Cytotoxic mechanisms of pemetrexed and HDAC inhibition in non-small cell lung cancer cells
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ABSTRACT: The cytotoxic effects of thymidylate synthase (TYMS) inhibitors, such as the multitarget antifolate pemetrexed (PMX), are not fully understood. Recent studies suggest that the combination of PMX with histone deacetylase inhibitors may be effective in the treatment of non- small cell lung cancer (NSCLC). To investigate this further, A549 NSCLC cells were exposed to various combinations of PMX and the HDAC inhibitor MS275 (entinostat) and Vorinostat (SAHA) and monitored for cell survival, cell cycle alterations and genotoxic markers. MS275 increased PMX sensitivity in A549 cells when added after preincubation with PMX. Both HeLa (p53 negative) and A549 (p53-naïve) exhibited robust activation of γH2AX upon treatment with the combination. Interestingly, CRISPR-Cas9 UNG knockout did not affect γH2AX activation or sensitivity to PMX. Proteomic analysis revealed that MS275 altered expression of known PMX targets, several proteins involved in pyrimidine metabolism, and several DNA repair proteins (RNaseH2, TOP1, RAD51C, PCNA). Contra to the prevailing model of antifolate toxicity mediated by uracil excision and repeated futile repair cycles caused by reincorporation of uracil, we propose that ribonucleotide incorporation in nuclear and mitochondrial DNA is a major contributor to cytotoxicity antifolates such as PMX, and likely also for fluorinated pyrimidine analogs. The DNA lesions induced by these agents and how their processing can be modulated by HDAC inhibition to potentiate cytotoxicity, warrants further investigation in clinical models.
INSTRUMENT(S): timsTOF Pro
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: Animesh Sharma
LAB HEAD: Tobias Iveland
PROVIDER: PXD051824 | Pride | 2024-12-18
REPOSITORIES: Pride
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