as well as hypoxic nonreplicating Mtb.

the HA tagged ubiquitin expression plasmid was transfected into H460 cells. After 24h, cells were treated with increasing concentrations of rapamycin from 1nM to 1uM for 48h. A co immunoprecipitation was performed using a Bad antibody. Bad ubiquitination was analyzed by Western blot using anti HA antibody. reveal that rapamycin induces Bosutinib a dose dependent ubiquitination of Bad, which is characterized as the typical higher molecular weight smear of the polyubiquitin chains on Bad protein. Total cell lysate was used as input control before co IP. These findings suggest that rapamycin induced reduction in the halflife of Bad may occur through its ubiquitination and degradation. Inhibition of rapamycin induced Bad phosphorylation by PD98059 or depletion of AKT sensitizes lung cancer cells to rapamycin Our findings suggest that rapamycin induced Bad phosphorylation may inactivate its proapoptotic function. Inhibition of rapamycin induced Bad phosphorylation may restore the proapoptotic activity of Bad and sensitize lung cancer cells to rapamycin. To test this hypothesis, H460 parental cells, H460 cells expressing Akt shRNA or control shRNA were treated with rapamycin Papillary thyroid cancer in the absence or presence of PD98059. reveal that inhibition of MAPK ERK1/2 by PD98059 specifically blocks rapamycin induced S112 site phosphorylation of Bad but has no significant effect on Bad phosphorylation at S136 or S155. By contrast, depletion of AKT by RNA interference using Akt shRNA specifically blocks rapamycin induced S136 site phosphorylation and has no effect on Bad phosphorylation at S112 or S155. Intriguingly, simultaneous shutdown of MAPK/ERK1/2 and Akt by PD98059 and Akt shRNA blocks rapamycin stimulated phosphorylation of Bad at both S112 and S136 sites, which additively enhances rapamycin induced growth inhibition of human Cilengitide lung cancer cells. To test whether ERK and Akt inhibition is effective to reverse rapamycin resistance, A549 P and A549 RR cells were treated with PD98059 and/or transfected with Akt shRNA in the presence or absence of rapamycin for 48h. show that simultaneous inhibition of ERK and Akt not only significantly sensitizes A549 P cells to rapamycin but also reverses rapamycin resistance of A549 RR cells, suggesting that inhibition of Bad phosphorylation at S112 and S136 by blocking ERK and Akt signal pathways can reverse rapamycin resistance. Suppression of rapamycin induced Bad phosphorylation by PD98059 or depletion of Akt enhances anti tumor efficacy of rapamycin in lung cancer xenografts To further test whether blockage of rapamycin enhanced Bad phosphorylation increases rapamycins anti tumor efficacy in vivo, we generated lung cancer xenografts using H460 cells or H460 cells expressing Akt shRNA. Xenograft mice were randomly grouped and treated with rapamycin or PD98059 or the combination for two weeks as described in .