Down-regulation of thymidylate synthase expression and its steady-state mRNA by oxaliplatin in colon cancer cells

Abstract

Recently, evidence has accumulated that weekly 24-h infusion of high-dose 5-fluorouracil (5-FU) with leucovorin (LV, folinic acid) biochemical modulation may improve the response rates compared with the bolus 5-FU regimens in colorectal cancer (CRC). Combining the infusional 5-FU/LV (iFL) regimens with oxaliplatin or irinotecan is widely adopted to further improve treatment efficacy. Either oxaliplatin-iFL or irinotecan-iFL may achieve an overall response rate of more than 50% in the first-line treatment. Intriguingly, in the salvage treatment for metastatic CRC patients who had failed iFL, only oxaliplatin-iFL may achieve a response rate of about 13-25%. In contrast, oxaliplatin alone or irinotecan-iFL had a very low response rate of 5% or less. To test if the oxaliplatin may reverse the iFL-related 5-FU resistance in CRC, we used DLD-1 colon adenocarcinoma cells as the in vitro study model. First, we revealed that oxaliplatin and 5-FU act synergistically on DLD-1 cells by MTT cytotoxicity assay and median drug effect analysis. Second, we treated the DLD-1 cells with serial concentrations of oxaliplatin (0.1-10 microM). Oxaliplatin treatment results in down-regulation of free thymidylate synthase (TS) protein expression by Western blotting. Further, we analyzed the TS mRNA level by reverse transcription and real-time quantitative polymerase chain reaction assay. Oxaliplatin treatment results in down-regulation of the TS mRNA level up to 40% (mean +/- SD of ratio to reference control = 0.60 +/- 0.21, range 0.42-0.84). In this study, our data provide important information explaining the reason why the combination of oxaliplatin and 5-FU results in a better objective response in 5-FU-resistant patients than oxaliplatin alone does. Our data also suggest that TS down-regulation happens at the transcriptional level. TS modulation and down-regulation had, thus, shed light on the useful potential strategy to achieve objective responses in 5-FU-resistant CRC patients.

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

“…Moreover, we also showed that TSGH cells could become more sensitive to 5-FU through gene silencing via TS-targeted siRNA method ( Figure 6B), which validated the important role of downregulated TS gene in rendering S3 cells more sensitive to 5-FU ( Figure 6C). Although the detailed mechanism for oxaliplatin-induced TS gene downregulation needs to be further elucidated, our result is concordant with two previous reports (Raymond et al, 2002;Yeh et al, 2004), which indicated that oxaliplatin could downregulate TS in cancer cells and thus potentiate the efficacy of 5-FU. As oxaliplatin and 5-FU have been shown to be highly synergistic not only in preclinical models (Raymond et al, 1997) but also in subsequent clinical trials (Rothenberg et al, 2003;Chao et al, 2004;Lordick et al, 2005;Schippinger et al, 2005;Cavanna et al, 2006), our data provide important information regarding why the combination of oxaliplatin and 5-FU results in better objective response than single use alone.…”

Biological predictive factors in rectal cancer treated with preoperative radiotherapy or radiochemotherapy

“…Moreover, evidence has accumulated that oxaliplatin treatment results in downregulation of TS expression, providing a possible explanation, although still speculative, for the higher response rate of the combination of oxaliplatin and FU, compared with oxaliplatin alone, in FU-resistant patients (Yeh et al, 2004). In addition, it has been shown that oxaliplatin remains highly cytotoxic in cells that overexpress TS.…”

Quercetin Induces Necrosis and Apoptosis in SCC-9 Oral Cancer Cells

“…6A). The ternary complex is an inactive form of TS, whereas the free form of TS is an active enzyme (27). Figure 6A shows that, whereas the expression of the free TS in untreated cells increased with time, quercetin inhibited free TS protein compared with the corresponding controls at all time points, with the 72-h treatment resulting in a 90% inhibition of free TS protein.…”

“…27. The lysates were sonicated and cleared by centrifugation at 14,000 g for 15 min at 4°C, and the supernatant (total cell lysate) was used or immediately stored at -80°C.…”