p66shc is a protein product of an mRNA isoform of SHC1 gene that has a pro-oxidant and pro-apoptotic activity and is implicated in the aging process. Mitochondria were suggested as a major source of the p66shc-mediated production of reactive oxygen species (ROS), although the underlying mechanisms are poorly understood. We studied effects of p66shc on oxidative stress induced by hydrogen peroxide or by serum deprivation in human colon carcinoma cell line RKO and in diploid human dermal fibroblasts (HDFs). An shRNA-mediated knockdown of p66shc suppressed and an overexpression of a recombinant p66shc stimulated the production of ROS in the both models. This effect was not detected in the mitochondrial DNA-depleted ρ0-RKO cells that do not have the mitochondrial electron transport chain (ETC). The p66shc-dependent accumulation of mitochondrial ROS was detected with HyPer-mito, a mitochondria-targeted fluorescent protein sensor for hydrogen peroxide. The fragmentation of mitochondria induced by mitochondrial ROS was significantly reduced in the p66shc deficient RKO cells. Mitochondria-targeted antioxidants SkQ1 and SkQR1 also decreased the oxidative stress induced by hydrogen peroxide or by serum deprivation. Together the data indicate that the p66shc-dependant ROS production during oxidative stress has mitochondrial origin in human normal and cancer cells.

An acquisition of increased sensitivity of cancer cells to viruses is a common outcome of malignant progression that justifies the development of oncolytic viruses as anticancer therapeutics. Studying molecular changes that underlie the sensitivity to viruses would help to identify cases where oncolytic virus therapy would be most effective. We quantified changes in protein abundances in two glioblastoma multiforme (GBM) cell lines that differ in the ability to induce resistance to vesicular stomatitis virus (VSV) infection in response to type I interferon (IFN) treatment. In IFN-treated samples we observed an up-regulation of protein products of some IFN-regulated genes (IRGs). In total, the proteome analysis revealed up to 20% more proteins encoded by IRGs in the glioblastoma cell line, which develops resistance to VSV infection after pre-treatment with IFN. In both cell lines protein-protein interaction and signaling pathway analyses have revealed a significant stimulation of processes related to type I IFN signaling and defense responses to viruses. However, we observed a deficiency in STAT2 protein in the VSV-sensitive cell line that suggests a de-regulation of the JAK/STAT/IRF9 signaling. The study has shown that the up-regulation of IRG proteins induced by the IFNα treatment of GBM cells can be detected at the proteome level. Similar analyses could be applied for revealing functional alterations within the antiviral mechanisms in glioblastoma samples, accompanying by acquisition of sensitivity to oncolytic viruses. The approach can be useful for discovering the biomarkers that predict a potential sensitivity of individual glioblastoma tumors to oncolytic virus therapy.

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