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


CONCLUSIONS
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.
Nannizzi
Sara
S
Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, 56126, Pisa, Italy.
Veal
Gareth J
GJ
Giovannetti
Elisa
E
Mey
Valentina
V
Ricciardi
Simona
S
Ottley
Christopher J
CJ
Del Tacca
Mario
M
Danesi
Romano
R
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005971:Glutamates / Q000008:administration & dosage; D006147:Guanine / Q000008:administration & dosage / Q000031:analogs & derivatives; D006801:Humans; D008856:Microscopy, Fluorescence; D009944:Organoplatinum Compounds / Q000008:administration & dosage; D000077150:Oxaliplatin; D000068437:Pemetrexed; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005971:Glutamates; D009944:Organoplatinum Compounds; D000068437:Pemetrexed; D000077150:Oxaliplatin; D006147:Guanine; D051057:Proto-Oncogene Proteins c-akt