Modulation Of The Mitochondrial Permeability Transition Proe On Multidrug Resistance In Human Hepatoma Cell Line

Background and Objective:Primary liver cancer, which consists predominantly of hepatocellular carcinoma (HCC), is the most common gastrointestinal tumor worldwide and the third most common cause of cancer mortality, with a high degree of malignancy and poor prognosis. A majority of patients are surgically unresectable at the time of diagnosis, and even for those where surgery is possible, the risk of recurrence is extremely high. Consequently, chemotherapy is an important treatment for most HCC patients. Multidrug resistance (MDR) is a major obstacle in the chemotherapy of cancer patients and HCC also responds poorly to chemotherapy owing to intrinsic MDR, but the mechanism of MDR remains unclear.MDR describes a phonmenon of cross-resistance of tumor cells to several structurally and functionally unrelated antitumor drugs after exposure to a kind of chemotherapeutic agents. At present, the mechanisms responsible for MDR remain complicated, There are two classes of transporter proteins at the cellular surface which are responsible for MDR in tumors. One is the adenosine triphosphate-binding cassette (ABC) transporter superfamily, which is an energy-requiring efflux pump with the function of extruding toxic chemotherapeutic drugs from the cancer cells. The other transporter superfamily, solute carrier transporters, which mediates the cellular uptake of anticancer drugs, and drug resistance may result from decreased activity of these transporters. However,cancer chemotherapy will be significantly developed by successfully overcome this MDR.Mitochondria is essential for maintaining cell life, which plays a major role in producing energy,regulating the oxidation-reduction, modulating the concentration of calcium and mediating cell signal transduction.Except that, the mitochondria have prossessed the key role of in cell apoptosis and necrosis. Moreover, apoptosis induction is the final common pathway for chemotherapeutic compounds. Recently, mitochondria mediated cell apoptosis has received more considerable attention, the mitochondria is supposed to be one of the new targets foe screening novel antineoplastic agents. Regulation of mitochondrial function can not only protect the normal cells from initiation of apoptosis, but also induce the release of apoptotic proteins that lead to apoptosis of tumor cells.The mitochondrial dysfunction is closely related to the states of mitochondrial permeability transition pore(mPTP), Mitochondrial permeability transition (mPT) caused by opening of mPTP is implicated as an important event in the control of cell death or survial and may causes in apotosis or necrosis. As the result of the mPTP`s important roles in cell death, the opening of mPTP made the cancer cell show the character of apoptosis by promoting the release of apoptotic protein family. So modulation of mPTP on MDR might provide new means for the reversal of multidrug resistance.In this study, we planned to establish a multidrug-resistant hepatoma cell line (SK-Hep-1/CDDP). Using SK-Hep-1 and established MDR cell line SK-Hep-1/CDDP as the in vitro model, we try to investigate the possible effect of mPTP on MDR of tumor By modulation of the mPTP.The results of this study may provide new means for the reversal of multidrug resistance in tumor cells.Methods:Part IThe CDDP-resistant subline SK-Hep-1/CDDP was induced by high-concentration, short-duration cisplatin (CDDP) treatment in vitro and were purified through single cell clone. Cell morphological observation, cell growth curve was painted and the doubling was accounted;The 50% inhibitory dose (IC₅₀) values and the resistance indexes (IC₅₀ SK-Hep-1/CDDP)/(IC₅₀ SK-Hep-1) for other chemotherapeutic agents and the growth curve of cells were also evaluated using cell counting kit-8 (CCK-8) assays; Flow cytometry was performed to determine the distribution of the cell cycle and apoptosis ratio;Expression of P-glycoprotein(MDR1, ABCB1), multidrug resistance-associated protein 1(MRP1, ABCC1) and Bax were detected by western blot. Expression and localization of MDR1/P-gp and MRP1 were compared with that in parent cells by laser scanning confocal microscopy. Part IISK-Hep-1 and SK-Hep-1/CDDP were treated by mPTP reactivator ATR and mPTP inhibitor CsA respectively;Effects of different treatment ways of CDDP on the IC₅₀ and RI of SK-Hep-1 and SK-Hep-1/CDDP cells were determined by CCK-8;Expressions of MDR1/P-gp and Bax were detected by Western blot. Apoptosis was assessed with FITC-Annexin V. Mitochondrial inner membrane potential (ΔΨm) were monitored with fluorescence dry JC-1.Results:1. Microscopic observation revealed that the SK-Hep-1/CDDP cells adopted a spindle shape, similar to that of the parent cells SK-Hep-1.2. As compared with the parent cells SK-Hep-1, SK-Hep-1/CDDP exhibited a prolonged doubling time and lower growth rate (p < 0.01).3. CCK-8 analysis revealed that the SK-Hep-1/CDDP cells (IC₅₀ = 70.61±1.06μg/mL) was 13.76 times more resistant to CDDP than the SK-Hep-1 cells (IC₅₀ = 5.13±0.09μg/mL), and CDDP-resistant cells also demonstrated cross-resistance to many anti-tumor agents such as doxorubicin, 5-fluorouracil and vincristine.4. The percentage of cells in S and G2/M phases were significantly arrested in SK-Hep-1/CDDP in comparison with those in SK-Hep-1 cells, while the percentage of cells in the G0/G1 phase decreased (p < 0.01).5. Laser scanning confocal microscopy examinations disclosed that p-gp(MDR1) and MRP1 proteins were localized in plasma membrances and cytoplasm In parent and CDDP-resistant cells.6. MDR1 and MRP1 expressions were significantly increased in SK-Hep-1/CDDP in comparison with those in SK-Hep-1 (p < 0.01), but Bax was lower than that in SK-Hep-1 (p < 0.01).7. Annexin-V binding and flow cytometry also showed that there is an decrease in the number of apoptotic cells after CDDP treatment with SK-Hep-1/CDDP in comparison with that of SK-Hep-1.8. ATR promotes the opening of mPTP, accelerates the decrease of mitochondrial membrane potential (ΔΨm), reduces Bax activity, and increases apoptosis in SK-Hep-1/CDDP cells.9. CsA inhibits mPTP opening, reduces and delays the decline of mitochondrial membrane potential, and increases Bax activity leading to increased tolerance of SK-Hep-1/CDDP cells to the apoptosis induction.Conclusions:1. A reliable multi-drug resistant human hepatoma cell line SK-Hep-1/CDDP is successfully established.2. Multiple drug resistance of multiple drugs in the human hepatoma cell line SK-Hep-1/CDDP was closely related to the overexpression of P-gp(MDR1), MRP1and lower expression of Bax and the attenuated cell apoptosis induced by chemotherapeutic agents.3. activation of mPTP may provide a new method to sensitize the tumor cells to chemotherapeutic drugs and reverse the multidrug resistance in tumor cells.