Mitochondrial Translocation Of Telomerase In Human Hepatocellular Carcinoma Cells With Multidrug Resistance

Background and Objective:Primary liver cancer, which consists predominantly of hepatocellular carcinoma(HCC), is the most common gastrointestinal tumor, with a high degree of malignancy andpoor prognosis. A majority of patients are surgically unresectable at the time of diagnosis.Consequently, chemotherapy is an important treatment for most HCC patients. Multidrugresistance (MDR) is a major obstacle in the chemotherapy of cancer patients and HCC alsoresponds poorly to chemotherapy owing to intrinsic MDR.MDR describes a phonmenon of cross-resistance of tumor cells to several structurallyand functionally unrelated antitumor drugs after exposure to a kind of chemotherapeuticagents. Mechanisms of chemotherapeutic drug resistance is very complicated, involvesincreased drug-effluxed, reduced drug-absorbed, altered target of anti-cancerdrugs,weakened the drug-activation,increased the ability to repair the damaged-DNA andchanged pathways of anti-apoptosis.Mitochondria are essential for maintaining cell life, which play a major role inproducing energy，regulating the oxidation-reduction, modulating the concentration ofcalcium and mediating cell signal transduction.Except that, mitochondria have prossessedthe key role in cell apoptosis. Moreover, apoptosis induction is the final common pathwayfor chemotherapeutic compounds. Recently, mitochondria are supposed to be one of thenew targets for screening novel antineoplastic agents.Telomerase is a ribonucleoprotein,which consisted of three main components–telomerase RNA, telomerase-associated protein and the catalytic protein, hTERT. Studieshave shown that expression of hTERT is consistent with the activity oftelomerase.So,hTERT is the major regulatory subunit of telomerase.Telomerase is expressed at varying levels in human cells and tissues. There is a highlevel of expression during early embryonic development,germ cells and cancer cells, but the expression is greatly down-regulated in most adult tissues. The main function of telomeraseis to elongate and maintain telomeres. hTERT over-expression involved in the originationand development of tumor.Furthermore, hTERT is also involved in the occurrence of MDRof cancer cells by antagonizing apoptosis,and hTERT over-expression may convey thesignal of resistance to chemotherapeutic drugs.Accumulating evidences suggest that telomerase has telomere-independent functions.Oxidative and chemotherapeutic stress can lead to hTERT translocation from the nuclei tothe mitochondria to protect the mitochondrial function (protection of mtDNA, reduction ofmitochondrial ROS, etc.) and prevent apoptosis,but can not protect telomeres fromshortening. Mitochondrial translocation of hTERT may form a new mechanism of MDR ofcancer cells.In this study, three drug-resistant human HCC cells derivatives(SK-Hep1/CDDP1,SK-Hep1/CDDP2and SK-Hep1/CDDP3) with different RIs were established. We try toinvestigate the possible effect of mitochondrial translocation of telomerase on MDR oftumor cells.Methods:Part IThree drug-resistant human HCC cells derivatives (SK-Hep1/CDDP1, SK-Hep1/CDDP2and SK-Hep1/CDDP3) with different RIs was induced by high-concentration,short-duration cisplatin (CDDP) treatment in vitro and were purified through single cellcloning.The Cells morphology was observed, cell growth curves were painted；The50%inhibitory doses(IC50) and the resistance indexes (IC50ofSK-Hep1/CDDP/IC50of SK-Hep1) for other chemotherapeutic agents and the growthcurves of the cells were also evaluated using cell counting kit-8(CCK-8) assays；Flow cytometry was performed to determine the distribution of the cell cycle.Part IIImmunofluorescence staining and Western blot assay perform the colocalisation ofhTERT in mitochondria and nuclei;Real-time PCR detected the oxidative damage of mitochondria DNA(mtDNA)andthe telomere length; Mitochondrial inner membrane potential was monitored with JC-1.Flow cytometry was performed to determine the apoptosis ratio of the cultured cells.Results:1.Microscopic observation revealed that the SK-Hep1/CDDP1,SK-Hep1/CDDP2andSK-Hep1/CDDP3cells adopted a spindle shape, similar to that of the parent cells SK-Hep1.2. As compared with the parent cells SK-Hep1, SK-Hep1/CDDP1,SK-Hep1/CDDP2and SK-Hep1/CDDP3cells exhibited a lower growth rate (p <0.05).3. CCK-8analysis revealed that SK-Hep1/CDDP1(IC50=40.72±0.06ug/mL),SK-Hep1/CDDP2(IC50=50.48±1.02μg/mL) and SK-Hep1/CDDP3(IC50=70.61±1.06μg/mL) were7.51,9.31and13.03times more resistant to CDDP than SK-Hep1(IC50=5.42±0.04μg/mL), and CDDP-resistant cells also cross-resistant to other anti-tumoragents such as doxorubicin and5-fluorouracil (P <0.01).4. The percentage in S and G2/M phases were significantly increased inSK-Hep1/CDDP1,SK-Hep1/CDDP2and SK-Hep1/CDDP3in comparison with those inSK-Hep1cells, while the percentage of cells in the G0/G1phases decreased (p <0.01).5. Immunofluorescence staining and Western blot assay demonstrated, with the gradualelevation in RI of the cultured cells, increasing hTERT translocated from the nuclei to themitochondria (P <0.05).6. Real-time PCR indicated the shortening of telomere length in drug-resistant cellsunder the chemotherapeutic stress and the reduction of damaged mtDNA with the increaseof RI (P <0.05).7. JC-1staining indicated the reduction of mitochondrial membrane potential indrug-resistant cells (P <0.05).8.The apoptosis ratio of SK-Hep1/CDDP1,SK-Hep1/CDDP2and SK-Hep1/CDDP3was decreased in comparison with that of SK-Hep1(P <0.01).Conclusions:Three drug-resistant human HCC cells derivatives(SK-Hep1/CDDP1,SK-Hep1/CDDP2and SK-Hep1/CDDP3) with different RIs were successfully established.Upon exposure of cancer cells to chemotherapeutic drugs, hTERT can translocate fromthe nuclei to the mitochondria. 3. Over-expression of hTERT in mitochondria of tumor may protects the mitochondriafounction and prevent apoptosis. Therefore, mitochondrial translocation of hTERT mayform a new mechanism of MDR of cancer cells.