Combined modalities of resistance in an oxaliplatin-resistant human gastric cancer cell line with enhanced sensitivity to 5-fluorouracil

Abstract

To identify mechanisms underlying oxaliplatin resistance, a subline of the human gastric adenocarcinoma TSGH cell line, S3, was made resistant to oxaliplatin by continuous selection against increasing drug concentrations. Compared with the parental TSGH cells, the S3 subline showed 58-fold resistance to oxaliplatin; it also displayed 11-, 2-, and 4.7-fold resistance to cis-diammine-dichloroplatinum (II) (CDDP), copper sulphate, and arsenic trioxide, respectively. Interestingly, S3 cells were fourfold more susceptible to 5-fluorouracil-induced cytotoxicity due to downregulation of thymidylate synthase. Despite elevated glutathione levels in S3 cells, there was no alteration of resistant phenotype to oxaliplatin or CDDP when cells were co-treated with glutathione-depleting agent, l-buthionine-(S,R)-sulphoximine. Cellular CDDP and oxaliplatin accumulation was decreased in S3 cells. In addition, amounts of oxaliplatin- and CDDP-DNA adducts in S3 cells were about 15 and 40% of those seen with TSGH cells, respectively. Western blot analysis showed increased the expression level of copper transporter ATP7A in S3 cells compared with TSGH cells. Partial reversal of the resistance of S3 cells to oxaliplatin and CDDP was observed by treating cell with ATP7A-targeted siRNA oligonucleotides or P-type ATPase-inhibitor sodium orthovanadate. Besides, host reactivation assay revealed enhanced repair of oxaliplatin- or CDDP-damaged DNA in S3 cells compared with TSGH cells. Together, our results show that the mechanism responsible for oxaliplatin and CDDP resistance in S3 cells is the combination of increased DNA repair and overexpression of ATP7A. Downregulation of thymidylate synthase in S3 cells renders them more susceptible to 5-fluorouracil-induced cytotoxicity. These findings could pave ways for future efforts to overcome oxaliplatin resistance.

Figure 1

Cellular GSH content and the expression level of GST-π in TSGH and S3 cells. (A) Total amount of GSH in TSGH and S3 cells was determined using GSH/GSSG ratio assay kit as described in Materials and Methods. The data are the means±s.d. of three independent experiments. (B) Analysis of the expression level of GST-π by using the Western blot analysis. α-Tubulin has been used as internal control. The results are the representatives of at least three independent experiments.

Figure 2

Cellular platinum accumulation, platinum-DNA adducts formation, and histone H₂AX phosphorylation between TSGH and S3 cells. (A) Cellular platinum accumulation and (B) Platinum–DNA adducts formation of TSGH and S3 cell lines after exposure to various concentrations (1, 10, 50, 100 μM) of either CDDP or oxaliplatin. Each data points are means from three independent experiments. (C) Induction of histone H₂AX phosphorylation. TSGH and S3 cells were exposed for 24 h to the indicated concentrations of platinum drugs, fixed and labelled with an antiphosphohistone H₂AX monoclonal antibody before flow cytometric analysis. γH₂AX positive cells were quantified and expressed as a function of drug concentration. Error bars show the standard deviations. ^* (P<0.05), significantly different for platinum accumulation, DNA adduct formation, and histone H₂AX phosphorylation between TSGH and S3 cells at equimolar concentration of platinum drugs.

Figure 3

Determination of the expression level of the copper transporters between TSGH and S3 cell. (A) Analysis of the expression level of copper uptake transporter CTR1 by using semiquantitative RT–PCR. GAPDH has been used as internal control. The results are the representatives of at least three independent experiments. (B) Analysis of the expression level of copper efflux transporters ATP7A and ATP7B by using Western blot analysis. α-Tubulin has been used as internal control. The results are the representatives of at least three independent experiments.

Figure 4

Effect of ATP7A-targeted siRNA on sensitivity of S3 cells towards oxaliplatin and CDDP. (A) Western blot analysis of S3 cells after treatment with an ATP7A-targeted siRNA. Cells were incubated in the absence or presence of ATP7A siRNA ON-TARGET plus SMART pool for the indicated times, and harvested and lysed for Western blot analysis. α-Tubulin as internal control. (B) Effect of ATP7A-targeted siRNA on sensitivity of S3 cells towards oxaliplatin and CDDP. S3 cells were transiently transfected with ATP7A-targeted siRNA for 9 h, then cells were exposed to oxaliplatin and CDDP for another 72 h. Cell growth was determined by methylene blue dye assay. Each value represents the mean of three independent experiments.

Figure 5

DNA repair ability between TSGH and S3 cells. (A) Enhanced DNA repair capacity of CDDP- and oxaliplatin-induced DNA damage by host cell reactivation assay. Percent of repair activity was measured using the undamaged vectors as control. This result is the representative of three independent experiments (mean±s.d.). (B) Evaluation of the DNA repairing proteins. The expression levels of ERCC-1 and XRCC-1 were examined in TSGH and S3 cells by Western blot analysis. α-Tubulin has been used as internal control. The results are the representatives of at least three independent experiments.

Figure 6

Enhanced sensitivity to 5-FU correlated with TS downregulation in S3 cells. (A) Analysis of TS protein and mRNA levels in TSGH and S3 cells by Western blotting and Northern blotting, respectively. α-Tubulin has been used as internal control in Western blotting. Beside, GAPDH has been used as internal control in Northern blotting. The results are the representatives of at least three independent experiments. (B) Western blot analysis of TSGH cells after treatment with a TS-targeted siRNA. Cells were incubated in the absence or presence of 0.1 nM of TS-targeted siRNA for the indicated times, and harvested and lysed for western blot analysis. (C) Effect of TS-targeted siRNA on sensitivity of TSGH cells towards 5-FU. TSGH cells were transiently transfected with TS-targeted siRNA at concentration of 0.1 nM for 12 h, then cells were exposed to 5-FU for another 72 h. Cell growth was determined by methylene blue dye assay. Each value represents the mean of three independent experiments.