Emodin enhances sensitivity of gallbladder cancer cells to platinum drugs via glutathion depletion and MRP1 downregulation
Introduction
Gallbladder carcinomas are highly malignant human cancers [1], [2]. For patients with unresectable tumors, the expected survival is dismal. Generally, the role of systemic chemotherapy in the palliation of gallbladder carcinomas is undefined [3]. But recently, a retrospective review suggests that adjuvant chemoradiotherapy using 5-fluorouracil concurrently with radiotherapy in patents who underwent surgical resection of gallbladder carcinoma may improve overall survival [4]. And the combination of gemcitabine and oxaliplatin has promising results in patients with advanced biliary tract carcinomas [5], [6]. Therefore, it is necessary to search effective chemotherapeutic agents or alternative chemotherapies for these gallbladder cancers, either alone or in combination.
The major platinum-containing drugs are cisplatin, carboplatin and oxaliplatin. They are widely used anticancer agents in the treatment of various solid tumors, but have not been proven effective for gallbladder cancers [7]. Insufficient sensitivity or resistance to platinum agents is usually the major obstacle for their effective application in cancers including gallbladder cancers. The mechanisms for platinum drug resistance have been referred to decrease the intracellular drug accumulation, increase the levels of cellular thiols and increase the nucleotide excision-repair activity [8], [9]. Reverse of these mechanisms may show benefits for enhancing platinum cytotoxicity in gallbladder cancers, however, effective synergistic approaches have not been studied adequately.
We have previously demonstrated that emodin (1,3,8-trihydroxy-6-methylanthraquinone), a kind of natural anthraquinone enriched in the traditional Chinese herbal medicines, facilitates arsenic trioxide-induced apoptosis in various cancer cell types, both in vitro and in vivo via the mechanism of generation of reactive oxygen species (ROS) [10], [11], [12]. We have also found that emodin sensitizes Du-145, a cell line derived from prostate carcinoma, to cisplatin in ROS-dependent manner [13]. In addition, we found that the multidrug resistance protein MDR1 is downregulated and its transcription factor, hypoxia inducible factor-1 (HIF-1), is inhibited. In the present study we investigated whether emodin could synergize with cisplatin and other two platinum drugs in gallbladder cancer cells. We found that emodin exerted enhancing action to anticancer efficacy of cisplatin, carboplatin and oxaliplatin via decrease of reduced glutathione, and inhibition of expression of MRP1, a drug efflux pump other than MDR1.
Glutathione (GSH) conjugation and transportation of GSH conjugates of anticancer drugs out of cells have been proposed for long time as a system in the detoxification of many anticancer drugs [14]. The major components of this system include GSH, GSH-related enzymes and GSH conjugate export pump (GS-X pump), and all of them are found increased or overexpressed in many drug-resistant cancer cells [14]. MRP1 has been reported to be a member of the GS-X pump based on evidence that MRP1 transports Leukotriene C4 [15]. Inhibition of this detoxification system is a reasonable strategy for modulation of drug resistance. Recently, some small molecule inhibitors of GSH have been shown to result in a significant enhancement in cisplatin cytotoxicity [16]. Our present study proposed that the small natural compound can target to MRP1, in addition to the depletion of GSH, to enhance cisplatin, carboplatin or oxaliplatin cytotoxicity in gallbladder cancer cells.
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Cells and reagents
The human gallbladder cancer cell line SGC996 was provided by Academy of Life Sciences, Tongji University (Shanghai, China). The prostate carcinoma-derived DU-145 cells were provided by American Type Culture Collection (ATCC, Rockville, MD, USA). SGC996 and DU-145 cells were maintained in RPMI-1640 medium (GibcoBRL, Gaitherburg, MD, USA). Normal human dermal fibroblasts (HDF), preserved in our laboratory, were maintained in DMEM medium (GibcoBRL, Gaitherburg, MD, USA). These media were
Emodin enhances CDDP-induced inhibition of cell viability in tumor cells but not in normal cells
To examine the synergistic effect of emodin on cell viability, human gallbladder cancer cells SGC996 were treated with CDDP or emodin alone, or co-treated. Although no obvious reduction in viable cell number was observed in emodin treated alone group, co-treatment with CDDP led to a significant reduction of cell viability at both 24 h and 48 h, compared with CDDP treatment alone (Fig. 1A).
The normal human dermal fibroblasts were selected as representative for nontumor cells. Notably, we do not
Discussion
As gallbladder carcinomas are highly resistant to drug treatment at the onset of therapy (intrinsic multidrug resistance), there is a real need to better understand the mechanism of drug resistance and to develop novel therapeutic strategies.
One of the mechanisms of drug resistance in cancer cells is associated with altered anticancer drug transport, mediated by members of the ABC superfamily of transport proteins [18] such as MDR1 [19], MRP1 [20] and MRP2 [21]. These ABC membrane transport
Acknowledgements
We thank Dr. Susan P.C. Cole, Queen's University at Kingston, Canada, for the construct of MRP1 plasmid. This work was supported by grants from Shanghai Bureau of the Health (2007049, J. Wang) and Renji Collaborative Project (PY07003, J. Wang and Y.P. Sun).
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