Autophagy Induced By Altered Oxygen Tension And Its Impact On The Behavior Of MDA-MB-231 Tumor Cells

ObjectiveTo investigate the autophagic activity induced by altered oxygen tension in the microenvironment and its impact on the survival,proliferation and migration of human breast carcinoma cell MDA-MB-231.MethodsMDA-MB-231 cells were cultured under hypoxic conditions for different periods of time and then returned to normoxic cultures.The autophagic activity was analyzed by measuring the levels of LC3-I?LC3-II and p62 using Western blotting.The mitochondrial content,ROS production and cell apoptosis were assessed by flow cytometry.Cell survival and proliferation was determined by colony formation assay.Transwell assay was performed to examine cell invasion capacity.Firstly,we monitored the changes in autophagy-associated markers following brief culture in EBSS.As expected,exposure to EBSS resulted in the elevation of LC3-II with a concomitant reduction of p62,verifying the reliability of the experimental systems.Subsequently,systemic analyses were performed to detect changes induced by reoxygenation following short-or long-term hypoxic cultures.Reoxygenation after short-term hopoxia led to elevated levels of autophagic activity,mitochondrial contents and ROS production,which was accompanied by increased cell apoptosis.Notably,treatment with rapamycin,apotent inducer of autophagy,improved cell survival.Increased levels of autophagy were also observed after long-term culture under hypoxic conditions,which gradually returned to basal levls after reoxygenation.Colony formation assay demonstrated that hypoxia-reoxygention caused a marked decrease in the capacity of colony formation.Such a capacity was further reduced in the presence of autophagy inhibitors.On the other hand,induction of autophagy was coupled with enhanced cell invasion.ConclusionsThe increased autophagic activity after short-term hypoxia-reoxygenation is probably a response to mitochondrial damage and overproduction of ROS and has a beneficial effect on cell survival.Long-term hypoxia by itself is able to induce high levels of autopahgy,which is suppressed by reoxygenation.While both short-and long-term hypoxia impairs colony formation,increased autophagic activity facilitates cell invasion.