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Synergistic cytotoxicity of gemcitabine, clofarabine and edelfosine in lymphoma cell lines.

ABSTRACT: Treatments for lymphomas include gemcitabine (Gem) and clofarabine (Clo) which inhibit DNA synthesis. To improve their cytotoxicity, we studied their synergism with the alkyl phospholipid edelfosine (Ed). Exposure of the J45.01 and SUP-T1 (T-cell) and the OCI-LY10 (B-cell) lymphoma cell lines to IC10-IC20 levels of the drugs resulted in strong synergistic cytotoxicity for the 3-drug combination based on various assays of cell proliferation and apoptosis. Cell death correlated with increased phosphorylation of histone 2AX and KAP1, decreased mitochondrial transmembrane potential, increased production of reactive oxygen species and release of pro-apoptotic factors. Caspase 8-negative I9.2 cells were considerably more resistant to [Gem+Clo+Ed] than caspase 8-positive cells. In all three cell lines [Gem+Clo+Ed] decreased the level of phosphorylation of the pro-survival protein AKT and activated the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) stress signaling pathway, which in J45.01 cells resulted in the phosphorylation and heterodimerization of the transcription factors ATF2 and c-Jun. The observed rational mechanism-based efficacy of [Gem+Clo+Ed] based on the synergistic convergence of several pro-death and anti-apoptotic signaling pathways in three very different cell backgrounds provides a powerful foundation for undertaking clinical trials of this drug combination for the treatment of lymphomas.

SUBMITTER: Valdez BC

PROVIDER: S-EPMC3913938 | biostudies-other | 2014 Jan

REPOSITORIES: biostudies-other

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Project description:PurposeOxaliplatin 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.MethodsHuman 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).ResultsA 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.ConclusionThese 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.

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Synergistic cytotoxicity of gemcitabine, clofarabine and edelfosine in lymphoma cell lines.

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