| Objective:Primary liver cancer (PLC) is one of the most common cancer that threats the human health in the world. It is the sixth most prevalent cancer and the third most frequent cause of cancer-related death, following with gastric cancer and esophageal carcinoma. More than500,000new cases of this malignant disease were diagnosed every year. PLC can be divided into three different types depending on the pathological types, hepatocellular carcinoma (90%), cholangiocellular carcinoma and mixed hepatocellular carcinoma. The main pathogenic factor includes viral hepatitis, cirrhosis, exposure to aflatoxin B1and nitrosamines. And the step from viral hepatitis, cirrhosis to hepatocellular carcinoma is considered to be one of the most important process for the hepatocarcinogenesis. Owing to the limit of diagnosis level and lack of opinion for health check, two per three patients were diagnosed for the advanced live cancer losing the value for operation. They can only be treated with local ablation therapy, radiofrequency ablation therapy, hepatic arterial chemoembolization, targeting therapy and biotherapy. Although systemic chemotherapy was used in treatment for hepatocellular carcinoma at early stage, the clinical application of the chemotherapy in hepatocellular carcinoma still remains stagnant because of the reason that low sensitivity to chemotherapy, high side effect and weak liver function. Therefore, it is still very important to search for new chemotherapeutics with low cell toxicity and the mechanisms of chemotherapeutics in hepatocellular carcinoma.There were two different proteins encoded by clusterin gene, one is secretory clusterin and another is nuclear clusterin. It has been suggested to be overexpressed in many tumor tissues, such as prostate cancer, kidney cancer, colorectal cancer, bladder cancer, breast cancer, lung cancer, ovarian cancer and cervical carcinoma, and involved in several basic biological events such as cell death, tumor progression and neurodegenerative disorders. Many reports document that regulating the expression of sCLU protein in prostate cancer, breast cancer and kidney cancer can increase the treatment sensitivity. For example, sCLU stabilizes cytosolic Ku70-Bax protein complex and inhibits Bax activation. sCLU protein also promotes prostate cancer cell survival by increasing NF-kB nuclear transactivation, acting as a ubiquitin-binding protein that enhances COMMDland I-kB proteasomal degradation through interaction with E3ligase family members. sCLU knockdown stabilized the COMMD1and I-kB, suppressing NF-kB translocation to the nucleus, and suppressing NF-kB regulated gene signatures. But there is still limited research about the role of sCLU proterin in hepatocellular carcinoma. We found that sCLU protein may be overexpressed in hepatocellular carcinoma tissues and low expressed in hepatic cells. And this finding was the same with the research group of KANG. This shows that sCLU proterin could be a new regulating target to increase the chemotherapy sensitivity in hepatocellular carcinoma. Our study aims to find the relationship between sCLU protein and chemotherapy resistance, and study the role in increasing chemotherapy sensitivity targeting the sCLU protein and using the oxaliplatin as well as the mechanisms.Methods:Five different hepatocellular carcinoma cells were chosed and the sCLU protein expression was detected in these five cells using Western blot. Four paired of oligos which can transcripted into shRNA targeting clusterin gene were designed and inserted into the pMAGic7.1vector to construct the interference vector. Four pMAGic7.1-based shRNA vectors (CLU1, CLU2, CLU3, CLU4), a scrambled shRNA vector were found. The sCLU fragment was amplified by PCR with a pair of primers, and inserted into the pIRES2-EGFP vector, which was termed pIRESs-EGFP/sCLU. These vectors were transfected into different cells with different expression of sCLU protein (Bel7402, SMMC7721and Bel7404) and the sCLU mRNA expression and protein expression were detected using real-time PCR and Western blot. The vector with the most efficient was found. We used CCK-8assay to quantitate changes of viabilities in the three selected cell lines after treated with various concentrations of oxaliplatin and used Flow Cytometry to test the changes of apoptosis induced by the oxaliplatin after regulation the expression of sCLU protein.To profile the gene expression associated with cell apoptosis, we employed the Human apoptosis RT2ProfilerTM PCR Array and searched for the significant target (AKT1). The expression of AKT1was detected after regulation the expression of sCLU protein using Western blot as well as the Bcl-2family protein in the mitochondrial apoptosis pathway. To find the relationship between the sCLU protein and PI3K/Akt pathway, the targeting Akt inhibitor (TCN) was used to treat cells.To detect the role of role of sCLU protein in vivo, six-week-old male athymic BALB/c nu/nu mice were used. Briefly,1×106of cells (Bel7402, Bel7404and SMMC7721) were subcutaneously injected into the back of the mice. Tumor volumes were estimated:π/6×a2×b. where a is the short axis and b the long axis. Two weeks later, the mice were assigned to respective groups receiving weekly intraperitoneal injection of200ul of either PBS or OXA at a dose of10mg/kg.Results:In these five selected hepatocellular carcinoma cell lines. sCLU expression level was in the following order (from high to low):Bel7402, SMMC7721, HepG2, SNU739and Bel7404. Transfection of CLU1, CLU2, CLU3and CLU4resulted in reduction of sCLU protein expression in Bel7402and SMMC7721cells. Similarly, quantitative real-time PCR analysis showed that CLU1, CLU2, CLU3and CLU4transfected cells had notable reductions in sCLU mRNA expression. Among the four shRNA vectors. CLU4displayed the strongest gene-silencing ability. Stable transfected cell lines were selected and the efficiency of interference was measured.Bel7402and SMMC7721cells with the regulation of sCLU expression had a significant reduction in IC50of OXA by68.9%and67.9%, from54.35uM,40.16uM to16.89uM,12.9uM. However, Bel7404cells had a significantly higher IC50of OXA by approximately threefold, from22uM to69uM, compared with parental Bel7404cells. Bel7402-sCLUlow, SMMC7721-sCLUl0W and Bel7404-sCLUhlgh cells had similar proliferation rates, compared with the respective parental cells, or Sc shRNA or empty vector-transfected controls. The above cells were incubated in the absence or the presence of OXA (16uM) for48h, and cell viability was measured. OXA alone significantly reduced the viability of Bel7402, SMMC7721and Bel7404cells. Depletion of sCLU significantly reduced, while overexpression of sCLU significantly increased, the viability of cells treated with OXA. Oxaliplatin and depletion of sCLU induced apoptosis of Bel7402cells at rates of9.2%and4.7%, respectively, which were significantly higher than that of untreated Bel7402cells (0.6%); and Bel7402-sCLUlow cells treated with OXA had an even higher apoptosis rate (20.0%). Similar results were obtained with SMMC7721cells. The untreated Bel7404-sCLUhigh cells had almost the same apoptosis rate as parental Bel7404cells. OXA treatment increased apoptosis of parental Bel7404cells, with a rate of31.5%, which was significantly higher than that of Bel7404-sCLUhigh cells treated with OXA (10.5%).Four up regulated genes (BNIP1, GADD45A, TNFRSF10A, TRADD) and one down regulated gene (AKT1) were obtained, using the RT2ProfileTMPCR Array Human Apoptosis array to detect the downstream pathways. Both Bel7404-OR and Bel7404-sCLUhigh cells had higher levels of sCLU than their parental cells. pAkt has been considered to be a cancer multidrug resistance locus; thus, its expression was detected. Both Bel7404-OR and Bel7404-sCLUhigh cells had higher levels of pAkt, while they had similar levels of Akt, compared with their parentals. The data drove us to detect expression of pAkt in HCC cells, but a correlation between endogenous expression of sCLU and Akt was not observed. Furthermore, specific inhibition of pAkt by TCN downregulated pAkt but had no effect on sCLU expression in Bel7404-OR cells. In addition, TCN was highly efficient in suppressing Akt phosphorylation in a dose-dependent manner, but did not affect sCLU expression in Bel7402cells. Bel7402-sCLUlow and SMMC7721-sCLUlow cells were cultured in the absence or the presence of OXA (16uM) for24h. Both OXA and sCLU knockdown upregulated Bax and downregulated Bcl-2, thus reducing the Bcl-2/Bax ratio; and the combination of OXA and sCLU knockdown further decreased the Bcl-2/Bax ratio. Overexpression of sCLU alone had almost no effect on the expression of Bcl-2/Bax ratio; the combination of OXA and sCLU overexpression significantly increased the Bcl-2/Bax ratio, compared with OXA alone.We further confirmed that TCN highly significantly reduced the level of pAkt, and increased the apoptosis rates of both parental Bel7404and Bel7404-sCLUhigh cells. Overexpression of sCLU itself had almost no effect on apoptosis rates but increased Akt phosphorylation and supressed TCN-induced inhibition of Akt phosphorylation, and inhibited the increased apoptosis of Bel7404cells induced by TCN. OXA had only a non-significant effect on, but TCN highly significant reduced, the viability of Bel7404-sCLUhigh cells. The combination of TCN and OXA resulted in even lower viability of the cells, compared with TCN alone. In vivo, OXA had an obvious antitumor effect by reducing tumour growth by45.1%compared to PBS in parental Bel7402tumours on day42. There was no significant difference in parental Bel7402and Bel7402-sCLUlow tumours treated with PBS, but Bel7402-sCLUlow tumours treated with OXA grew significantly slower than OXA-treated parental Bel7402tumours. In contrast, OXA demonstrated even stronger anti-tumor activity in parental Bel7404tumours by reducing tumor growth by61.6%compared with that in PBS-treated parental Bel7404tumours.Conclusions:1. sCLU protein could be overexpressed in hepatocellular carcinoma, and it may be a significant regulating target as a function of cytoprotection.2. Regulating the sCLU protein could influence the sensitivity of hepatocellular carcinoma cells to oxaliplatin in hepatocellular carcinoma.3. Protein expression, especially the phosphorylated p-Akt level, could be regulated depending on the different expression of sCLU protein. The increased sensitivity to oxaliplatin by sCLU knockdown in hepatocellular carcinoma cells is also mediated by mitochondrial apoptosis pathway.4. In hepatocellular carcinoma, sCLU protein could be an efficient target to increase the chemotherapy sensitivity.Significance:1. We revealed the relationship between sCLU and the key proteins in mitochondrial apoptosis pathway in hepatocellular carcinoma. 2. We demonstrated that sCLU influences the cells sensitivity to oxaliplatin and reported the related pathway.3. We provided a new candidate target, sCLU, for the treatment of hepatocellular carcinoma. |
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