الفهرس 
Possible modulation of necroptosis by cisplatin and a tyrosine kinase inhibitor in hepatocyte G2 cancer cell line
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Abstract
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Systemic chemotherapy such as cisplatin and multi-targeted receptor tyrosine kinase inhibitors (RTKs), including sunitinib, has marginal activity and frequent toxicity and is not associated with improved survival. Recently, triggering necroptosis has been proposed to be a novel therapeutic strategy in HCC. The purpose of this study is to evaluate the effect of combining sunitinib and cisplatin on HepG2 cells regarding its cytotoxicity and the implicated intracellular pathways. Materials and methods: The half-maximal inhibitory concentration (IC50) values of cisplatin, sunitinib, and their combination were determined by Sulforhodamine-B (SRB) assay. Apoptotic markers Bcl2 and Bax protein levels were determined using western blot analysis. Enzyme-linked immunosorbent assay (ELISA) technique was used to measure total receptor-interacting protein kinase 3 (RIPK3), phosphorylated RIPK3 (pRIPK3), and total extracellular signal-regulated kinase (ERK), phosphorylated ERK (pERK), caspase-9, caspase-8. Malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH peroxidase). Results: Cisplatin-sunitinib combination (4 μg/ml cisplatin + 2.8 μg/ml sunitinib) exhibited a superior cytotoxic effect on HepG2 cells with lower IC50 values than either of the drug alone (cisplatin and sunitinib). Moreover, the combination showed significant Bcl2 downregulation with a 53% and Bax upregulation with a 3.2-fold increase in protein expression yielding a Bax/Bcl2 ratio of 1.6 in Western Blot (WB) analysis. Combined treatment also produced a 1.9-fold increase in total RIPK3 while reducing pRIPK3 by 48.7% with lowered pRIPK3/RIPK3 by 74% (p < 0.05) compared to control. Significant reduction of total ERK by 33.5% and increase of pERK by 2.8 folds (P<0.05) with significant increase in pERK/ERK by 3.9 folds over the normal control was also demonstrated. Moreover, combined treatment produced a significant 4- and 4.6-folds increase in caspase-9 and -8 levels respectively. An increase in MDA level by 1.3 folds, a decrease in the intracellular GSH level by 63%, and an increase in GSH peroxidase level by 1.17 folds were demonstrated. Conclusion: Sunitinib modulated cisplatin effect on cytotoxicity, oxidative stress, apoptosis, necroptosis and MAPK pathways. Sunitinib enhanced cisplatin-induced apoptosis and increased oxidative stress, but decreased necroptosis. Combined cisplatin and sunitinib might be promising for treating advanced HCC.