Study On The Effect Of Astragaloside â…¡ And â…£ Reversing Drug-resistant In Human Hepatic Carcinoma BEL-7402/ 5-FU Cell Line And Its Mechanism

Background and objectiveMultidrug resistance refers to the drug resistance of tumor cells to a kind of antitumor drug, simultaneously, cross resistance also occurs to other antitumor drugs with different structure and mechanism. The mechanism of multidrug resistance which is the synthetic action result of multi gene and many steps[1], is very complex, in addition to connect with multidrug resistance gene (mdrl) and its encoded product P-glycoprotein-mediated classical mechanism, but also refer to apoptosis related genes-mediated (such as Bcl-2, Ras, P53) non-classical mechanism[2,3]. Multidrug resistance has now become the primary reason for the failure of clinical cancer chemotherapy, how to solve this problem is currently the hot spot of anti-cancer research, including looking for multidrug resistance reversal agents or chemosensitizer agents are one of the means to solve MDR problem.Astragalus which commonly used in tonify Chinese crude drug, has the efficacies of invigorate vital energy and invigorating splenic yang, yi wei-solid form. Common clinical form of medication is Astragalus injection, Astragalus-based rough preparation or complex prescription with Astragalus-based. In previous study, we observed that the total Astragaloside can inhibit the growth of tumor cells of HepA and S180 in mice and the growth of HeLa cells in vitro[4]. It has been reported that Astragalus injection enhanced chemotherapy sensitivity of drug-resistant hepatocellular carcinoma cell line BEL/FU, decreased the expression and drug efflux function of P-gp[5]. AstragalosideⅡandⅣare the active ingredients which are extracted from Astragalus, having wide pharmacologic action. The total Astragaloside have the effects of anti-tumor, synergia and attenuation, so AsⅡand IV which are the active ingredients of the total Astragaloside whether having the effects of synergia and attenuation is not clear. We aim to study the effect of AsⅡandⅣon reversing drug resistant hepatoma cells, as well as to explore the molecular mechanism of AsⅡand IV reversing drug resistance through the mRNA levels of mdrl, Bax, Bcl-2 and expression of P-glycoprotein.Methods 1.70%ethanol circumfluence extraction was used. Total Astragaloside loside was enriched and purified by extraction refine through water saturated of n-buta nol and D101 Mcroporous Adsorption resin. Different components of Astragaloside were isolated by silica gel laminar analysis.2. Identification by using TLC Method and determination the contents by using HPLC-RID Method.3. BEL-7402 cell of hepatoma cell line sensitive and BEL-7402/5-FU cell of heaptoma cell line resistant were selected as experimental cell. By MTT measurement, the sensi-tivity of three chemotherapy drugs (5-FU, ADR, MMC) on cells were determined as well as resistant index.4. Using MTT measurement, the dose-response curve of (0.04,0.08,0.16,0.32,0.64) mg/mL AS II and ASIV on BEL-7402 cell and BEL-7402/5-FU cell was drawn, deter-mined reversal of drug resistance dose in accordance with the above-mentioned experi mental results.5. Select non-cytotoxic AS II and ASIV dose on BEL-7402/5-FU cells, with MTT method to detect cell survival rate and the relative reversal fold.6. RT-PCR was adopted to detect the mRNA level of mdrl gene and immunocytochem-istry method was applied to detect P-gp protein expression.7. Intracellular Rho123 accumulation and apoptosis were determined by flowcytometry8. With the RT-PCR method, the mRNA level of Bcl-2, Bax gene was determined.Results 1. Six compounds were isolated from total Astragaloside (AS-Ⅰ～AS-Ⅵ), AS-Ⅰwhich content is 29.4% is mainly contained Astragaloside II by identification. AS-Ⅲwhich content is 22.82% is mainly contained Astragaloside IV by identification. The rest four compounds are required to be identified.2. To all these three chemotherapy drugs (5-FU, ADR, MMC), BEL-7402/5-FU cell showed resistance, and their resistance index were 19.64,1.98,1.92.3. (0.04,0.08 mg/mL) AS II and AS IV could increase 5-FU cytotoxicity on BEL-7402/5-FU cells, relative reversal folds were 1.49,1.81 and 1.70,1.86.4. After BEL-7402/5-FU cells treated with (0.08,0.16) mg/mL AS II, the mdrl/ GAPDH ratio of BEL-7402 group, BEL-7402/5-FU group, BEL-7402/5-FU+5-FU group and BEL-7402/5-FU+5-FU+ASII groups were respectively 0.219±0.03,0.367±0.08,0.568±0.09,0.321±0.06,0.116±0.01; while cells treated with (0.08,0.16) mg/mL ASⅣ, the mdrl/GAPDH ratio of BEL-7402/5-FU+5-FU+ASIV groups were 0.200±0.03,0.148±0.02.5. With optical microscopy, the positive response of P-gp protein showed brown-yellow, mainly located in cytoplasm, showing uniform fine granular distribution. The expression of drug-resistant cell BEL-7402/5-FU group increased, the color deepened, expression rate was (0.566±0.02)%. After the treatment of (0.08,0.16) mg/mL ASⅡon drug-resistant cells, P-gp protein expression rate were (0.401±0.02)%, (0.319±0.01) %; while afer the treatment of (0.08,0.16) mg/mL ASⅣon drug-resistant cells, P-gp protein expression rate were (0.364±0.02)%, (0.287±0.01)%, the color became shallow, the brown-yellow granules in cytoplasm reduced significantly.6. In Rho123 accumulation experiment, the Rho123 fluorescence was very weak in BEL-7402/5-FU cell. After the treatment of (0.08,0.16) mg/mL ASⅡand ASⅣ, the intracellular fluorescence markedly increased and the peak shifted to right.7. RT-PCR results showed that:after the treatment of (0.08,0.16) mg/mL ASⅡon BEL-7402/5-FU cells, the Bax/Bcl-2 mRNA ratio of BEL-7402 group, BEL-7402/ 5-FU group, BEL-7402/5-FU+5-FU group and BEL-7402/5-FU+5-FU+ASⅡgroups were respectively 0.727±0.11,0.501±0.07,0.439±0.07,0.856±0.05,0.993±0.08; while after the treatment of (0.08,0.16) mg/mL ASⅣon cells, the Bax/Bcl-2 mRNA ratio of BEL-7402/5-FU+5-FU+ASIV groups were 0.580±0.09,1.259±0.03.8. Different concentrations of AS II and AS IV increased the DNA sub-G1 peak in BEL-7402/5-FU and BEL-7402 cells. Random Software by flow cytometry analysis showed that, cells treated with (0.08,0.16,0.32) mg/mL of ASⅡ, apoptotic rates were (2.75±0.25)%, (4.75±0.25)%, (6.05±0.21)%, (5.95±0.35)%, (9.3±0.2)%, (14.8±0.42); cells treated with (0.08,0.16,0.32) mg/mL of ASIV, apoptotic rates were (3.15±0.29)%, (4.90±0.35)%, (7.85±0.21)%, (4.5±0.1)%, (6.95±0.17)%, (19.1±0.2)%, P<0.01. Compared to the control group, they were statistically significant.Conclusion The way that the Total Astragaloside which used 70%ethanol circumfluence extraction was enriched and purified by extraction refine through water saturated of n-butanol and D101 Mcroporous Adsorption resin and Different compo nents of Astragaloside were isolated by silica gel laminar analysis is good for separating and purifying Astragaloside; AS II and ASⅣcould partially reverse drug resistant of BEL-7402/5-FU cell and ite mechaim may be related to reduced expression of mdrl mRNA and inhibit function and expression of P-gp, also associated with increased Bax/Bcl-2 mRNA ratio and promote apoptosis.