Epidermal growth factor and serum activate distinct pathways to inhibit the BH3 only protein BAD in prostate carcinoma LNCaP cells.

A better understanding of pathways involved in survival of prostate cancer cells is the key to develop effective and target-selective therapies. Presence of serum or epidermal growth factor in the culture medium of LNCaP cells decreases apoptosis induced by the inhibition of phosphatidylinositol 3-kinase with LY294002. However, intracellular pathway(s) involved in this survival signaling are not well defined. Here, we investigated the mechanism(s) involved in serum or epidermal growth factor-mediated inhibition of LY294002-induced death in LNCaP cells. Cell death was assessed by the percentage of cells in sub-G1 phase and caspase 3 activity. Phosphorylation status of BAD, ERK1/2 and RSKs were assessed by Western blot. Specific gene expression knock down of BAD, BAX, RSK1 and RSK2 were performed using siRNA transfections. Our results demonstrate that cell death induced by LY294002 is mediated by translocation of BAD and BAX proteins from the cytosol to the mitochondria. Whereas, epidermal growth factor activates a MAPK/ERK/RSK1 module leading to inactivation of BAD via Ser(75) phosphorylation, the presence of serum, on the other hand, induces a nonconducive intracellular environment for mitochondrial translocation of dephosphorylated BAD. Taken together, these results indicate that phosphorylation of BAD or inhibition of its translocation to the mitochondria are critical phosphatidylinositol 3-kinase-independent survival pathways in LNCaP cells.

Activation of the RacGTPase inhibits apoptosis in human tumor cells.

The small GTP-binding protein Rac is a downstream effector of the oncogene product p21-ras. Rac is involved in actin polymerization, Jun kinase activation, and intracellular superoxide anion production, through distinct pathways in tumor cells. Here we investigated the role of activated Rac in the response of tumor cells to apoptosis triggered by anti-cancer drugs or the cell surface death receptor CD95. Using M14 melanoma cells stably transfected with a constitutively active form of Rac1, we show that activated Rac inhibits tumor cell response to apoptosis. The inhibitory effect of activated Rac on apoptotic signaling is mediated by the interaction of Rac with intracellular oxidase and the subsequent production of superoxide, which is supported by experiments performed with M14 and NIH3T3 cells transiently transfected with the loss-of-function mutants of Rac in an activated RacV12 background. Consistent with these findings, we also demonstrate that inhibition of the Rac pathway in the HaRas-expressing T24 bladder carcinoma cell line induces a decrease in superoxide anion concentration, and results in a significant increase in tumor cell sensitivity to apoptosis. These findings demonstrate the existence of a novel Rac-dependent survival pathway mediated by intracellular superoxide in tumor cells.