Concurring with these results, tMSC showed the lowest levels of the active form of glutathione, the form of glutathione able to provide antioxidant power. Overall, these data indicate that transformation of MSC leads to a global transcriptional selleck chemical Z-VAD-FMK down regulation of the cellular antioxidant program. Nrf2 is repressed during cellular transformation via activation of RASRAFERK pathway Western blot experiments confirmed suppression of Nrf2 expression and its downstream target NQO1 that correlated with ST and H RasV12 induced activation of ERK and AKT pathways. To investigate the mechanism of Nrf2 repression during transformation, we focused in the last transformation step where the more pronounced down regulation of Nrf2 and ARE containing genes occurred.

We studied the roles of RAS and some RAS downstream effectors by expressing con stitutive active mutants of H RAS, RAF 1, and AKT in immortal MSC4. We found that activation of RAS and RAF, but not AKT, led to decreased expression of Nrf2 and NQO1. Recent reports showed that Nrf2 Inhibitors,Modulators,Libraries expression was de creased in certain human breast cancer cells and breast tumors when compared with normal mammary epithe lial cells or normal breast tissue. Interestingly, we found a reduction in Nrf2 and NQO1 expression when normal human mammary epithelial cells were transformed using the same oncogenic elements that we employed to transform MSC, suggesting that this mechanism for Nrf2 regulation is not restricted to adult MSC. Next we used chemical inhibitors to address whether Nrf2 expression is transcriptionally regulated via ERK or PI3KAKT pathways in the breast cancer cell lines MDA MB 231 and Inhibitors,Modulators,Libraries MCF 7.

While cell survival was not affected by the concentration of Inhibitors,Modulators,Libraries inhibitors used in this assay, treatment with the ERK inhibitor U0126 led to a significant increase in the transcription of Nrf2 and NQO1. How ever, inhibition of AKT with GSK690693, or PI3K with LY294002 and wortmannin did not induce expression Inhibitors,Modulators,Libraries of Nrf2 nor NQO1. The effect of these in hibitors on ERK and PI3KAKT Inhibitors,Modulators,Libraries pathways is shown in Figure 3E, where a modest but consistent activation of the Nrf2 pathway could be detected following only 16 hours treatment with U0126. Overall our data indicate that the RASRAFERK pathway mediates Nrf2 repres sion in these cancer cells. Nrf2 activity was found suppressed in tumor cells due to increased expression of the ubiquitin ligase Cul3 that, together with Keap1, targets Nrf2 for degradation by the proteasome.

However, expression of Keap1 and Cul3 did not increase in transformed MSC. Nrf2 protein stabilization by means of tert butylhydroquinone impairs MSC transformation To investigate whether Nrf2 down regulation contributes to increased ROS, we induced Nrf2 in tMSC by phosphatase inhibitor TBHQ, a chemical that stabilizes Nrf2 protein by impairing its pro teasomal degradation.