Studies On Multidrug Resistance Reversal Activity And Molecular Mechanisms Of A Cyclolanstane Triterpenoid Acerinol

The ABCB1transporter is one of the members of ATP-binding cassettesuperfamilies, frequently overexpressed in cancer cells and leads to multidrugresistance (MDR). Over-expressing of ABCB1in cancer cells is one of the mostcommon reasons for chemotherapeutic failure. During the past three decades, thedirect inhibition of ABC transportors has been considered to be the most effectiveway to overcome MDR. Notably, some of the third generation ABCB1modulatorshave been shown to be efficacious in clinic test, but face a tough challenge in respectof adverse effects. Currently, no MDR modulators have been approved for use inclinic. In fact, it is a viable alternative to synthetic compounds that natural productsprovide diverse and novel compounds as sources of effective, relative non-toxicMDR 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 theanti-tumor activity of acerinol. In the present study, we evaluate its MDR reversalactivity and the underlying molecular mechanisms, which will provide the evidencesfor the development of acerinol to be a new natural drug candidate for MDRmodulation.Objective:The objective of this study is to evaluate the MDR reversal activities of acerinoland explore the underlying mechanisms.Methods:(1) The evaluation of the MDR reversal activity of acerinol:①MTT assaywas used to determine the cytotoxicity of acerinol in HepG2/ADM, MCF-7/ADRand their parental sensitive cell lines HepG2and MCF-7.②The effect of acerinolon the chemosensitivity of cancer cells was evaluated by MTT assay.③Therelative enzymatic activity of CYP3A4was measured using P450-Glo assaysystems. (2) The investigations of underlying molecular mechanisms of acerinol:①MTT assay was used to determine the viability of cancer cells co-treated cisplatinwith acerinol.②Intracellular accumulation of doxorubicin affected by acerinolwas detected by flow cytometry.③The retention of ABCB1specific substraterhodamine-123in cancer cells was detected by flow cytometry.④RT-PCR wasused to determine ABCB1mRNA levels in the presence or absence of acerinol.⑤Western blot analysis was used to detect ABCB1expression in HepG2/ADM andMCF-7/ADR cells treated with acerinol.⑥ABCB1siRNA transfected cells wasincubated with VCR in the presence or absence of acerinol, cell viability wasdetermined by MTT assay.⑦The vanadate-sensitive ATPase activity of ABCB1in the membrane vesicles was measured using Pgp-Glo ATPase assay kit.⑧The duration effect of acerinol was analyzed by MTT assay.⑨Docking analysiswas performed to explore the binding sides of acerinol and ABCB1.Results:(1) MDR reversal activity of acerinol:①Acerinol was not toxic to HepG2,HepG2/ADM, MCF-7and MCF-7/ADR at a concentration of4μM. Theconcentrations of2μM and4μM were used in the following MDR reversalexperiments.②Acerinol increased chemosensitivity of doxorubicin, vincristineand pacletaxel in HepG2/ADM and MCF-7/ADR cells.③Acerinol had little effecton CYP3A4.(2) MDR reversal mechanisms of acerinol:①Acerinol can not sensitizedcytotoxicity of cisplatin to HepG2/ADM and MCF-7/ADR cells.②Acerinolsignificantly increased the accumulation of doxorubicin in HepG2/ADM andMCF-7/ADR cells.③Acerinol induced intracellular retention of rhodamine-123inHepG2/ADM and MCF-7/ADR cells.④Acerinol did not alter the expressionlevels of ABCB1mRNA and protein.⑤Acerinol reversed the resistance ofMCF-7/ADR to VCR depending on ABCB1.⑥Acerinol activated ABCB1ATPaseactivity.⑦The reversal effect of acerinol on MCF-7/ADR was no longer present incells following the washout of acerinol.⑧Acerinol binds to ABCB1and thebinding position is partially overlap with verapamil. Conclusion:Our present data show that acerinol can potently reverse the ABCB1-mediatedMDR in cancer cells by inhibiting the efflux function of ABCB1and increasingintracellular retention of anticancer drugs in MDR cells. It has been found thatacerinol stimulates ABCB1ATPase activity, without affecting the expression levelsof ABCB1mRNA and protein.