Activation of ER stress and apoptosis by hydrogen peroxide in HeLa cells: Protective role of mild heat preconditioning at 40 °C

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) during stress conditions causes activation of the unfolded protein response (UPR). If this adaptive response cannot restore ER homeostasis, cells undergo ER-mediated apoptosis. This study determines whether thermotolerance developed at a mild temperature (40 degrees C) can alter induction of ER-mediated stress and apoptosis by H2O2 in HeLa cells. Protein expression of PERK, p-PERK, eIF2 alpha and p-eIF2 alpha was increased in thermotolerant compared to non-thermotolerant cells. Thus, mild thermotolerance enhanced pro-survival effects of the PERK/eIF2 alpha branch of the UPR. A short exposure (15 min) of cells to H2O2 (15-50 mu M) activated the UPR: expression of p-PERK, p-eIF2 alpha and p-IRE1 alpha increased, and ATF6 cleavage occurred. Longer exposure (1-3 h) to H2O2 induced ER-mediated apoptosis, whereby CHOP expression increased, and enzymatic activity of calpain, caspase-7, -4, -12 and -9 also increased. These pro-apoptotic events and clonogenic cell killing were all diminished in thermotolerant cells. Activation of caspases-4/-12 was decreased by the calcium chelator BAPTA-AM, and by inhibitors of calpain and caspase-7, confirming the roles of calcium, calpain and caspase-7 in activation of ER-mediated apoptosis by H2O2. In thermotolerant cells with decreased levels of PERK by siRNA, there was partial reversal of resistance to H2O2-induced apoptosis. Hence, a causal connection exists between the ER stress response and resistance to H2O2-induced apoptosis. Mild thermotolerance plays a protective, anti-apoptotic role by increasing the threshold for induction of ER-mediated apoptosis by H2O2. Moreover, the adaptive response (UPR) dominates during milder H2O2 stress, whereas ER-mediated apoptosis occurs during more severe stress. (C) 2011 Elsevier B.V. All rights reserved.