Role And Mechanism Of 5-aza-2'-deoxycytidine On Telomerase In Hepatocellular Carcinoma Cells

Human primary hepatocellular carcinoma (HCC) is one of the highly prevalent malignant diseases worldwide, particularly in China and some other parts of Asia.,which carries a very poor prognosis and high recurrence. Chemotherapy is one of the three major means for treatment of primary liver cancer, especially for those patients who is not suited to surgury. But the curative chemotherapy is far from satisfaction, because hepatocellular carcinoma is more resistant to chemotherapeutics. It is significant to find a new anticancer target and increase hepatocellular carcinoma sensitive to chemotherapy to improve HCC therapy and patients'survival. Recently, growing evidence show that telomerase and DNA methylation were involved in tumorigenesis and targeting them was the potential approach for a mechanism-based anticancer therapy.Telomerase is a rib nucleoprotein enzymatic complex that adds repeated telomere sequences (TTAGGG) onto the chromosome ends, compensates for the telomeric loss that occurs with cell division and stabilizes, telomerase consists of three components: telomerase reverse transcriptase (hTERT), an RNA template-telomerase RNA component (hTR), another subunit is telomerase associate protein (TEP1). The hTR and TEP1 is ubiquitously exist in both normal and malignant tissues and cells, but the hTERT component is undetectable in most normal human tissues and somatic cells but almost expressed in human cancer cells/immortally cell lines and malignant tissues. So the expression of hTERT is thought to be paralleled with telomerase activity and considered as a rate-limiting determinant of enzymatic activity. Data of our lab revealed more than 85% HCC with much stronger telomerase activity than cirrhosis. Based on the other spurring observation that normal hepatocellular tissue doesn't telomerase activity, many researchers propose that telomerase plays an important role in the initiation and development of HCC.DNA methylation is the best-known epigenetics marker and has critical roles in the control of gene activity. It involves addition of a methyl group to the carbon 5 position of the cytosine ring. This reaction is catalyzed by DNA methyltransferase in the contex of the sequence 5'-CG-3', which is also referred to as a CpG dinucleotide. Numerous reports have shown that promoter DNA methylation leads to genes silencing. Three possible mechanisms have been proposed to account for transcriptional repression by DNA methylation:The first mechanism involves direct interference with the binding of specific transcription factors to their recognition sites in their respective promoters; A second potential mechanism for methylation induced silencing is through the direct binding of specific transcriptional repressors to methylated DNA,such as MeCP; A third mechanism by which methylation may mediate transcriptional repression is by altering chromatin structure. Hypermethylation of tumor-suppressor genes and hypomethylation of overall genomic are common findings in tumorigenesis. Studies show that methylation status of p21, p15, p16, WTI and E2F-1 was significantly associated with HCC.5-Aza-2'-deoxycytidine(DAC) was first demonstrated to have a wide range of antimetabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. Then researchers find that it is a demethylating agent with which genes silenced by hypermethylation can be reactivated. Recent studies have examined the effect of DAC on telomerase activity in prostate cancer cell lines and found that DAC inhibits telomerase activity through transcriptional downregulation of hTERT. However, the mechanisms responsible for downregulation of hTERT remain unclear.The telomerase catalytic subunit hTERT is generally repressed in normal cells and upregulated in immortal cells, suggesting that hTERT is the primary determinant for the enzyme activity. Sequence analysis indicates that the hTERT promoter has no TATA or CAAT boxes but is highly GC-rich. The GC-rich region forms a large CpG island around the ATG, suggesting that methylation may be involved in regulation of hTERT expression, even though no consistent results have been reported. However the abundance of those potential transcription factor binding sites in the promoter region suggests that the regulation of hTERT expression may be subject to multiple levels of control by different factors in different cellular contexts.Our preliminary experiments find that CpG island methylator phenotype associates with upregulated telomerase activity in hepatocellular carcinoma. To approach the role of DNA methyaltion on telomerase activity, this study proceeded from the following three aspects:1. examined the effect of DAC on telomerase activity and hTERT expression in hepatocellular carcinoma cell lines; 2. investigate the role of DNA methylation through which DAC effect on telomerase activity; 3. compare the effect DAC in combination with chemotherapeutic agent on hepatocellular carcinoma cells with that of agent alone.Partâ… :effect of demethylating agent DAC on telomerase activity in hepatocellular carcinoma cellsFirst of all, treat hepatocellular carcinoma cells SMMC-7721 and HepG2 with chemotherapeutic agents DAC of varied concentrations for different times, and then observe the effect of the agent on cell survival and the relation between the dose, exposed time and cytotoxicity of DAC; next, select appropriate concentration and exposed time, according to the previous result, to treat hepatocellular carcinoma cells SMMC-7721 and HepG2, using the method TRAP-ELISA, real time RT-PCR and Western blot respectively, examine the telomerase activity and hTERT expression in the levels of mRNA and protein. The result shows that different telomerase activity and hTERT expression are detected in hepatocellular carcinoma cells SMMC-7721 and HepG2. Under some certain concentrations and exposed time, DAC inhibits telomease activity in a dose-and time-dependent manner.Moreover, hTERT expression is also down-regulated coinciding with telomerase activity. The result suggests that DAC inhibitis telomease activity through downregulated hTERT expression in hepatocellular carcinoma cells.Partâ…¡:Role of DNA methylation in the downregulation of hTERT expression by DACSince hTERT expression is regulated by various transcription factors as well as DNA methyaliton, study in this part aimed to solve the problem that which mechanism mediates the downregulation of hTERT expression by DAC in hepatocellular carcinoma cancer cells.Evidences show that the promoter of the hTERT gene was methylated in several cancer cell lines, and hTERT gene expression is positively regulated by promoter methylation, opposing to the traditional views:gene silencing by DNA methyaltion. we wondered whether treatment with the demethylating agent DAC could revert the methylation status of the hTERT promoter region and re-establish its expression. Using methylation-specific PCR, we detect the methylation status of hTERT in hepatocellular carcinoma cancer cells untreated and treated by DAC. It was observed that the promoter of hTERT is unmethyalted in HepG2, and DAC doesn't affect the methylation status of hTERT promoter in a dose-and time-dependent manner. The data indicates that methylation is not the sole regulator of hTERT gene expression and DAC downregulating hTERT gene expression may be not through directly changing the methylation pattern of hTERT promoter.Transcriptional regulation of hTERT is believed to be the major mechanism of telomerase regulation in human cells, and the hTERT promoter contains binding sites for many transcription factors that may be involved in its regulation. Such as activitors:c-myc, Spl, human papillomavirus 16 E6, Steroid hormones, and repressors:Mad1, p53, p15, p16, p21, pRB, E2F1, Wilm's tumor 1 tumor suppressor (WT1). Our preliminary experiments in HCC tissue shows the hypermethylation of p21, p15, p16, WT1 and E2F1 and that the upregulated telomerase activity in hepatocellular carcinoma is associated with these genes methylator phenotype. So we observed the effect of DAC on these genes expression. The result shows that c-myc and WT1 are overexpressed in hepatocellular carcinoma cells, while p16 expression is low or lost, compared to the normal liver cells. After treated by DAC for certain time, c-myc expression is downregulated and p16 expression is upregulated. Because DAC is demethylating reagent, it is possible that treatment with DAC alters p16 expression level. We therefore examined the changes in p16 promoter methylation level during exposure to DAC. The data reveals that DAC revert the methylation status of the promoter region and re-establish p16 expression. All of the data suggest that DAC inhibits telomerase activity via transcriptional repression of hTERT, in which p16 and c-myc may play a key role. Since p16 is a negative regulator of c-myc expression, the downregulation of c-myc by DAC may be mediated by p16 upregulation.Partâ…¢:DAC Sensitize hepatocellular carcinoma cells to chemotherapeutic agentTo determine whether DAC sensitizes cells to chemotherapeutic drugs, hepatoma cells SMMC-7721 and HepG2 were cultured in complete medium additioned with chemotherapeutic drug cisplatin in the presence or absence of DAC. while dramatic morphological changes of revealed detachment shrinkage and floatation were caused by combination of DAC and cisplatin, there was much less in the preasence of chemotherapeutic drugs alone. Then we performed an MTT assay to determine the IC50 of cisplatin in the presence or absence of DAC in two human hepatocellular carcinoma cell lines. The mean IC50 of cisplatin under normal culture condition was 20 and 22.5 uM in SMMC-7721 and HepG2, respectively. DAC administered at the lowest concentration (0.2 uM) reduced the IC50 of cisplatin significantly, and the impact was greater at the concentration of 2uM. More over, DAC and the cisplatin each caused dosage dependent cell growth suppression of hepatocellular carcinoma cells.Cisplatin have been reported to induce apoptosis via mitochondrial pathways, so we hypothesized DAC may sensitize hepatoma cells to chemotherapeutic agents-induced cell death by enhancing apoptosis. To test this hypothesis, we compared the percentage of apoptotic cells induced by cisplatin or DAC alone and combined chemotherapy.3 days after treatment, nuclear morphology was examined using DAPI staining, and the percentage of apoptotic cells was analyzed by flow cytometry. Data show that more cells are induced to apoptosis by combined chemotherapy than in the presence of cisplatin or DAC alone. These results demonstrated that DAC could sensitize hepatocellular carcinoma cells to cisplatin.To sum up, we conclude as follows:1. The demethylating agent DAC inhibitis telomease activity and downregulates hTERT expression in hepatocellular carcinoma cells.2. DAC downregulates the expression of hTERT through reactiviting p16, which is silenced by hepermethylation in hepatocellular carcinoma cells, and repressing c-myc expression, which is overexpressed in hepatocellular carcinoma cells.3.. DAC enhance the chemosensitivity of hepatocellular carcinoma cells to cisplatin, indicating that tageting telomerase and DNA methyaltion is the potential approach for a mechnism-based anticancer therapy.