Cellular and molecular mechanisms for the synergistic cytotoxicity elicited by oxaliplatin and pemetrexed in colon cancer cell lines

Abstract

Purpose: Oxaliplatin effect in the treatment of colorectal cancer is improved upon combination with thymidylate synthase (TS) inhibitors. Pemetrexed is polyglutamated by the folylpolyglutamate synthase (FPGS) and blocks folate metabolism and DNA synthesis by inhibiting TS, dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study evaluates the pharmacological interaction between oxaliplatin and pemetrexed in colorectal cancer cells.

Methods: Human HT29, WiDr, SW620 and LS174T cells were treated with oxaliplatin and pemetrexed. Drug interaction was studied using the combination index method, while cell cycle was investigated with flow cytometry. The effects of drugs on Akt phosphorylation and apoptosis were studied with ELISA and fluorescence microscopy, respectively. RT-PCR analysis was performed to assess whether drugs modulated the expression of pemetrexed targets and of genes involved in DNA repair (ERCC1 and ERCC2). Finally, platinum-DNA adduct levels were detected by ultra-sensitive multi-collector inductively coupled plasma mass spectrometry (ICP-MS).

Results: A dose-dependent inhibition of cell growth was observed after drug exposure, while a synergistic interaction was detected preferentially with sequential combinations. Oxaliplatin enhanced cellular population in the S-phase. Drug combinations increased apoptotic indices with respect to single agents, and both drugs inhibited Akt phosphorylation. RT-PCR analysis showed a correlation between the FPGS/(TS x DHFR x GARFT) ratio and pemetrexed sensitivity, as well as a downregulation of ERCC1, ERCC2, TS, DHFR and GARFT after drug exposure. In addition, pretreatment with pemetrexed resulted in an increase of oxaliplatin-DNA adducts.

Conclusion: These data demonstrate that oxaliplatin and pemetrexed synergistically interact against colon cancer cells, through modulation of cell cycle, inhibition of Akt phosphorylation, induction of apoptosis and modulation of gene expression.

Fig. 1

Dose-response cytotoxicity of oxaliplatin (L-OHP), pemetrexed (MTA) and their combinations in HT29, WiDr, SW620 and LS174T colon cancer cells. Data represent the mean percentage of proliferating cells (±SD) from three independent experiments. In combination experiments the values refer to oxaliplatin concentration

Fig. 2

a Percentage of cells with apoptotic morphology evaluated following treatment with oxaliplatin (L-OHP), pemetrexed (MTA) and their combinations. Data represent mean values ±SD from three independent experiments. For single drug incubations, *P < 0.05, **P < 0.01 versus control; for drug combinations *P < 0.05, **P < 0.01, ***P < 0.001 versus oxaliplatin and pemetrexed alone. b Reduction of P-Ser473 Akt by oxaliplatin (L-OHP) and pemetrexed (MTA) in HT29, WiDr, SW620 and LS174T colon cancer cells. Data represent mean values ±SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 with respect to control

Fig. 3

Gene expression analysis of selected genes involved in the activity of oxaliplatin and pemetrexed. a modulation of ERCC1 and ERCC2 mRNA expression by pemetrexed, at IC₅₀ levels. b Modulation of ERCC1 and ERCC2 mRNA expression by 5-fluorouracil (5-FU), methotrexate, LY309887, at IC₅₀ levels, in HT29 cells. c FPGS, TS, DHFR and GARFT mRNA modulation after oxaliplatin exposure at IC₅₀ levels. Data represent mean values ±SD from three independent experiments*P < 0.05, **P < 0.01, ***P < 0.001 with respect to control. In particular, the results of the repeated measures ANOVA followed by the Tukey’s test were 0.001, 0.007, 0.033 and 0.045 for the modulation of ERCC1 and ERCC2 after pemetrexed exposure in the HT29, WiDR, SW620 and LS174T cells, respectively; 0.023 for the modulation of ERCC1 and ERCC2 after 5-FU exposure in the HT29 cells; and 0.023, 0.003 and <0.001 for the modulation of FPGS, TS, DHFR and GARFT after oxaliplatin exposure, in the WiDr, SW620 and LS1574T cells, respectively

Fig. 4

Formation of oxaliplatin–DNA adducts in SW620 cells. Cells were treated with oxaliplatin (L-OHP) at a concentration of 300 μM for 2 h, either alone or following a 24 h preincubation with pemetrexed (MTA) at the IC₅₀ concentration, 10× below IC₅₀ or 10× above IC₅₀. Data represent mean values ±SD from three independent experiments. *P < 0.05 with respect to control (oxaliplatin alone)