The ABCB1 transporter is one of the members of ATP-binding cassette superfamilies,frequently overexpressed in cancer cells and leads to multidrug resistance (MDR).Over-expressing of ABCB1 in cancer cells is one of the most common reasons for chemotherapeutic failure.During the past three decades, the direct inhibition of ABC transportors has been considered to be the most effective way to overcome MDR.Notably, some of the third generation ABCB1 modulators have been shown to be efficacious in clinic test, but face a tough challenge in respect of adverse effects.Currently, none of MDR modulators have been approved for use in clinic.In fact, it is a viable alternative to synthetic compounds that natural products provide diverse and novel compounds as sources of effective, relative non-toxic MDR modulators.Acerinol, as a representative cyclolanstane triterpene of the Cimicifuga species, was isolated from Cimicifuga acerina in our laboratory.There is still no report on the anti-tumor activity of acerinol.In the present study, we evaluate its MDR reversal activity and the underlying molecular mechanisms, which will provide the evidences for the development of acerinol to be a new natural drug candidate for MDR modulation.Objective: The objective of this study is to evaluate the MDR reversal activities of acerinol and explore the underlying mechanisms.Methods: (1) The evaluation of the MDR reversal activity of acerinol: firstly, MTT assay was used to determine the cytotoxicity of acerinol in HepG2/ADM,MCF-7/ADR and their parental sensitive cell lines HepG2 and MCF-7.The effect of acerinol on the chemosensitivity of doxorubicin, vincristine, paclitaxel and cisplatin on cancer cells was also evaluated by MTT assay.On the other hand, the relative enzymatic activity of CYP3A4 was measured using P450-GloTM assay systems.(2) The investigations of underlying molecular mechanisms of acerinol:Intracellular accumulation of doxorubicin affected by acerinol was detected by flow cytometry.The retention of ABCB 1 specific substrate rhodamine-123 in cancer cells was also detected by flow cytometry.RT-PCR was used to determine ABCB1 mRNA levels in the presence or absence of acerinol.Western blot analysis was used to detect ABCB1 expression in HepG2/ADM and MCF-7/ADR cells treated with acerinol for 48 h.ABCB1 siRNA transfected cells was incubated with VCR in the presence or absence of acerinol, cell viability was determined by MTT assay.The vanadate-sensitive ATPase activity of ABCB1 in the membrane vesicles was measured using Pgp-GloTM ATPase assay kit.The duration effect of acerinol was analyzed by MTT assay.Docking analysis was performed to explore the binding manner and bingding sides ofacerinol and ABCB 1.Results: (1) MDR reversal activity of acerinol: acerinol was not toxic to HepG2,HepG2/ADM, MCF-7 and MCF-7/ADR at a concentration of 4 μM.Thus, the concentrations of 2 μM and 4 μM were used in the following MDR reversal experiments.Acerinol increased chemosensitivity of doxorubicin, vincristine and pacletaxel in HepG2/ADM and MCF-7/ADR cells.However, acerinol can not sensitized cytotoxicity of cisplatin to HepG2/ADM and MCF-7/ADR cells.Besides,acerinol had little effect on CYP3A4.(2) MDR reversal mechanisms of acerinol: acerinol significantly increased the accumulation of doxorubicin and induced intracellular retention of rhodamine-123 in HepG2/ADM and MCF-7/ADR cells.Acerinol did not alter the expression levels of ABCB 1 mRNA and protein in HepG2/ADM and MCF-7/ADR cells for 48 h.Acerinol reversed the resistance of MCF-7/ADR to VCR depending on ABCB1, while the reversal activity of acerinol attenuated after expression of ABCB 1 was interfered by siRNA.Further mechanistic investigation found that acerinol could activate ABCB1 ATPase activity, concentration-dependently.The reversal effect of acerinol on MCF-7/ADR was no longer persist after cells following the washout of acerinol.In docking analysis, acerinol binds to ABCB1 and the binding position is partially overlap with verapamil.Conclusion: Our present data show that acerinol can potently reverse the ABCBl-mediated MDR in cancer cells by stimulating ABCB1 ATPase activity,subsequently, inhibite the efflux function of ABCB1 and increase intracellular retention of anticancer drugs in MDR cells.In conclusion, the present study is the first to show that acerinol from Cimicifuga acerina significantly enhances the cytotoxicity of chemotherapeutic drugs by modulating the function of ABCB1.It is hopeful to develop acerinol as a new MDR reversal agent.