We next tested whether ROS scavenging by TBHQ affected the transforming capabilities of tMSC. TBHQ significantly impaired the growth of tMSC, but not that of immortal MSC3. Furthermore, selleckchem Ruxolitinib treatment with TBHQ Inhibitors,Modulators,Libraries decreased anchorage independent growth of both tMSC and tHMEC measured by soft agarose colony formation. These results suggest that loss of Nrf2 expression contri butes to both accumulation of intracellular ROS, and to MSC in vitro transformation. Restoration of Nrf2 expression in tMSC induces the cellular antioxidant response and impairs in vivo tumor growth To validate the observed effect of TBHQ in our model, we genetically over expressed Nrf2 in transformed MSC. tMSC over expressing Nrf2 exhibited increased transcrip tion of ARE containing genes and antioxidant Inhibitors,Modulators,Libraries enzymes.
Activation of the Nrf2 pathway was con firmed by increased expression of Nrf2 and NQO1 pro teins. Furthermore, tMSC over Inhibitors,Modulators,Libraries expressing Nrf2 showed an increase in the pool of reduced gluta thione and a decrease in intracellular ROS. Next, we investigated how Nrf2 mediated reduction in ROS levels affected the transformation capability of tMSC. Over expression of Nrf2 led to a slight, but significant reduction in tMSC viability and soft agarose growth when compared with tMSC expressing empty vector. Next we questioned whether these cells could respond differentially when they encounter physiological conditions in vivo. Hence we inoculated tMSC over expressing Nrf2 or empty vector into nude mice. While all mice from the empty vector group showed rapidly growing tumors, only three out of six mice from the Nrf2 group produced tumors, and these after a significantly longer latency.
Nrf2 over expression sensitizes tMSC to apoptosis and diminishes the angiogenic response by destabilization Inhibitors,Modulators,Libraries of HIF 1 and VEGF repression Due to the different responses observed in vitro and in vivo, we challenged the cells to a variety of stressors in order to mimic aspects of the in vivo tumor microenviron ment. We found that tMSC over expressing Nrf2 exhibited more apoptotic cells when compared with control Inhibitors,Modulators,Libraries cells after double staining with Annexin V and Propi dium Iodide. Furthermore, Nrf2 sensitized cells to apoptosis induced by the DNA damaging agent camptothecin as mea sured by staining with Annexin V and Propidium Iodide, by accumulation of cleaved PARP protein, and by increased caspase 3 and 7 activity.
Like wise, cells over expressing Nrf2 showed increased cyto further information toxicity following treatment with the apoptotic inducers etoposide and the ATP competitive kinase inhibitor staurosporine. ROS are implicated in the response to hypoxia through a mechanism involving stabilization of hypoxia inducible factor 1. Interestingly, tMSC over expressing Nrf2 were not able to stabilize HIF 1 at 1% O2 concentra tion.