Vedam K, Nishijima Y, Druhan LJ, Khan M, Moldovan NI, Zweier JL, Ilangovan G. Role of heat shock factor-1 activation in the doxorubicin-induced heart failure in mice.

Transcriptional activation of p53 target genes, due to DNA damage, causes either apoptosis or survival by cell cycle arrest and DNA repair. However, the regulators of the choice between cell death and survival signaling have not been completely elucidated. Here, we report that human adenocarcinoma cells (MCF-7) survive UV-induced DNA damage by heat shock protein 27 (Hsp27)-assisted Akt/p21 phosphorylation/translocation. Protein levels of the p53 target genes, such as p21, Bcl-2, p38MAPK, and Akt, showed a positive correlation to Hsp27 level during 48 hours postirradiation, whereas p53 expression increased initially but started decreasing after 12 hours. Hsp27 prevented the G 1 -S phase cell cycle arrest, observed after 8 hours of post-UV irradiation, and PARP-1 cleavage was inhibited. Conversely, silencing Hsp27 enhanced G 1 -S arrest and cell death. Moreover, use of either Hsp27 or Akt small interference RNA reduced p21 phosphorylation and enhanced its retention in nuclei even after 48 hours postirradiation, resulting in enhanced cell death. Our results showed that Hsp27 expression and its direct chaperoning interaction increases Akt stability, and p21 phosphorylation and nuclear-to-cytoplasm translocation, both essential effects for the survival of UV-induced DNA-damaged cells. We conclude that the role of Hsp27 in cancer is not only for enhanced p53 proteolysis per se, rather it is also a critical determinant in p21 phosphorylation and translocation. Mol Cancer Res; 8(10); 1399-412. ©2010 AACR.

Heat-shock factor 1 (HSF-1), a transcription factor for heat-shock proteins (HSPs), is known to interfere with the transcriptional activity of many oncogenic factors. In the present work, we have discovered that HSF-1 ablation induced the multidrug resistance gene, MDR1b, in the heart and increased the expression of P-glycoprotein (P-gp, ABCB1), an ATP binding cassette that is usually associated with multidrug-resistant cancer cells. The increase in P-gp enhanced the extrusion of doxorubicin (Dox) to alleviate Dox-induced heart failure and reduce mortality in mice. Dox-induced left ventricular (LV) dysfunction was significantly reduced in HSF-1 −/− mice. DNAbinding activity of NF-κB was higher in HSF-1 −/− mice. IκB, the NF-κB inhibitor, was depleted due to enhanced IκB kinase (IKK)-α activity. In parallel, MDR1b gene expression and a large increase in Pgp and lowering Dox loading were observed in HSF-1 −/− mouse hearts. Moreover, application of the P-gp antagonist, verapamil, increased Dox loading in HSF-1 −/− cardiomyocytes, deteriorated cardiac function in HSF-1 −/− mice, and decreased survival. MDR1 promoter activity was higher in HSF-1 −/− cardiomyocytes, whereas a mutant MDR1 promoter with heat-shock element (HSE) mutation showed increased activity only in HSF-1 +/+ cardiomyocytes. However, deletion of HSE and NF-κB binding sites diminished luminescence in both HSF-1 +/+ and HSF-1 −/− cardiomyocytes, suggesting that HSF-1 inhibits MDR1 activity in the heart. Thus, because high levels of HSF-1 are attributed to poor prognosis of cancer, systemic down-regulation of HSF-1 before chemotherapy is a potential therapeutic approach to ameliorate the chemotherapy-induced cardiotoxicity and enhance cancer prognosis.dilated cardiomyopathy | oxidative stress | mouse model of cardioprotection | chemotherapeutics A nthracyclines such as doxorubicin (Dox) and its derivatives are used as antineoplastics, either alone or in combination with other drugs, to treat many types of cancer. Although Dox is an effective chemotherapeutic drug, it has long been known to cause severe cardiotoxicity, leading to dilated cardiomyopathy and congestive heart failure (1). Despite recent improvements of the drug, clinical manifestation of cardiac dysfunction among Dox-treated cancer patients still persists as a serious side effect. Other classes of chemotherapeutics, including tyrosine kinase inhibitors (TKIs) such as imatinib mesylate and humanized antibody-based therapeutics such as trastuzumab have also been found to cause significant cardiotoxicity (2, 3). In the case of Dox, the cardiotoxicity has been considered to be primarily due to oxidative-stress-induced death of cardiomyocytes and subsequent irreversible myocardial remodeling. However, many clinical trials testing the effectiveness of antioxidants indicated no significant cardioprotection during Dox treatment (4). Thus, alternate strategies, based on other identified pathways, are warranted to overcome this deleterious effect. Recent studies have identified several pathways of D...

In the present work, we have observed that exposure of bovine aortic endothelial cells (BAECs) to extreme hypoxia (1-5% O 2) attenuates cellular respiration by a mechanism involving heat shock protein 90 (Hsp90) and endothelial nitric oxide (NO) synthase (eNOS), so that the cells are conditioned to consume less oxygen and survive in prolonged hypoxic conditions. BAECs, exposed to 1% O 2, showed a reduced respiration compared with 21% O 2-maintained cells. Western blot analysis showed an increase in the association of Hsp90-eNOS and enhanced NO generation on hypoxia exposure, whereas there was no significant accumulation of hypoxia-inducible factor-1␣ (HIF-1␣). The addition of inhibitors of Hsp90, phosphatidylinositol 3-kinase, and NOS significantly alleviated this hypoxiainduced attenuation of respiration. Thus we conclude that hypoxiainduced excess NO and its derivatives such as ONOO Ϫ cause inhibition of the electron transport chain and attenuate O 2 demand, leading to cell survival at extreme hypoxia. More importantly, such an attenuation is found to be independent of HIF-1␣, which is otherwise thought to be the key regulator of respiration in hypoxia-exposed cells, through a nonphosphorylative glycolytic pathway. The present mechanistic insight will be helpful to understand the difference in the magnitude of endothelial dysfunction.oxygen; electron paramagnetic resonance oximetry; heat shock protein 90; endothelial nitric oxide synthase IN TISSUES, HYPOXIA is the state of insufficient O 2 , caused by inadequate transport or an excess consumption of O 2 . Duration, frequency, and severity of hypoxia strongly influence whether the effect is detrimental or beneficial (20, 43). Interestingly, hypoxia-exposed cells do not always undergo cell death or diminish ATP levels (11), therefore leading to cell survival and normal cell function. Many factors that are activated upon hypoxia, such as the hypoxia-inducible transcription factor (HIF-1), heat shock protein 90 (Hsp90), nitric oxide (NO) synthase (NOS), and reactive oxygen species (11, 13), determine whether the cells survive after salvage or succumb to death (2). Particularly, the role of NO during and after hypoxia in the endothelium has been found to be important in maintaining oxygen metabolism (31). NO is a multifaceted endogenous factor and is involved in many pathophysiological processes in cells. In vitro studies have shown that NO production is increased in hypoxic conditions (9, 31). NO has been found to inhibit the mitochondrial electron transport chain (ETC) (8, 14, 15). Thus it is an endogenous modulator of cellular respiration in different pathogenic conditions. While NO restrains cytochrome-c oxidase (CcO) by competing for the oxygen binding site at the heme of the enzyme, its derivative, peroxynitrite (ONOO Ϫ ), blocks the other ETC complexes mainly by S-nitrosation (25). Moreover, intermittent or acute hypoxia exposure is known to cause an increase in Hsp90 binding to endothelial NOS (eNOS), to facilitate Ser1177 phosphorylation (4, 13, 43). Thus...

Uncoupling of NO production from NADPH oxidation by endothelial nitric-oxide synthase (eNOS) is enhanced in hyperglycemic endothelium, potentially due to dissociation of heat shock proteins 90 (Hsp90), and cellular glucose homeostasis is enhanced by a ROS-induced positive feed back mechanism. In this study we investigated how such an uncoupling impacts oxygen metabolism and how the oxidative phosphorylation can be preserved by heat shock (42°C for 2 h, hyperthermia) in bovine aortic endothelial cells. Normal and heat-shocked bovine aortic endothelial cells were exposed to normoglycemia (NG, 5.0 mM) or hyperglycemia (30 mM). With hyperglycemia treatment, O 2 consumption rate was reduced (from V O 2 max ‫؍‬ 7.51 ؎ 0.54 to 2.35 ؎ 0.27 mm Hg/min/10 6 cells), whereas in heat-shocked cells, O 2 consumption rate remained unaltered (8.19 ؎ 1.01 mm Hg/min/10 ؋ 10 6 cells). Heat shock was found to enhance Hsp90/endothelial NOS interactions and produce higher NO. Moreover, ROS generation in the hyperglycemic condition was also reduced in heat-shocked cells. Interestingly, glucose uptake was reduced in heat-shocked cells as a result of decrease in Glut-1 protein level. Glucose phosphate dehydrogenase activity that gives rise to NADPH generation was increased by hyperthermia, and mitochondrial oxidative metabolism was preserved. In conclusion, the present study provides a novel mechanism wherein the reduced oxidative stress in heat-shocked hyperglycemic cells down-regulates Glut-1 and glucose uptake, and fine-tuning of this pathway may be a potential approach to use for therapeutic benefit of diabetes mellitus. Heat shock proteins (Hsps)2 play critical roles in endothelial function in the hyperglycemic state, although their exact function is not clearly established. Along with hyperglycemia, the production of superoxide, insulin resistance, and a decline in vascular bioavailability of nitric oxide (NO) contribute to extreme rates of morbidity and mortality (1-4). Various factors regulate endothelial nitric-oxide synthase (eNOS) signaling in endothelial cells by modulating its phosphorylation/de-phosphorylation dynamics and the coupling/uncoupling of its redox reactions. Protein kinase B (Akt) phosphorylates eNOS on serine 1177 and increases the production of NO (5), and it is found to be very critical in overall glucose metabolism and cell survival (6). Hsp90 binding to eNOS was found to be a prerequisite for successive Akt-mediated stimulation of eNOS, and indeed, Hsp90 can be considered as a scaffold between eNOS and Akt (5,7,8). Vascular endothelial growth factor enhances the recruitment of Hsp90 and facilitates Akt-dependent phosphorylation of eNOS, which results in increased NO production. NO generation is reduced under diabetic conditions due to NOS uncoupling (i.e. incomplete redox transformation of NADPH, arginine, and O 2 to NO, citrulline, and H 2 O, leading instead to the generation of reactive oxygen species) resulting in the generation of more superoxide, and Hsp levels, especially Hsp90, are reduced in hypergly...