Abstract
BackgroundNon-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed, a multi-target folate antagonist, has demonstrated efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS) expression. Among many other potential targets, histone deacetylase inhibitors (HDACi) modulate TS expression, potentially sensitizing to the cytotoxic action of anti-cancer drugs that target the folate pathway, such as pemetrexed. Since high levels of TS have been linked to clinical resistance to pemetrexed in NSCLC, herein we investigated the molecular and functional effects of combined pemetrexed and ITF2357, a pan-HDACi currently in clinical trials as an anti-cancer agent.ResultsIn NSCLC cell lines, HDAC inhibition by ITF2357 induced histone and tubulin acetylation and downregulated TS expression at the mRNA and protein level. In combination experiments in vitro ITF2357 and pemetrexed demonstrated sequence-dependent synergistic growth-inhibitory effects, with the sequence pemetrexed followed by ITF2357 inducing a strikingly synergistic reduction in cell viability and induction of both apoptosis and autophagy in all cell line models tested, encompassing both adenocarcinoma and squamous cell carcinoma. Conversely, simultaneous administration of both drugs achieved frankly antagonistic effects, while the sequence of ITF2357 followed by pemetrexed had additive to slightly synergistic growth-inhibitory effects only in certain cell lines. Similarly, highly synergistic growth inhibition was also observed in patient-derived lung cancer stem cells (LCSC) exposed to pemetrexed followed by ITF2357. In terms of molecular mechanisms of interaction, the synergistic growth-inhibitory effects observed were only partially related to TS modulation by ITF2357, as genetic silencing of TS expression potentiated growth inhibition by either pemetrexed or ITF2357 and, to a lesser extent, by their sequential combination. Genetic and pharmacological approaches provided an interesting link between the autophagic and apoptotic pathways, and showed that sequential pemetrexed/ITF2357 causes a toxic form of autophagy with consequent activation of a caspase-dependent apoptotic program. In vivo experiments in NSCLC xenografts confirmed that sequential pemetrexed/ITF2357 is feasible and results in increased inhibition of tumor growth and increased mice survival.ConclusionsOverall, these data provide a strong rationale for the clinical development of sequential schedules employing pemetrexed followed by HDACi in NSCLC.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-230) contains supplementary material, which is available to authorized users.
Highlights
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide
The sequence of ITF2357 followed by pemetrexed had synergistic growth inhibitory effects in H1299 and Calu-1 cells (H1299: combination index (CI) = 0.54; Calu-1: CI = 0.37), whereas it had an effect comparable to that of ITF2357 alone in A549 cells (Additional file 1: Figure S1A,B)
Sequential treatment with ITF2357 enhances pemetrexedinduced apoptosis in NSCLC models we explored the putative mechanisms of synergistic growth-inhibitory interactions induced by pemetrexed followed by ITF2357 in the A549 and H1299 representative cell lines
Summary
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. It has been proposed that patients with NSCLC might benefit from combined treatment with epigenetic drugs [14,15] In this context, histone deacetylase inhibitors (HDACi) represent a promising class of antitumor agents, developed to reverse the silencing of critical regulatory pathways [16,17]. The cellular response to treatment with HDACi shows pleiotropic effects involving cell cycle arrest, induction of apoptosis/autophagy and differentiation, modulation of microtubule function, DNA repair, and angiogenesis [18,19,20] Based on their ability to activate the apoptotic and autophagic pathways, HDACi may have interest in combination with conventional chemotherapeutic agents to enhance tumor cell chemosensitivity [14,18,21,22]. The effects of drugs that critically rely on TS inhibition to exert their cytotoxic action, such as 5Fluorouracil (5FU), raltitrexed, and pemetrexed [23,24] can be potentially increased by HDACi [25,26]
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