasis and intratumoral tumor associated macrophage (TAM) infiltration, whereas knockdown of VersicanV1 reduced FoxQ1-mediated HCC metastasis and intratumoral TAM infiltration. Depletion of macrophages using clodronate liposomes dramatically decreased FoxQ1-enhanced HCC metastasis. In human HCC tissues, FoxQ1 expression was positively correlated with ZEB2 and VersicanV1 expression, and intratumoral TAM infiltration. Patients with positive co-expression of FoxQ1 and ZEB2, FoxQ1 and VersicanV1, or FoxQ1 and intratumoral TAMs were associated with poorer prognosis. Conclusion: FoxQ1 promoted HCC metastasis by trans-activating ZEB2 and VersicanV1 expression, resulting in the induction of EMT and the recruitment of macrophage infiltration. (Hepatology 2013;).
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.