Cancer Letters

Cancer Letters

Volume 291, Issue 1, 1 May 2010, Pages 76-82
Cancer Letters

Silencing MRP4 by small interfering RNA reverses acquired DDP resistance of gastric cancer cell

https://doi.org/10.1016/j.canlet.2009.10.003Get rights and content

Abstract

A cisplatin (DDP) resistant cell line (SGC7901/DDP) from a Chinese gastric cancer cell line (SGC7901) was established by step-increasing DDP treatment, and the resultant cell line showed an over 21.9-fold increased resistance to DDP. To identify the mechanism of DDP resistance, the differential gene expression panel was examined by Affymetrix microarray. Among the identified differential genes, 681 genes expression were increased and 1139 genes were decreased. To confirm these gene changes furtherly, one of the upregulated gene, MRP4 was identified with increased mRNA and protein level of SGC7901/DDP by RT-PCR and Western-blot analysis compared with its parental cell line. By using the small interfering RNA (RNAi) to decrease the MRP4 expression, the DDP resistance phenotype of SGC7901/DDP was reversed. These data suggest that MRP4 is a DDP resistance candidate gene of SGC7901 gastric cancer cell line.

Introduction

Gastric cancer is the second mostly frequent cause of cancer-related mortality worldwide and the incidence is higher at Asia compared with other geographic areas [1], [2], [3], [4], [5]. Radical surgery remains the standard form of therapy with curative intent, but recurrence appears in approximately 50–70% of patients with advanced disease [1]. As a systemic approach to control cancer, more and more studies of chemotherapy on gastric cancer are forthcoming, and the existing evidence seems to support that chemotherapy, especially when the disease are in their advanced stage, is commonly used and effective [6]. The overall benefits of chemotherapy should be depended on patient status and chemotherapy regimens. Directing to individual management of patients, to identify the subgroup that actually benefit from chemotherapy is necessary. Using biologic markers has been pursued in other kinds of cancer, such as HER-2/neu over-expression of breast cancer, while this kind of research of gastric cancer is limited [5].

Several randomized studies comparing different regimens have demonstrated that chemotherapy increases the survival of gastric cancer and CF (cisplatin (CDDP)/5-fluorouracil (5-FU)) may be considered as a reference regimen to date [1]. Recently, several novel chemotherapeutic agents, including Taxanes, Irinotecan, and S-1 have demonstrated activity in gastric cancer and offered hope for improving patient outcome when administrated with platinum-based regimens. Nevertheless chemoresistance is a common phenomenon of treatment failure, which can be intrinsical or acquired during treatment. Furthermore, in the process of acquiring resistance, the tumor may become cross-resistance to a range of reagents and progress to multi-drug resistance and ultimate treatment failure [7]. The problem of drug resistance is complex and a lot of factors affect drug sensitivity, including drug efflux, drug activation and inactivation, alterations in drug target, etc. [8]. Based on recent advances in the understandings of the mechanisms of actions and resistance of platinum compound, two emerging areas are of special importance: (1) the role of transporters and exporters in imparting the special selectivity of platinum drugs for specific tumors, and (2) the relevance of inactivated DNA repair pathways [9].

In this study, a DDP resistant gastric cancer cell line SGC7901/DDP was firstly established by using step-increasing DDP treatment of SGC7901 and its gene expression panel was compared with its parental cell line by using Affymetrix gene chip. As a member of human multi-drug resistance-associated protein (MRP) and a kind of cell membrane transporter protein, an over-expression gene MRP4 was selected and identified as a candidate DDP resistance associated gene of SGC7901 by the subsequent experiments.

Section snippets

Cell culture

Human moderately differentiated gastric adenocarcinoma cell line SGC7901 was kindly gifted by Prof. Daiming Fan [10]. Cells were cultured in RPM11640 medium (Gibco) supplemented with 10% fetal calf serum in a CO2 incubator as reported [11]. SGC7901 cells were treated with stepwise-increasing concentrations of DDP (Qilu Pharm, China) up to 1 μg/ml. The DDP-resistant cell line SGC7901/DDP were selected and subcultured in medium mentioned above with 0.5 μg/ml DDP.

Drug sensitivity assay

Cells (5 × 104/ml) were seeded onto 96

Sensitivity of cell lines to DDP and differential expression gene profile

To obtain DDP-resistant cell lines, SGC7901 cells were treated with increasing concentrations of DDP up to 1 μg/ml and SGC7901/DDP clones were selected after 8 months cultivation. The SGC7901/DDP cell line showed 21.9-fold acquired resistance to DDP based on IC50 (4.60 ± 0.039 μg/ml vs. 0.21 ± 0.023 μg/ml).

A differential gene expression profile between SGC7901/DDP and SGC7901 was identified. Six hundred and eighty one genes were upregulated and 1113 genes were downregulated in the DDP resistance cell

Discussion

DDP shows promising activity against a number of cancers and is approved for treatment of gastric cancer. However, the clinical response rates of DDP for some patients have been lower and varied with therapy. The sensitivity of DDP is partly related to decreased accumulation of cisplatin in resistant cells, increased intra-cellular trapping of cisplatin, increased repair of DNA damage or increased tolerance of DNA damage [9], [14]. Mechanisms for transporting platinum drugs were not known until

Conflicts of interest

None declared.

Acknowledgments

This study was supported by Shanghai Natural Science Foundation and Xuhui Central Hospital.

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