Reversal Of Multidrug Resistance Of Hepatocellular Carcinoma Cells BEL-7402 By Ginsenosides Rg3

ObjectiveHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, causing 500,000 deaths yearly(1).The development of the markers of AFP and imaging of liver cancer make it is possible to find more sub-clinical liver cancer or liver cancer that can be treated with surgery, but most of liver cancer is advanced and associated with liver cirrhosis. The comprehensive treatment is used for these patients, including surgery, chemotherapy, Regional Perfusion et cetera. Regional Perfusion is in an important position.But the effect of chemotherapy is still not ideal.Recent study found that the resistance of tumor cells was related to the over-expression of multidrug resistant gene. It is a hot topic to research mechanisms of MDR in HCC and to seek methods to overcome them. The resistance of tumors occurs not only to a single cytotoxic drug used, but also occurs as a cross-resistance to a whole range of drugs with different structures and cellular targets. This phenomenon is called multidrug resistance (MDR) (2). Multidrug resistance of tumor cells is one of the reasons for the failure of chemotherapy, the drug toxicity and side effects of the current MDR modulators limit their clinical application, therefore, drugs extracted from natural plant with no side effects were found to reverse drug resistance and gradually become be more attentioned.Ginsenoside Rg3 with lower toxicity is extracted from the monomer composition of ginseng. A number of studies have confirmed that ginsenoside Rg3 induced tumor cell apoptosis, inhibited tumor cell proliferation, invasion and metastasis, Furthermore it has the synergistic and attenuated effect with other chemotherapy drugs(3). Therefore, we select the innated-resistant HCC cells BEL-7402 for the experiment,explore the effect and mechanism of ginsenoside Rg3 on reversal of drug resistance(4).It can provide the theoretical basis and experimental evidence for discovering and researching MDR modulators. Methods(1) Detecting the absorbance value (OD value ) of BEL-7402 human hepatoma cells which was disposed by Ginsenoside Rg3, and then calculating the cell growth inhibiting rate;drawing the growth inhibiting curve of the cells line changing with the drug concentration,furthermore calculating out their IC50, so as to offer the suitable drug concentration in the following experiment.(2) Researching the effect of Ginsenoside Rg3 on reversal of human hepatoma cells BEL-7402, Detecting the absorbance value (OD value ) of human hepatoma cells BEL-7402 with different concentrations of Ginsenoside Rg3 alone or combinating with VCR, then calculating the reversal multiples.(3)Culturing hepatocellular carcinoma BEL-7402 cells with Ginsenoside Rg3 (40.0μg/mL) for 72h, detecting MDR1, MRP protein in liver cells by immunohistochemistry.(4) Culturing human hepatocellular carcinoma BEL-7402 cells with Ginsenoside Rg3 (40.0μg/mL) at different times (24h, 48h, 72h). Studying the reversal mechanisms of Ginsenoside Rg3 and detecting changes of the expression of mdr1 mRNA in hepatocellular carcinoma BEL-7402 by RT-PCR.Result(1) Ginsenoside Rg3 in different concentrations obviously inhibited the growth of BEL-7402 cells.And the inhibiting effect increased with the increasing concentration of Ginsenoside Rg3.There had obvious dose and time effect relationship between the cell growth and the concentration of drug; We calculated out the IC50 of Ginsenoside Rg3 was 66.9μg/ml.(2) The experiment found that the rates of inhibition in cells with different concentrations of ginsenoside Rg3 In combination with VCR, were higher than the drug alone (P <0.05), CDI <1, The two drug combination had the synergistic inhibition effect. And found that the effect of ginsenoside Rg3 <40.0μg/mL combinated with VCR in liver cancer cells BEL-7402 was not obvious,the effect was significal when liver cancer cells BEL-7402 by Ginsenoside Rg3> 40.0μg/mL combinated with the VCR (P <0.01). then selected 40.0μg/mL as the trial concentration of Ginsenoside Rg3 on the development experiment (3)The reversal multiple of Ginsenoside Rg3 is 6.27,while the reversal multiple of the normal reversal drug VRP is 11.18 after 72 hours.(4) Ginsenoside Ginsenoside Rg3 could significanty inhibit the expression of MDR compared with the control group,P<0.01.while there were no significant changes to the expression of MRP compared with the control group.(5)Liver cancer BEL-7402 cells treated with Ginsenoside Rg3 (40.0μg/mL) for different times (24h, 48h, 72h). The expression of MDR1 mRNA detected by RT-PCR was significantly decreased compared with the control group ,which had statistical significance (P < 0.05).Conclusion1. This experiment showed that Ginsenoside Rg-3 remarkablly inhibited the growth of BEL-7402 cell lines in vitro,there had the time and dose-effect relationship between the cell growth and the drugs.2. The combination of Ginsenoside Rg3 and VCR had synergistic inhibition of BEL-7402 to reverse the resistance on VCR.3. Through immunohistochemical method (SABC) detection, Ginsenoside Rg3 could inhibit expression of P-glycoprotein on BEL-7402. There was no effection to MRP.4. Reverse transcription-PCR (RT-PCR) detected that Ginsenoside Rg3 could inhibit the expression of MDR1 mRNA on BEL-7402 cells.