Abstract 1206: Superoxide-mediated regulation of Na+/H+ exchanger set point and increase in intracellular BAD kinase activity: two redox-dependent survival pathways independent of the survival kinase Akt
Authors: Goh Shijie , Ping Yueh Shyang , Clement Marie-Veronique 
Published: February 2011

Abstract
The LNCaP cell line is the most widely used in vitro model of prostate cancer. LNCaP cell line contains a frameshift mutation in the PTEN gene leading to constitutive activation of Akt in the absence of growth factor stimulation. However, inhibition of the PI3K/Akt pathway by PI3K inhibitor LY294002 (LY) in presence of 10% FBS does not induce significant apoptosis in LNCaP cells. Apoptosis is only detected when PI3K/Akt pathway is inhibited in serum-free medium, suggesting the existence of a serum-dependent PI3K-independent survival pathway. However the nature of such a pathway is still unclear. Two important hallmarks of tumor cells are: an acquired defect in the death execution pathway(s) and a pro-oxidant intracellular milieu. Our laboratory has shown that these two characteristics are not coincidental but may be tightly linked. We found that modulation of intracellular levels of reactive oxygen species; in particular superoxide anion (O2−) regulates cellular response to apoptosis. Using diphenyleneiodonium (DPI), an efficient inhibitor of intracellular O2− production, we investigated if a decrease in O2− could revert the serum-dependent resistance to apoptosis induced by LY in the prostate cancer cell line LNCaP. Our results demonstrate that indeed DPI sensitizes LNCaP cells to LY-mediated apoptosis in the presence of serum. The sensitization was associated with a decrease in the set point of Na+/H+ exchange sensing and dephosphorylation of the BH3 only protein BAD. Decrease in the set point of Na+/H+ exchange sensing led to a dramatic intracellular acidification at steady state as well as following LY treatment. Increase in the inhibition of BAD phosphorylation on ser75 was assessed using specific BADser75 kinase assay in total cell lysate. The role of BAD in LNCaP cell death upon exposure to DPI and LY was shown by inhibition of BAD expression using BAD specific siRNA. Silencing of BAD in LNCaP prevented apoptosis induced by LY in the presence or absence of DPI. This demonstrates the critical role of BAD dephosphorylation in the induction of apoptosis in serum-free medium as well as in DPI containing medium in the presence of LY. Maintenance of intracellular level of O2− upon exposure of the cells to DPI and LY using the inhibitor of Cu/Zn SOD, diethyldithiocarbamate (DDC) prevented the decrease in the set point of Na+/H+ exchange sensing, the acidification of the intracellular milieu as well as the decrease in BAD kinase activity and apoptotic cell death. Finally, supporting the role of DPI on Na+/H+ exchange, EiPa, a Na+/H+ exchanger inhibitor also prevented the inhibition of apoptosis by serum upon exposure of the cells to LY. Taken together, these results suggest that regulation of Na+/H+ pH sensing and level of BAD kinase activity are novel targets for redox-mediated survival in a PI3K/Akt independent manner.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1206.