The multidrug resistance of tumour cells was reversed by tetrandrine in vitro and in xenografts derived from human breast adenocarcinoma MCF-7/adr cells

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Abstract

Multidrug resistance (MDR) is one of the main obstacles limiting the efficacy of chemotherapy treatment of tumours. One of the main causes of MDR is linked to the overexpression of P-glycoprotein (P-gp). This study aimed to characterise tetrandrine (Tet), a potent inhibitor of P-gp mediated MDR. Cytotoxicity was determined by the tetrazolium (MTT) assay. A MCF-7/adr cell xenograft model was established to investigate the effect of Tet on reversing MDR in vivo. Mechanistic experiments were conducted to examine the uptake, efflux and accumulation of doxorubicin (Dox) and Fura-2, and to assess lipid membrane fluidity. Tet potentiated the cytotoxicity of Dox; a 20.4-fold reversal of resistance was achieved in the presence of 2.5 μmol/l of Tet. Accumulation and efflux studies with the P-gp substrates, Dox and Fura-2, demonstrated that Tet inhibited the P-gp-mediated drug efflux. In addition, Tet lowered cell membrane fluidity in a concentration-dependent manner. In mice bearing the MDR MCF-7/adr cell xenografts, coadministration of Tet potentiated the antitumour activity of doxorubicin without a significant increase in toxicity. Tet was an extremely potent MDR modulator both in vitro and in vivo, without apparently enhancing the toxicity of the co-administered drugs. Hence, Tet holds great promise as a MDR modulator for the treatment of P-gp-mediated MDR cancers.

Introduction

Multidrug resistance (MDR) is a major obstacle for successful chemotherapy treatment, MDR is often the result of overexpression of a 170 kD plasma membrane glycoprotein known as P-glycoprotein (P-gp). P-gp belongs to the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) transporters and actively pumps out a wide range of structurally and functionally diverse amphipathic anticancer drugs from the inside of tumour cells thereby decreasing their intracellular accumulation [1]. Recent studies showed that tumour cells expressing MDR-associated protein (MRP), lung resistant protein (LRP) and mutation of DNA topoisomerase II are likely to be MDR 2, 3.

A plethora of agents are able to reverse the MDR phenotype in vitro4, 5, 6. Many natural and synthetic products of various structures, including calcium channel blockers (e.g. verapamil, nifedipine), calmodulin antagonists (e.g. trifluoperazine, chlorpromazine), quinolines (e.g. chloroquine, quinidine), immunosuppressive drugs (e.g. cyclosporin A, rapamycin) and yohimbine alkoids (e.g. reserpine, yohimbine) have been shown to partly or competitively inhibit the function of P-gp.

In vivo, combined therapy with MDR-related cytotoxins and modulators could shrink tumours and prolong the life-span in animal models. Unfortunately, the data regarding clinical efficacy are not yet available from the early clinical trials. Moreover, the MDR reversal agent may expose the patient to unacceptable side-effects or toxicity at the doses required for effectiveness and/or affect the pharmacokinetics of the anticancer drug 1, 7. These limitations have spurred on efforts to search for new, more effective compounds. New drugs developed specifically to inhibit P-gp and modulate MDR are currently one of the most important strategies in the field of cancer chemotherapy.

Tetrandrine (Tet), one of the bisbenzylisoquinoline alkaloids, has been used as an antifibrotic drug to treat the lesions of silicosis in China since the 1960s. It is relatively non-toxic to humans, even at the administration of 180 mg, intramuscularly (i.m.) three times daily (t.i.d.) [8]. Our previous studies showed Tet was able to reverse MDR in vitro[9]. The aim of this study was to determine whether Tet can enhance the efficacy of doxorubicin (Dox), both in vitro and in nude mice bearing tumours, and to probe the mechanism of the reversal of MDR.

Section snippets

Materials

Tet was generously provided by Dr Pan XP (Kuming Institute of Botany, Chinese Academy of Sciences). The powder of Tet, C38H42O6N2, with a purity of >98% was obtained by chromatography. Tet was freshly made before use. Fura 2-AM, Fura 2, 1,6-diphenyl-1,3,5-hexatriene (DPH) and Dox were purchased from Sigma Chemical Co. Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco BRL.

Cell lines and cell culture

The MDR cell lines MCF-7/adr and the parental sensitive cell line MCF-7 were generously provided by Prof.

Tet itself inhibits cell growth in the MCF-7 and MCF-7/adr cells

The cytotoxic effect of Tet after a 72-h treatment is shown in Fig. 1. The IC50 of Tet to MCF-7 and MCF-7/adr cells was 9.3±1.7 and 11.5±2.4 μmol/l, respectively. Cell survival was more than 90% in both the MCF-7/adr cells and their parental sensitive MCF-7 cells using Tet concentrations of ⩽2.5 μmol/l. So Tet concentrations of 2.5, 1.25, 0.625 μmol/l were used to study the reversal of MDR.

Reversal of MDR in vitro by Tet

The cells were incubated with various concentrations of Tet and a full range of concentrations of the

Discussion

Simultaneous genetic knockout of mdr1a and mdr1b resulted in healthy mice, indicating that P-gp is not essential for basic physiological functions [16]. This suggested that the combination of anticancer drugs with a non-toxic and potent P-gp inhibitor may be a promising approach to solve the problem of resistance. A large number of compounds, such as verapamil and cyclosporin A, can reverse the MDR phenotype mediated by P-gp in vitro17, 18. However, these agents are relatively weak P-gp

Acknowledgements

This work was supported in part by 990B82 Key Science and Technology Project Research Grant of Guangdong Province, China and No. 9800182, natural science foundation of Guangdong province, China and No. 9905 Excellent Youth Grant of the Ministry of Health of China.

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