engleski

The role of de novo expressed sirtuin 3 in triple negative human breast cancer cells upon oxidative stress

Hyperoxic treatment (inducer of reactive oxygen species) was shown to support some tumorigenic properties, but finally suppresses growth of certain mammary carcinoma cells. While estrogen receptor α cancers are more receptive to hormonal therapy, triple negative breast cancers (TNBC) are characterized by an aggressive behaviour and the lack of targeted therapeutic strategies. Sirtuin 3 (Sirt3), a major mitochondrial NAD+-dependent deacetylase, has bifunctional role in cancer tumorigenesis, acting as both oncogene and tumor suppressor, depending on the tissue and cancer-type specific metabolic programs. Due to strikingly reduced Sirt3 level in many breast cancer cells, we hypothesized it would have tumor suppressive effect in TNBC cells. Therefore, we stably transfected MDA-MB-231 cells with Flag- tagged Sirt-3 or empty plasmid, as confirmed by real time PCR, western blot analysis and confocal microscopy. We found the optimal duration of hyperoxic treatment which induces DNA damage in the cells but macroscopically does not influence the vitality of the cells. We are using western blot to monitor the expression of proteins involved in mitochondrial biogenesis, glycolysis, metabolic regulation and antioxidant defence. To assess the differences in the growth rate and metabolic activity upon expression of Sirt3 and normoxic/hyperoxic treatment, we are using MTT test and colony-forming cell assay. Furthermore, we are checking the mitochondrial function through cellular respiration and FACS analysis, by monitoring the mitochondrial mass, mitochondrial potential and ROS production. The surprising finding that Sirt-3 markedly promoted growth of MDA-MB-231 cells in normoxic conditions, whereas hyperoxia markedly inhibited the growth of the Sirt3- overexpressing cells compared to control cells gave us a rationale for more detailed studies on Sirt3 and hyperoxia as an adjuvant tumor therapy in TNBC cells.

hyperoxia ; MDA-MB-231 ; oxidative stress ; breast cancer ; sirtuin 3

nije evidentirano