Studies On ABCB1-mediated Multidrug Resistance Reversal Activity And Molecular Mechanisms Of Ganoderenic Acid B

Liver cancer is one of the common malignant tumor, its mortality is just less than lung cancer in China. The antineoplastic drugs(doxorubicin, vincristine, paclitaxel) have effective curative, but cancer cells which were treated with these drugs for long time are prone to multidrug resistance(MDR). According to the American society of clinical oncology(ASCO) statistics, MDR is contribute to more than 90% death rate of the tumor patients. The main cause of MDR is over-express ABCB1 in cancer cells. Currently, none of reversal agent is approved by the Food and Drug Administration of American. Thus finding reversal agents, which are efficient and non-toxic, from the traditional Chinese medicine are hotspots of current research.Ganoderenic acid B(GAB), a lanostane-type triterpene isolated from Ganoderma lucidum, exhibited antineoplastic activity in a variety of cancer cells. However, the cellular and molecular mechanisms of its anti-cancer action is less reported. So far as we know, there was no report on its MDR reversal activity. In this report, we demonstrated that GAB could sensitize ABCB1-overexpressing MDR cells Hep G2/ADM, and revealed the mechanisms of action for its MDR reversal activity. Objective: This study was aims to evaluate reversal activity of GAB and explore its reversal mechanism, and provide scientific basis for developing the ganoderenic acid B to be a potential reversal agent. Methods:(1) The evaluation of the MDR reversal activity of constituents isolated from ganoderma lucidum were determined by MTT assay;(2) The inhibition effect of GAB was tested by MTT assay in Hep G2, MCF-7 and Hep G2/ADM, MCF-7/ADR cells;(3) The MDR reversal activity of GAB was determined by MTT assay in vitro;(4) The influence of GAB to rhodamine B-123 accumulation and excretion in Hep G2/ADM cells was determined by microplate reader;(5) Whether the reverse effect of GAB is dependent on the ABCB1 level by disturbing the expression of ABCB1;(6) Western blot was used to determine ABCB1 levels in the presence or absence of GAB;(7) P-gp Glo TM ATPase kits was used to measured ABCB1 ATPase activity treated with GAB;(8) The sustainability of GAB was determined by MTT;(9) Docking analysis of GAB binding with ABCB1;(10) The relative enzymatic activity of CYP3A4 was measured by P450-Glo? kit. Results:(1) This screening results showed that the GAB have potential reversal acticity;(2) The GAB of 10 μM exhibited non-toxic to Hep G2/ADM, Hep G2, MCF-7/ADR and MCF-7 cells, so the concentration of 10 μM and 5 μm were selected to evacuted its reversal activity and explored its reversal mechanism;(3) This reseach results showed that the GAB can significantly increase toxicity of DOX, VCR, paclitaxel in Hep G2/ADM cells, but had no obvious effect in Hep G2 cells, moreover, GAB have no significant effect toxicity of cisplatin in Hep G2/ADM and Hep G2 cells;(4) The GAB increased the accumulation of Rhm-123 and inhibited its efflux in Hep G2/ADM cells;(5) The reversal effect of GAB depends on the level of ABCB1;(6) The expression of ABCB1 had no significant influence by GAB;(7) GAB did not alter the activity of ABCB1 ATPase;(8) The reverse function of GAB was not continuous;(9) The binding position of GAB to ABCB1 were not overlap with VRP;(10) The activity of CYP3A4 enzyme had no significant influence by GAB. Conclusion: The GAB significantly reversed the ABCB1 mediated MDR; The GAB did not influence the expression of ABCB1, also did not alter the ABCB1 ATPase activity, but GAB influenced ABCB1 function to reverse MDR.