SINP Triennial Report - Saha Institute of Nuclear Physics

Biophysical Sciences Including Chemistry 27
Akt. Furthermore, exposure to SB203580 also blocked the increase in Hsp70 and MnSOD levels and
the elevated SOD activity brought about by chronic heat stress. Heat shock factor 1 (HSF1)
transcriptional activity and nuclear translocation of HSF1 were prominently augmented by chronic heat
stress, and this amplification is markedly reduced by concomitant exposure to SB203580. Also,
activations of p38MAPK and Akt and upregulations of Hsp70 and MnSOD were observed on exposure
to heat shock for a single exposure of longer duration (40 min). siRNA against p38MAPK notably
reduced Akt phosphorylation by single exposure to heat stress and drastically diminished the rise in
Hsp70 and MnSOD levels. Similarly, siRNA against Akt also eliminated the augmentation in Hsp70
and MnSOD levels but p38MAPK levels remained unaffected. Heat stress produced reactive oxygen
species (ROS) in V79 fibroblasts. N-acetyl cysteine blocked the increase in phosphorylation of
p38MAPK, amplification of Hsp70, and MnSOD levels by heat stress. Therefore, we conclude that
heat stress-activated p38MAPK which in turn activated Akt. Akt acted downstream of p38MAPK to
increase Hsp70 and MnSOD levels.
Concise summary: Thermal injury of the skin over a long period of time has been associated with
development of cancerous lesions. Also, in many cancers, the cytoprotective genes Hsp70 and MnSOD
have been found to be overexpressed. Therefore, we considered it important to identify the signaling
elements upstream of the upregulated survival genes in heat stress. We conclude that heat stress activated
p38MAPK which in turn activated Akt. Akt mediated an augmentation in Hsp70 and MnSOD levels
working downstream of p38MAPK.
Mustafi, Soumyajit Banerjee; Chakraborty, Prabir Kumar; Dey, Rakhi Sharma; Raha, Sanghamitra
Pro-survival effects of repetitive low-grade oxidative stress are inhibited by simultaneous exposure
to Resveratrol
V79 lung fibroblasts were subjected to repetitive oxidative stress in culture through exposures to 30
mu M H2O2 for 4 weeks. Repetitively stressed cells were found to be significantly resistant to apoptosisinducing
agent such as ultraviolet radiation (UVR). Concurrent treatment with Resveratrol completely
restored the normal apoptotic response after UVR. p38MAPK became dually phosphorylated during
the stress period. Akt also became phosphorylated on Ser(473) in cells subjected to repetitive oxidative
stress. In these cells, NF kappa B p65 became phosphorylated and appreciable nuclear localization of
p65 was observed. NF kappa B transcriptional activity also became augmented during repetitive stress.
Treatment of the repetitively stressed cells concurrently with Resveratrol or SB203580, a p38MAPK
inhibitor, robustly blocked activation of p38MAPK, NF kappa B transcriptional activity, phosphorylation
and nuclear localization of p65, and Akt phosphorylation. Pre-exposure to short interfering RNA (si
RNA) to p38MAPK, resulted in a blockage of the Akt and NF kappa B p65 phosphorylation. However,
inhibition of Akt activity through P13 kinase inhibitor LY294002 did not result in obstruction of
p38MAPK phosphorylation by H2O2. Also, Resveratrol was effective as an antioxidant in counteracting
a rise in reactive oxygen species (ROS) and p38MAPK activation by H2O2 was completely blocked
by antioxidant N-acetyl cysteine (NAC). We conclude that Resveratrol acts as an antioxidant and
completely reverses the anti-apoptotic effects of repetitive stress by blocking oxidative stress-induced
p38MAPK activation which is the key regulatory step for the activation of down-stream survival elements
Akt and NF kappa B.
Chakraborty, Prabir Kumar; Mustafi, Soumyajit Banerjee; Raha, Sanghamitra