The Role of Telomerase in Insensitivity of Hepatocellular Carcinoma to Chemotherapy

hepatocellular carcinoma (HCC) is one of the most common malignancies; the incident rate of it is growing every year in our country. The most common way to cure it is undergoing combined therapy including chemotherapy. But the curative chemotherapy is far from satisfaction, because hepatocellular carcinoma is more resistant to chemotherapeutics. How to increase hepatocellular carcinoma sensitive to chemotherapy is more significant to hepatocellular carcinoma (HCC) therapy and improving patients" survival. Although there are a lot of drug-resistant mechanisms about HCC, it is still out of fully understanding. Recently, growing evidence show that telomerase was involved in tumor drug resistance and many induced drug-resistant cell line has high telomerase activity.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), an other 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 cell but almost expressed in human cancer cell/immortally cell line 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 without telomerase activity, many researcher propose that telomerase plays an important role in the initiation and development of HCC. In the preliminary experiment, we find that low concentrate cisplatin not only can’t kill HCC cell line but also up-regulate telomerase and induce drug insensitivity to cisplatin. So our object is to study the role of up-regulation of telomerase in insensitivity of hepatocellular carcinoma to cisplatin.K Low concentrate cisplatin induce insensitivity of HCC to cisplatinIn this part. HCC cell line 7721 was continuously grown in the presence of low concentrate cisplatin (2ug/ml) for up to one week. Then we treat those cell and an other untreated cell (as control) with high concentrate cisplatin(10ug/ml).cell viability was measured by MTT assay,cell morphous change was detected under phase contrast microscope and apoptosis cell was stained with PI buffer and detected by FACS. We found that treated 7721 cell has high viability, low number of morphous-changed cell and low apoptosis rate than untreated 7721 cell. Those data show that treated group has more resistant to cisplatin than control group.2% cisplatin induce drug insensitivity of HCC to cisplatin accompanying with up-regulation of telomerase activity and hTERT protein.This part .we use TRAP assay to test and compare telomerase activity of treated group and control group; since expression of hTERT was the determinant of telomerase activity ,we also use western blot to identify hTERT protein. Our result reveal that treated group has high telomerase activity and more hTERT protein than control group. Those data demonstrate that low concentrate cisplatin induce drug insensitivity of HCC to cosplatin accompaning with up-regulation of telomerase activity and hTERT protein. 3-. cisplatin improve hTERT gene expression through transcriptional level.The regulation of telomerase activity occurs at various levels. Substantial experimental data demonstrate that the transcriptional regulation of hTERT expression represents the primary and rate-limiting step in the activation of telomerase activity in most cells, so this part ,we use RT-PCR to test hTERT mRNA level of treated group and control group, and then we also use hTERT promoter-luciferas reporter system to test promoter activity of both groups. Our data indicate treated group has high level of hTERT mRNA and promoter activity; this result reveals that cisplatin can improve hTERT gene expression through transcriptional level.4, Inhibitions of hTERT by adenovirus -mediated siRNA enhance HCC drug sensitivity.Many finding indicate inhibition of hTERT protein can eliminate telomerase activity.so we use adenovirus -mediated siRNA to inhibit hTERT of treated group, then we treated hTERT-inhibited group and other three different groups with high concentrate cisplatin(10ug/ml).those three groups include control group, hTERT-uninhibited treated group and treated group delivering only with empty adenovirus. Apoptosis cell was stained with PI buffer and analyzed by FCAS. We found hTERT-inhibited group had