Elsevier

Phytomedicine

Volume 21, Issues 8–9, July–August 2014, Pages 1110-1119
Phytomedicine

Tetrandrine and fangchinoline, bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

https://doi.org/10.1016/j.phymed.2014.04.029Get rights and content

Abstract

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.

Introduction

Multidrug resistance (MDR) is considered as an important obstacle for an effective clinical cancer chemotherapy (Johnstone et al. 2000). MDR often emerges as a result of overexpression of ABC transporters, which obtain energy from ATP hydrolysis to actively export lipophilic drugs across extra- and intra-cellular membranes (Robey et al. 2008). Presently, 49 human ABC transporters with subfamilies A to G have been recorded in the human genome. ABC transporter can export xenobiotics which entered a cell by diffusion with broad substrate specificity (Leonard et al. 2003). ABC transporters decrease drug accumulation in multidrug-resistant cells which can thus mediate the development of resistance of cells to anticancer drugs. In particular, subfamily B member 1 [ABCB1/MDR1 gene; P-glycoprotein (P-gp)] was the first studied protein of ABC transporters (Lautier et al. 1996). P-gp is a polypeptide dimer (with 1280 amino acid residues); it represents a pore forming membrane protein that can export many types of drugs in MDR cells (Leselle et al. 2005).

A possibility to overcome the problem of ABC transporter-mediated MDR is the development of efficient inhibitors, which either down-regulate the expression of transporter proteins or have a synergistic effect with chemotherapeutic agents by inhibiting the efflux function of ABC transporters (Liu et al. 2013).

Medicinal plants are rich sources of secondary metabolites which can be used to treat human diseases and illnesses (Van Wyk and Wink 2004). Many natural compounds and phytopharmaceuticals have been discovered that can inhibit P-gp and re-sensitize resistant tumour cells in vitro (review in Wink et al., 2012, Wagner and Ulrich-Merzenich, 2009). We have screened several natural compounds having a complementarity effect with chemotherapeutic agents used in treating cancer cells. Successful examples include the isolation of limonin and other secondary metabolites from Citrus species as P-gp inhibitors (EI-Readi et al. 2010).

Tetrandrine and fangchinoline are bisbenzylisoquinoline alkaloids, occurring in roots of the creeper Stephania tetrandra Moore (Wu et al. 2010). In China, the alkaloids are employed to decrease portal venous pressure and blood pressure (Kim et al. 1999). In recent years, tetrandrine and fangchinoline have been found to exert anti-inflammatory (Choi et al. 2000) and antitumor activities (Yang et al. 2007), inhibiting proliferation and inducing apoptosis of hepatoma, breast cancer, lung cancer and leukaemia cells (Chen et al. 2009). However, we are not aware on any report whether tetrandrine and fangchinoline have the ability to reverse ABC transporter-mediated MDR.

In this study a series of experiments were carried out to investigate the effect of tetrandrine and fangchinoline alone or in combination with doxorubicin to the reverse P-gp mediated MDR. The direct inhibition of P-gp by the alkaloids was studied in rhodamine 123 competition experiments. In addition, we could show that both alkaloids can down-regulate the expression of P-gp.

Section snippets

Chemicals and reagents

Doxorubicin, dimethyl sulfoxide (DMSO), rhodamine 123 (Rho123), verapamil hydrochloride and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoleum) were purchased from Sigma–Aldrich GmbH, Germany. The polyclonal antibody against MDR1/ABCB1 and HRP-conjugated rabbit IgG antibody were purchased from Boster Co. (China). RPMI-1640 and DMEM medium were products of Gibco, Karlsruhe, Germany. FBS (foetal bovine serum) was from BioChrom KG, Berlin, Germany.

Identification of terandrine and fangchinoline by LC–MS

Tetrandrine (C38H42O6N2) and fangchinoline

Cytotoxicity of tetrandrine and fangchinoline

The cytotoxicity of tetrandrine or fangchinoline was investigated using two pairs of resistant (Caco-2, CEM/ADR5000) and sensitive cell models (HCT-116, CEM/CCRF). IC50 values of the tetrandrine and fangchinoline are documented in Table 1. The two compounds showed significant antitumor effects against human colon and leukaemia cell lines, but their cytotoxicities are lower than that of doxorubicin except against CEM/ADR5000 cells. HCT-116 and CEM/CCRF cells are more sensitive for both alkaloids

Discussion

The overexpression of ABC transporters is considered as a primary cause for the failure of cancer chemotherapy. Several MDR reversal agents from natural sources have been investigated, as many secondary metabolites and phytotherapeutics show low side effects and good tolerability (Wang et al., 2004, Wink et al., 2012). To date, several secondary metabolites have been found to strongly inhibit the function of ABC transporters (review in Wink et al. 2012). However, the potential of tetrandrine

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgement

The authors thank the Germany Federal Ministry of Education and Research for providing a DAAD (German Academic Exchange Service) scholarship for Yan Fang Sun.

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