Aleksandra Barac, Goran Mitulovic, Seth Hallström, Sonja Zehetmayer, Matthaeus Ch Grasl, Boban M Erovic

Despite significant advances in diagnosis and therapy, the rate of survival of patients with oral cancers still remains poor as an appropriate treatment has not been found yet, due to side effects of chemo/radiotherapy. This study aimed to identify molecular mechanisms of cell death of oral cancer cells caused by treatment with a nonselective Cox-2 inhibitor in combination with a low-dose chemotherapeutic drug. Squamous cell carcinoma (SCC) cells SCC9 and SCC25 were subjected to mono- and combination therapy with nimesulide and cisplatin. Fluorescence-activated cell sorting (FACS), immunohistochemistry, high-pressure liquid chromatography (HPLC), microarray gene chips, and isobaric tags for a relative and absolute quantitation (iTRAQ) system were used. Increased numbers of apoptotic and necrotic SCC9/SCC25 cells were detected after combined exposure. ATP levels and the energy charge of SCC9 cells were significantly decreased after both individual and combined treatment. We detected and quantified a responsible gene, keratin 6a, and 540 relevant proteins. In SCC25 cells, ATP levels significantly decreased only after combination therapy. After combined treatment of SCC9 cells, significant upregulation of Histon-H2A/H2B/H4 was found, with a local discovery false rate of 0.003 for Histon-H2A and 0.0027 for Histon-H2B, respectively. Compared to the single-drug treatment, combined treatment of the oral cancer cells with nimesulide and cisplatin increases and induces necrosis and apoptosis through different pathways. A significant effect of the cytoplasmic increase was also observed in histones of cell lines SCC9 and SCC25 that were previously treated with combined nimesulide and cisplatin therapy.