Rifampicin and verapamil induce the expression of P-glycoprotein in vivo in Ehrlich ascites tumor cells

Abstract

The effect of an in vivo treatment with two commonly employed drugs that are P-glycoprotein substrates, verapamil and rifampicin, on Ehrlich ascites carcinoma cells, was evaluated.

Ehrlich ascites carcinoma cells were inoculated i.p. in CD-1 mice and animals were orally treated for 10 days with rifampicin (60 mg/kg/day) or verapamil (6 mg/kg/day). In the harvested cells the transcripts for mdr1a and mrp1, but not those for mdr1b, mrp2 and CYP3A, were detected, and treatment with verapamil or rifampicin did not modify the levels of the transcripts. On the contrary, an increased expression of P-glycoprotein was observed at the protein level with Western blot. The intracellular uptake of doxorubicin, a P-glycoprotein and MRP substrate, was significantly lower in cells obtained from treated animals in comparison with cells obtained from controls; in addition, the uptake was increased by a pretreatment with verapamil. The survival time of control animals implanted with untreated cells was similar to that of animals inoculated with cells obtained from rifampicin treated animals, however, the antineoplastic effect of doxorubicin was significanly higher in control animals.

A treatment with rifampicin or verapamil in Ehrlich ascites tumor confers resistance to the antineoplastic drug doxorubicin, probably through an increased expression of P-glycoprotein.

Introduction

One of the major obstacles to the successful treatment of many human cancers is the development of multidrug resistance. The most important mechanism of resistance is linked to a reduced drug concentration at the target site, due to enhanced cellular efflux of antitumor compounds. This pheno menon is usually related to the expression of ATP-driven efflux pumps, among which P-glycoprotein and Multidrug Resistance-related Proteins (MRPs). P-glycoprotein is a 170 kDa protein, whose substrates include a huge number of lipophylic, naturally occurring substances [1]; the protein is encoded by the MDR1 gene in humans and by the mdr1a and mdr1b genes in rodents. The MRP family comprises at least nine members that transport drugs conjugated with glutathione, glucuronide, sulfate or unconjugated amphiphilic anions [2]; MRP1 and MRP2 confer resistance to a wide variety of chemotherapeutic agents [2]. The overexpression of these proteins in tumors after chemotherapy is usually attributed to induction or to selection of preexisting multidrug resistant cells. Various factors can influence the expression of P-glycoprotein and MRP, such as environmental stresses [3], physiological inducers [4], hormones [5] and drugs among which anticancer agents [6], [7] as well as drugs employed in non neoplastic diseases [7], [8], [9].

It is now widely recognized that P-glycoprotein or MRP overexpression is associated with clinical evidence of drug resistance and treatment failure [10]. Considerable effort has therefore been made to apply chemotherapeutic protocols that could prevent the development of P-glycoprotein or MRP expression by combining multiple drugs and delivering them at optimal doses and intervals. On the contrary, little attention has been played to the possibility that the expression of these proteins may be affected by drugs that are administered to cancer patients for other pathologies.

Among the substances that induce P-glycoprotein and MRP in normal and tumor cells, are two commonly employed pharmacological agents such as verapamil [8], widely used in cardiovascular therapy, and rifampicin [9], [11], [12], [13], a first line agent in the chemotherapy of tuberculosis. It is not unlikely that cancer patients are affected by cardiovascular diseases, and they are often immunocompromised as a consequence of the disease or of the antineoplastic therapy; therefore these drugs can be employed in cancer patients and also in subjects in which a tumor is growing but is not yet clinically evident.

It may be hypothesized that these compounds, positively tested in tumor cell lines in vitro, may also exhert their induction potential in vivo. The aim of the present study was therefore to investigate whether a chronic treatment with rifampicin or verapamil could induce the expression of P-glycoprotein, MRP1 or MRP2 in Ehrlich ascites tumor cells in vivo, and could consequently modify the sensitivity of this tumor to doxorubicin.