- Aim: To enhance the anticancer activity of vinorelbine, cisplatin and ionizing radiation (IR) combination against non-small cell lung cancer (NSCLC) cells by co-administration of sodium valproate (VPA), a histone deacetylase inhibitor, and to elucidate molecular events underpinning treatment efficacy. Materials and Methods: The NSCLC A549 cell line was treated with cisplatin (0.2 μg/ml), vinorelbine (2 nM), VPA (1 mM) and IR (2.5 Gy) alone, or in combination. Cell proliferation, cell- cycle distribution, apoptosis, and levels of DNA double- strand breaks, activated DNA damage checkpoint kinases pCHK1, pCHK2, cell-cycle inhibitors p21 CIP1/WAF1 and p27 KIP1 were assessed. Results: VPA markedly enhanced the DNA-damaging effect of the cisplatin-vinorelbine-IR combination and induced increased DSBs, and expression of pCHK2, pCHK1, p21 CIP1/WAF1 and p27 KIP1 . These molecular changes led to cell-cycle arrest and increased apoptosis and consequently markedly curtailed cancer cell growth. Conclusion: VPA markedly enhances the anticancer activity of cisplatin-vinorelbine-IR combination. This finding has translational implications for enhancing the efficacy of anticancer treatment and for reducing side-effects by reducing doses of radiation and drugs. Non-small cell lung cancer (NSCLC) accounts for 80%-85% of all lung cancer cases (1). A considerable number (up to 30%) of patients with NSCLC present with locally advanced, unresectable stage III disease (2). For these patients, concomitant chemoradiotherapy was shown to be the best strategy (3). Vinorelbine, a vinca alkaloid, in association with a platinum compounds such as cisplatin or carboplatin, together with thoracic radiotherapy is considered a relatively safe treatment regimen (4). However, treatment outcomes of NSCLC with concurrent chemoradiotherapy remain poor. Therefore, the search for new anticancer drug combinations for concurrent chemoradiotherapy of inoperable locally advanced NSCLC is mandatory and remains a key issue of public health concern. Pre-clinical research has demonstrated that histone deacetylase (HDAC) inhibitors exhibit anticancer activity against NSCLC cell lines when used alone or in combination with conventional chemotherapeutic drugs or radiotherapy (5- 7). Sodium valproate (VPA), a widely used anti-epileptic drug, also exhibits HDAC inhibitory activity and like other HDAC inhibitors exerts antitumor effects (8). The ability of VPA to sensitize cancer cells to the damaging effect of ionizing radiation (IR) and of other anti-neoplastic agents (9, 10) appears to be the most promising anticancer feature of this drug. The main aim of the present pre-clinical study was to assess whether concomitant VPA administration enhances the anticancer activity of the conventional cisplatin-vinorelbine- IR treatment. The study was performed using the human NSCLC cell line A549. To appraise the efficacy of the suggested combination, we determined (i) cell proliferation, cell-cycle distribution profile and apoptosis; (ii) the DNA damage response (DDR) by measuring levels of DNA double-strand breaks (DSBs) and of activated CHK1 and CHK2 checkpoint kinases; and (iii) assessed the expression levels of the cell-cycle regulatory proteins cyclin-dependent kinase inhibitors p21 CIP1/WAF1 and p27 KIP1 .