FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway

The fucosyltransferase (FUT) family is the key enzymes in cell-surface antigen synthesis during various biological processes such as tumor multidrug resistance (MDR). The aim of this work was to analyze the alteration of FUTs involved in MDR in human hepatocellular carcinoma (HCC) cell lines. Using mass spectrometry (MS) analysis, the composition profiling of fucosylated N-glycans differed between drug-resistant BEL7402/5-FU (BEL/FU) cells and the sensitive line BEL7402. Further analysis of the expressional profiles of the FUT family in three pairs of parental and chemoresistant human HCC cell lines showed that FUT4, FUT6 and FUT8 were predominant expressed in MDR cell lines. The altered levels of FUT4, FUT6 and FUT8 were responsible for changed drug-resistant phenotypes of BEL7402 and BEL/FU cells both in vitro and in vivo. In addition, regulating FUT4, FUT6 or FUT8 expression markedly modulated the activity of the phosphoinositide 3 kinase (PI3K)/Akt signaling pathway and MDR-related protein 1 (MRP1) expression. Inhibition of the PI3K/Akt pathway by its specific inhibitor wortmannin, or by Akt small interfering RNA (siRNA), resulted in decreased MDR of BEL/FU cells, partly through the downregulation of MRP1. Taken together, our results suggest that FUT4-, FUT6- or FUT8-mediated MDR in human HCC is associated with the activation of the PI3K/Akt pathway and the expression of MRP1, but not of P-gp, indicating a possible novel mechanism by which the FUT family regulates MDR in human HCC.     

Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

TRAIL (TNF-related apoptosis-inducing ligand) is one member of TNF superfamily[1]. It is unique, for it could specifically induce the apoptosis of tumor cells or virus-infected cells but have no cytotoxic effects onnormal cells[1,2]. Owing to this characteristic, it has become a promising candidate molecule for biological therapy for tumor. Many factors could affect the sensitivity towardsTRAIL-induced apoptosis, including cytokines, virus infection, drugs, radials, etc. Studies show tha…     

Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study preliminary molecular mechanisms for its effects. In order to set up a modelin vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test, caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL receptors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 μg/L TRAIL was 41.4%±7.2%, significantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly confirms the effects of HBx on TRAIL-induced apoptosis from two different points and it is not related with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.     

PEI-CS/siRNA复合颗粒对肝癌耐药细胞BEL7402/5-FU中MRE11表达的影响

目的:探讨聚乙烯亚胺-壳聚糖(PEI-CS)/si RNA复合颗粒对肝癌耐药细胞BEL7402/5-FU中MRE11表达的影响。方法:采用复凝聚法将PEI-CS(100μg/m L)与不同浓度的MRE11 si RNA-FAM形成PEI-CS/si RNA复合颗粒,并转染BEL7402/5-FU细胞,用荧光显微镜和Real-time PCR检测转染效率和沉默效率。结果:荧光显微镜观察结果显示:转染细胞48 h后,3.125、6.25、12.5、25、50μg/m L的si RNA与PEI-CS形成的复合颗粒的转染率分别为62.31%、76.09%、79.99%、86.49%、96.59%。转染细胞48、72、96 h后,12.5μg/m L的si RNA与PEI-CS形成的复合颗粒的转染率分别为78.22%、55.76%、42.85%,25μg/m L的si RNA与PEI-CS形成的复合颗粒的转染率分别为83.67%、74.23%、67.45%。Real-time PCR检测结果显示:25μg/m L的si RNA与PEI-CS形成的复合颗粒转染48小时后,对BEL7402/5-FU细胞中MRE11基因的沉默效率为35.4%。结论:聚乙烯亚胺-壳聚糖/si RAN复合颗粒能有效转染肝癌耐药细胞Bel7402/5-FU,并对BEL7402/5-FU细胞中MRE11基因表达有一定抑制作用。     

Reversal effect of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on multi-drug resistance in resistant human hepatocellular carcinoma cell line BEL-7402/5-FU

Multi drug resistance (MDR) is a major obstacle in the chemotherapeutic treatment of many human cancers. 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), a chalcone, isolated from the buds of Cleistocalyx operculatus, has been shown to have antitumor effects on human carcinoma SMMC-7721 cells in vitro and in vivo. In this paper, we studied the reversal effect and the mechanism of DMC on human hepatocellular carcinoma drug-resistant cells BEL-7402/5-FU in vitro. Administration of DMC reversed the multi-drug resistance of human hepatocellular carcinoma BEL-7402/5-FU cells significantly. DMC enhanced the sensitivity of BEL-7402/5-FU cells to 5-fluorouracil (5-FU) and doxorubicin (DOX). Staining with Hoechst 33258 and flow cytometric analysis showed that DMC has apoptosis-inducing effect on BEL-7402/5-FU cells. It could also increase the concentration of 5-FU in the resistant multi-drug-resistant cells. We also observed that over-expression of the multi-drug resistance-associated protein (MRP1) and of the glutathione S-transferase π (GST-π) contributed to MDR in BEL-7402/5-FU cells. The mRNA expressions of MRP1 and GST-π and the protein expression of MRP1 were decreased by DMC. These data demonstrated that DMC could effectively reverse MDR in BEL-7402/5-FU cells.     

Proteomic analysis on multi-drug resistant cells HL-60/DOX of acute myeloblastic leukemia

Multi-drug resistance (MDR) is an important factor that causes treatment failure in acute leukemia. However, the full development mechanisms of MDR still await [corrected] investigation. The purpose of this study is to investigate differentially expressed proteins in the multi-drug resistant acute myeloblastic leukemia (AML) cell line HL-60/DOX and the drug sensitive cell line HL-60, and to identify new potential multi-drug resistant related molecules with the proteomic approach. Two-dimensional gel electrophoresis (2-DE) maps of the proteins, extracted from two AML cell lines, HL-60/DOX and HL-60, were established respectively. The extracted proteins were digested by enzymes and identified with the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The data of the peptide mass fingerprinting (PMF) was matched with databases of proteomics available on the Internet. Results showed that 16 proteins were identified to be differentially expressed between HL-60/DOX and HL-60 cells. They involved the protein disulfide isomerase precursor (PDI), the proteasomes alpha1 and other proteins which are related to drug resistance or cell metabolism, but their functional significances are required further investigation. Nevertheless, it is clear that this proteomic approach for studing the biology and development of MDR is a prerequisite in leukemia.     

VEGF is essential for the growth and migration of human hepatocellular carcinoma cells

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF induces new vessel formation and tumor growth by inducing mitogenesis and chemotaxis of normal endothelial cells and increasing vascular permeability. However, little is known about VEGF function in the proliferation, survival or migration of hepatocellular carcinoma cells (HCC). In the present study, we have found that VEGF receptors are expressed in HCC line BEL7402 and human HCC specimens. Importantly, VEGF receptor expression correlates with the development of the carcinoma. By using a comprehensive approaches including TUNEL assay, transwell and wound healing assays, migration and invasion assays, adhesion assay, western blot and quantitative RT-PCR, we have shown that knockdown of VEGF165 expression by shRNA inhibits the proliferation, migration, survival and adhesion ability of BEL7402. Knockdown of VEGF165 decreased the expression of NF-κB p65 and PKCα while increased the expression of p53 signaling molecules, suggesting that VEGF functions in HCC proliferation and migration are mediated by P65, PKCα and/or p53.     

健脾化瘀中药提高胞嘧啶脱氨酶/单纯疱疹病毒胸苷激酶基因治疗肝细胞癌的实验研究

目的:观察健脾化瘀中药提高胞嘧啶脱氨酶/单纯疱疹病毒胸苷激酶基因治疗肝细胞癌的作用。方法:脂质体lipofectamine将含有双自杀基因的腺病毒载体pAd-CD/TK导人293细胞,收集病毒上清转染人肝癌细胞BEL7402,MTT法测定BEL7402细胞存活率。裸鼠人肝癌模型转染CD/TK双自杀基因后,给予5-FC500mg/kg,GCV 100mg/kg腹腔注射,同时予健脾化瘀中药960复方灌胃。观察肿瘤生长情况。结果:给予前体药物5-FC和GCV后,CD/TK转染细胞被杀死。并表现出较强的旁观者效应。转染细胞比例达到10%即表现出较强的杀伤作用(P<0.01)。健脾化瘀中药960复方具有提高旁观者效应作用,1.67ml/kg和2.5ml/kg960复方含药血清组细胞存活率显著低于对照组(P<0.01)。转染基因组应用5-FC和GCV治疗后,裸鼠肝癌的生长明显受到抑制(P<0.05),抑瘤率39.42%,单用中药组抑瘤率18.04%,中药与CD/TK 5-FC/GCV联合运用组,较单纯CD/5-FC/HSV-tk/GCV对裸鼠肿瘤模型的生长抑制作用更加明显(P<0.05),抑瘤率55.10%。结伦:腺病毒介导CD/TK自杀基因可有效地杀死人肝癌BEL7402细胞,健脾化瘀中药960复方具有显著提高CD/TK双自杀基因对人肝癌细胞的抑杀作用。     

Proteome analysis of multidrug resistance in vincristine-resistant human gastric cancer cell line SGC7901/VCR

In order to elucidate the mechanisms of multidrug resistance (MDR) of vincristine-resistant human gastric carcinoma cell line SGC7901/VCR, 2-DE was used to separate the total proteins of SGC7901/VCR and its parental cell line SGC7901. PDQuest software was applied to analyze 2-DE images, and the differential protein spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Then the differential expressional levels of partially identified proteins were determined by Western blot analysis and real-time RT-PCR. Furthermore, the association of heat shock protein (HSP27), one of the highly expressed proteins in sgc7901/vcr, with MDR was analyzed using antisense inhibition of HSP27. In this study, the well-resolved, reproducible 2-DE patterns of SGC7901/VCR and SGC7901 were established, and yielded about 1100 protein-spots each. All the 24 differential proteins between the two cell lines were identified, and the differential expression levels of the partial proteins were confirmed. The suppression of HSP27 expression by HSP27 antisense oligonucleotides could enhance vincristine chemosensitivity in sgc7901/vcr and induce the cells to exhibit apoptotic morphological features after vincristine treatment. The differentially expressed proteins could be divided into six groups based on their functions: calcium-binding proteins, chaperones, proteins involved in drug detoxification or repair of DNA damage, metabolic enzymes, proteins related to cellular structure, and proteins relative to signal transduction, some of which may contribute to MDR of human gastric carcinoma cell line SGC7901/VCR. These data will be valuable for further study of the mechanisms of MDR in human gastric cancer.     

Quantitative proteome analysis of ovarian cancer tissues using a iTRAQ approach

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Here, we comparatively analyzed the proteome profiles of ovarian cancer tissues and normal ovarian epithelial tissues. Using the high‐throughput proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ)‐coupled with two‐dimensional‐liquid chromatography‐tandem mass spectrometry, 1,259 unique proteins were identified. Of those, 205 were potentially differentially expressed between ovarian cancer and normal ovarian tissues. Several of the potentially differentially expressed proteins were validated by Western blotting and real‐time quantitative RT‐PCR analyses. Furthermore, up‐regulation of KRT8, PPA1, IDH2, and S100A11 were validated in ovarian tissue microarrays by immunohistochemistry. Silencing of S100A11 expression suppressed the migration and invasion properties of ovarian cancer cells in vitro. Our study represents the successful application of iTRAQ technology to an investigation of ovarian cancer. Many of the potentially differentially expressed proteins identified had not been linked to ovarian cancer before, and provide valuable novel insights into the underlying mechanisms of carcinogenesis in human ovarian cancer. J. Cell. Biochem. 113: 3762–3772, 2012. © 2012 Wiley Periodicals, Inc.     

Quantitative Proteomic Analysis Reveals That Anti-Cancer Effects of Selenium-Binding Protein 1 In Vivo Are Associated with Metabolic Pathways

Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways.     

Ganoderic acid produced from submerged culture of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> induces cell cycle arrest and cytotoxicity in human hepatoma cell line BEL7402

Ganoderic acid (GA), produced by submerged culture of Ganoderma lucidum, at 500 μg/ml, caused nearly a 70% inhibition of the growth of human hepatoma cell line BEL7402 but not of a normal human liver cell line L02. Flow cytometry analyses showed that GA blocked the BEL7402 cell cycle at the transition from G₁ to S phase.     

Proteomic analysis of cellular protein alterations using a hepatitis B virus-producing cellular model

Hepatitis B virus (HBV) is one of the major etiological factors responsible for acute and chronic liver disease and for the development of hepatocellular carcinoma (HCC). To determine the effects of HBV replication on host cell-protein expression, we utilized 2-DE and MS/MS analysis to compare and identify differentially expressed proteins between an HBV-producing cell line HepG2.2.15 and its parental cell line HepG2. Of the 66 spots identified as differentially expressed (+/- over twofold, p <0.05) between the two cell lines, 62 spots (corresponding to 61 unique proteins) were positively identified by MS/MS analysis. These proteins could be clearly divided into three major groups by cluster and metabolic/signaling pathway analysis: proteins involved in retinol metabolism pathway, calcium ion-binding proteins, and proteins associated with protein degradation pathways. Other proteins identified include those that function in diverse biological processes such as signal transduction, immune regulation, molecular chaperone, electron transport/redox regulation, cell proliferation/differentiation, and mRNA splicing. In summary, we profiled proteome alterations between HepG2.2.15 and HepG2 cells. The proteins identified in this study would be useful in revealing the mechanisms underlying HBV-host cell interactions and the development of HCC. This study can also provide some useful clues for antiviral research.