Sodium Selenite as a Survival Agent: Signaling Mediated by PI3K, Akt and Rac1

• Main Authors: Yu-Chi, Lee, 李玉琦
• Other Authors: Ann-Ping Tsou
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/57387159993177795944

碩士 === 國立陽明大學 === 遺傳學研究所 === 89 === Reactive oxygen species (ROS) has been implicated as key molecules in the pathophysiology of many diseases and in various cellular responses. Selenium, which is an essential component of the selenoproteins behaves as both anti-oxidant and anti-cancer agent. Previously, selenium has been shown to sustain the growth of selected human hepatocellular carcinoma (HCC) cell lines in serum-free condition, but the detailed mechanism was undetermined. During last two years, our laboratory found that sodium selenite (Na2SO3, Se) can function as a survival-signaling molecule. Se (10-7 M) protects HuH7, a HCC cell line, from serum deprivation-induced apoptosis and also supports the long-term growth of HuH7 up to 15 months. The anti-apoptotic effect of Se involves a PI3K-dependent activation of Akt, suppression of caspases and inhibition of ASK1. Suppression of ASK1 activity appears to be a thiol redox-regulated mechanism. How Se achieves its anti-apoptotic activity, however, remained undetermined. The specific aim of my thesis is to elucidate the molecular mechanism(s) with which Se modulates the survival signaling upon oxidative stress in HuH7 cells. I assayed the activation of several key kinases and intracellular ROS upon oxidative stress. I also overexpressed several Rac1 mutants to explore the possibility that Rac1 activates PI3K under the influence of Se. Important observations from my thesis research strongly support that in the presence of Se, Rac1 acts as a key molecule in activating the Akt-directed survival signaling. In HuH7 cells, oxidative stress induced by serum-deprivation generated elevated amount of H2O2, which was rapidly reduced by Se treatment with a concomitant activation of Akt. Activation of Akt can be detected from 15 min to 24 hr post selenium treatment. Meanwhile, a fluctuation of ERK1/2 activation was noticed. Using antibody specific to phospho-Akt, phospho-ERK and phospho-Raf(S259), I observed a positive correlation between Akt activation and phosphorylation of Raf(S259) as well as an inverse correlation between the ERK activation and phosphorylation of Raf(S259). My findings thus suggested that Akt activation during Se treatment inactivated Raf kinase and resulted in a selective suppression of the ERK signaling pathway. It is intriguing, however, HuH7 cells conditioned to Se showed no sign of Akt activation but a sustained activation of ERK1/2. Whether the elevated ERK1/2 activity is essential for serum-independent growth of HuH7 remains to be determined. One critical downstream targets of Akt is Bad. Its phosphorylation at S136 inhibits further transmission of death signal to caspases. Using specific antibody to phosph-S136 of Bad, I detected a direct correlation of Akt activity and phosphorylation of Bad at S136, which further strengthened the notion of anti-apoptotic function of Akt in Se-treated cells. HuH7 cells expressing Rac1(N17) mutant failed to respond to Se-mediated Akt activation. In those cells, activation of Akt was only detected with high concentration of H2O2 (15 mM), suggesting an involvement of ROS in activating the PI3K-Akt pathway. One-day after serum-deprivation, selenium supplement strongly enhanced the GTP-binding of Rac-1 in HuH7 cells transiently transfected with WT-Rac1. Under the same culture condition, more endogenous Rac1 was also detected in a complex with activated PI3K. The active state of PI3K was revealed by tyrosine phosphorylation of the regulatory subunit of p85. An interesting finding from this study is that the Rac1-overexperessed HuH7 cells did not show exceedingly high level of ROS. My preliminary results seem to suggest a possibility that selenium is able to redirect Rac1 from activating NADPH-oxidase for ROS production to interact with PI3K for its activation. Such interaction allows the subsequent activation of Akt and the consequential anti-apoptotic signaling for cell survival. My thesis work further supported the fact that selenium can function as a signal transduction-mimetic element in regulating signaling kinases, anti-apoptosis and cell proliferation. Hepatitis B virus infection remains to be one of the highest risk factors for hepatocarcinogenesis in certain parts of the world. Accumulating evidence suggests that local, elevated concentration of cytokines such as TNFa and interferon-g can contribute to chronic, low-dose and/or sub-lethal damage for the hepatocytes of those chronic carriers. The Se-HuH7 cell system established in our laboratory thus provides a unique tool to address the biological roles of Se as a chemopreventive agent and a cell growth regulator.