Cytotoxic effects of pemetrexed in gastric cancer cells

Abstract

Pemetrexed is a newly developed multitargeted antifolate with promising clinical activity in many solid tumors including gastric cancer. The aim of the present study was to evaluate the cytotoxicity of pemetrexed and its mode of interaction with cisplatin in gastric cancer cell lines, and to identify genes associated with sensitivity to pemetrexed. The cytotoxic activity of pemetrexed was assessed by tetrazolium-based colorimetric assay (MTT assay) and the interaction between pemetrexed and cisplatin was evaluated by the isobologram method. Western immunoblotting and real time RT-PCR analysis of thymidylate synthase (TS), folylpoly-γ γ γ γ-glutamate synthetase (FPGS) and reduced folate carrier (RFC1) were performed in order to determine whether sensitivity to pemetrexed would be predictable by protein or mRNA expression levels. Pemetrexed was more cytotoxic than 5-fluorouracil, with IC 50 between 17 and 310 nM in most of the gastric cancer cell lines examined and the pemetrexed/cisplatin combination resulted in additive or synergistic interaction. The protein expressions of TS, FPGS, and RFC1 were significantly associated with IC 50 for 5-fluorouracil, but no such association was found for pemetrexed chemosensitivity. The mRNA expressions of RFC1, FPGS and other target and resistance related genes revealed no significant association with pemetrexed sensitivity. In conclusion, pemetrexed is active against gastric cancer cell lines and the pemetrexed/cisplatin combination showed a synergistic or additive interaction, supporting its clinical use in gastric cancer. Drug sensitivity toward pemetrexed could not be predicted by the expressions of TS, RFC1, or FPGS and we suggest that it is determined by interactions between multiple genes. (Cancer Sci 2005; 96: 365-371) P emetrexed disodium is a newly developed antifolate that targets multiple enzymes involved in pyrimidine and purine synthesis.(1-3) It primarily inhibits thymidylate synthase (TS), but at higher levels pemetrexed also inhibits additional folate-dependent enzymes such as dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT), which participate in de novo purine synthesis.(4) Its multiple sites of enzyme inhibition are the basis for its characterization as a 'multitargeted' antifolate. Pemetrexed gains entry to cells via reduced folate carrier (RFC) and is converted to polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS). These polyglutamated forms are more negatively charged than pemetrexed and concentrate intracellularly. Whereas pemetrexed only moderately inhibits TS, the pentaglutamate form is 100-fold more potent than pemetrexed, making pemetrexed one of the most potent folate-based TS inhibitors known today. However, the mechanism of resistance to pemetrexed is not fully understood. Studies on cells with acquired resistance suggest that the mechanism of resistance involves impaired membrane transport, reduced polyglutamation or increased and/or structurally altered target enzymes.(6) Studies on antifolat...

Thymidylate Synthase and Excision Repair Cross-Complementing Group-1 as Predictors of Responsiveness in Mesothelioma Patients Treated with Pemetrexed/Carboplatin

“…Conversely, they did not find a significant correlation between TS expression and outcome in MPM patients who were not treated with pemetrexed. However, TS mRNA and protein expression levels might also play a prognostic role, as reported in patients with NSCLC (24)(25)(26), whereas the sensitivity of pemetrexed was not predicted by the expression levels of TS in gastric cancers (27).…”

Enzastaurin, a Protein Kinase Cβ Inhibitor, Suppresses Signaling through the Ribosomal S6 Kinase and Bad Pathways and Induces Apoptosis in Human Gastric Cancer Cells

“…Tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT; Sigma] assays were used to evaluate the growth inhibitory effects of enzastaurin, as described in prior studies (20,21). Cells were seeded on 96-well plates, incubated for 24 h, and then treated with drugs for 72 h at 37jC.…”

“…Analyses of drug interactions were performed by constructing an envelope of additivity using the isobologram method of Steel and Peckham, as described previously (21,22). Based on available dose-response curves, the combined effects of enzastaurin and other cytotoxic chemotherapeutic agents were analyzed at IC 50 .…”

Phase II Study on Pemetrexed-based Chemotherapy in Treating Patients with Metastatic Gastric Cancer not Responding to Prior Palliative Chemotherapy

“…Pemetrexed demonstrated activity in a variety of tumor types based on previous reports, including non-small cell lung cancer, malignant pleural mesothelioma, pancreas, colorectal, gastric, bladder, breast, head and neck cancers (Martin, 2006;Bajetta et al, 2003). Pemetrexed is reported to be more active than 5-FU against several human gastric cancer cell lines (Kim et al, 2005).…”