KRCC1: A potential therapeutic target in ovarian cancer.

Using a systems biology approach to prioritize potential points of intervention in ovarian cancer, we identified the lysine rich coiled-coil 1 (KRCC1), as a potential target. High-grade serous ovarian cancer patient tumors and cells express significantly higher levels of KRCC1 which correlates with poor overall survival and chemoresistance. We demonstrate that KRCC1 is predominantly present in the chromatin-bound nuclear fraction, interacts with HDAC1, HDAC2, and with the serine-threonine phosphatase PP1CC. Silencing KRCC1 inhibits cellular plasticity, invasive properties, and potentiates apoptosis resulting in reduced tumor growth. These phenotypes are associated with increased acetylation of histones and with increased phosphorylation of H2AX and CHK1, suggesting the modulation of transcription and DNA damage that may be mediated by the action of HDAC and PP1CC, respectively. Hence, we address an urgent need to develop new targets in cancer.

Characterization of an umbilical cord blood sourced product suitable for allogeneic applications.

Aim: Umbilical cord blood (UCB) sourced allografts are promising interventions for tissue regeneration. As applications of these allografts and regulations governing them continue to evolve, we were prompted to identify parameters determining their quality, safety and regenerative potential. Materials & methods: Flow-cytometry, mass-spectrometry, protein multiplexing, nanoparticle tracking analysis and standard biological techniques were employed. Results: Quality attributes of a uniquely processed UCB-allograft (UCBr) were enumerated based on identity (cell viability, immunophenotyping, proteomic profiling, and quantification of relevant cytokines); safety (bioburden and microbiological screening), purity (endotoxin levels) and potency (effect of UCBr on chondrocytes and mesenchymal stem cells derived exosomes). These attributes were stable up to 24 months in cryopreserved UCBr. Conclusion: We identified a comprehensive panel of tests to establish the clinical efficacy and quality control attributes of a UCB-sourced allograft.

Cytokines in umbilical cord blood-derived cellular product: a mechanistic insight into bone repair.

AIM: Umbilical cord blood (UCB) finds frequent applications in regenerative medicine. We evaluated the role of cytokines present in a uniquely processed, UCB-derived cellular allograft product (UCBp). MATERIALS & METHODS: Luminex multiplex assay and standard cell biology methods were employed. RESULTS: Study with allografts from 33 donors identified 44 quantifiable cytokines in the UCBp derived conditioned media (CM). The UCBp-CM elevated proliferation and migration rates of mesenchymal stem cells (MSCs) and bone marrow stromal cells. Moreover, UCBp-CM induced secretion of VEGF-A and osteoprotegerin, which promoted angiogenesis of endothelial cells and positively influenced the osteogenic differentiation of MSCs, respectively. CONCLUSION: Cytokines in UCBp stimulate cellular processes important for bone regeneration, making UCBp an excellent candidate for potential applications in orthopedic procedures like bone non-union and spinal fusion.

Biochemical characterization of pure dehydrated binate amniotic membrane: role of cytokines in the spotlight.

AIM: Placental allografts used for tissue regeneration differ in membrane compositions and processing techniques. A uniquely folded dehydrated binate amniotic membrane (DBAM) was biochemically characterized to evaluate its potential role in wound healing. METHODS: Histology, Luminex-based immunoassay and standard in vitro cell biology techniques were employed. RESULTS: Histological staining confirmed that the DBAM was chorion free with epithelial cell layer of the respective amnion membranes facing outward. DBAM had quantifiable levels of relevant cytokines that induced proliferation and migration while bolstering secretory activity of the cells. DBAM retained biological efficacy at a broad range of temperatures. CONCLUSION: Cytokines in DBAM stimulate bone marrow stromal and stem cells that may lead to tissue regeneration and wound healing in a clinical setup.

Biomonitoring role of some cellular markers during heat stress-induced changes in highly representative fresh water mollusc, Bellamya bengalensis: Implication in climate change and biological adaptation.

Owing to increasing concern of global climate-change, temperature rise is of great interest which can be primarily evaluated from the seasonal variations in some organisms. Aquatic environment can be extremely stressful to its inhabitants because most of them are poikilothermous. In the present study, attempt was made to evaluate the biological effects of oxidative-stress and adaptive/antioxidant capacities during temperature variations (36-40 °C for 24hrs to 72hrs) in Bellamya bengalensis both in environmental and laboratory conditions by testing some biomarkers like HSP70, catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH) and glutathione reductase (GR). The biomarker potency of the molecules and the anti-oxidative metabolic-network was postulated and extrapolated to find its resemblance to the climate-change associated organismal variations. In a natural and eco-restored environment in the Eastern part of India, 10-20 fold increases in CAT, SOD and HSP70 protein expressions (Western blot results) were noticed in Bellamya paralleling to their increased enzymatic activities (gel zymogram studies) due to the seasonal (summer versus winter) temperature variation. It is evident from the consecutive three years' study that this variation resulted in the unfavorable physico-chemical changes of water quality parameters like dissolved oxygen, biochemical oxygen demand, alkalinity and consequently decreased the animal density in summer. And that was revived due to their higher reproduction-rate in post rainy/winter season when temperature normalizes resulting in a restoration of favorable environment. In laboratory condition, the reduced GR and increased GPx indicated the oxidative damage as evident by higher tissue MDA level following to higher mortality. Changes in SOD and CAT activities suggest activation of physiological mechanism to scavenge the ROS produced during heat stress. However, when mortality increased at different time points (36 °C - 72 h and 38 °C - 72 h), these enzyme activities also decreased as they failed to save the tissues from ROS. The results suggest that temperature variation does alter the active oxygen metabolism by modulating antioxidant enzyme activities, which can be used as biomarker to detect sub-lethal effects of climate change-associated pollution. The parity in environmental and laboratory experimental results may justify this laboratory experiment as model heat-stress experiment and indicate temperature as a universal stressor which alone or in combination with other water parameters initiates a consistent adapting behavior. The Bellamya bengalensis being the highest faunal representative in its habitat may serve as a good bioindicator species.

Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

Deregulation of mitochondrial morphogenesis, a dynamic equilibrium between mitochondrial fusion and fission processes, is now evolving as a key metabolic event that fuels tumor growth and therapy resistance. However, fundamental knowledge underpinning how cancer cells reprogram mitochondrial morphogenesis remains incomplete. Here, we report that cystathionine ß-synthase (CBS) reprograms mitochondrial morphogenesis in ovarian cancer (OvCa) cells by selectively regulating the stability of mitofusin 2 (MFN2). Clinically, high expression of both CBS and MFN2 implicates poor overall survival of OvCa patients, and a significant association between CBS and MFN2 expression exists in individual patients in the same data set. The silencing of CBS by small interfering RNA or inhibition of its catalytic activity by a small molecule inhibitor creates oxidative stress that activates JNK. Activated JNK phosphorylates MFN2 to recruit homologous to the E6-AP carboxyl terminus' domain-containing ubiquitin E3 ligase for its degradation via the ubiquitin-proteasome system. Supplementation with hydrogen sulfide or glutathione (the catalytic products of CBS enzymatic activity), anti-oxidants, or a JNK inhibitor restores MFN2 expression. In CBS-silenced orthotopic xenograft tumor tissues, MFN2 but not MFN1 is selectively downregulated. In summary, this report reveals a role for deregulated mitochondrial morphogenesis in OvCa, suggests one of the mechanisms for this deregulation, and provides a way to correct it through modulation of the metabolic enzyme CBS.-Chakraborty, P. K., Murphy, B., Mustafi, S. B., Dey, A., Xiong, X., Rao, G., Naz, S., Zhang, M., Yang, D., Dhanasekaran, D. N., Bhattacharya, R., Mukherjee, P. Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

Evaluating the Mechanism and Therapeutic Potential of PTC-028, a Novel Inhibitor of BMI-1 Function in Ovarian Cancer.

BMI-1, also known as a stem cell factor, is frequently upregulated in several malignancies. Elevated expression of BMI-1 correlates with poor prognosis and is therefore considered a viable therapeutic target in a number of malignancies including ovarian cancer. Realizing the immense pathologic significance of BMI-1, small-molecule inhibitors against BMI-1 are recently being developed. In this study, we functionally characterize PTC-028, an orally bioavailable compound that decreases BMI-1 levels by posttranslational modification. We report that PTC-028 treatment selectively inhibits cancer cells in clonal growth and viability assays, whereas normal cells remain unaffected. Mechanistically, hyperphosphorylation-mediated depletion of cellular BMI-1 by PTC-028 coupled with a concurrent temporal decrease in ATP and a compromised mitochondrial redox balance potentiates caspase-dependent apoptosis. In vivo, orally administered PTC-028, as a single agent, exhibits significant antitumor activity comparable with the standard cisplatin/paclitaxel therapy in an orthotopic mouse model of ovarian cancer. Thus, PTC-028 has the potential to be used as an effective therapeutic agent in patients with epithelial ovarian cancer, where treatment options are limited. Mol Cancer Ther; 17(1); 39-49. ©2017 AACR.

MICU1 drives glycolysis and chemoresistance in ovarian cancer.

Cancer cells actively promote aerobic glycolysis to sustain their metabolic requirements through mechanisms not always clear. Here, we demonstrate that the gatekeeper of mitochondrial Ca2+ uptake, Mitochondrial Calcium Uptake 1 (MICU1/CBARA1) drives aerobic glycolysis in ovarian cancer. We show that MICU1 is overexpressed in a panel of ovarian cancer cell lines and that MICU1 overexpression correlates with poor overall survival (OS). Silencing MICU1 in vitro increases oxygen consumption, decreases lactate production, inhibits clonal growth, migration and invasion of ovarian cancer cells, whereas silencing in vivo inhibits tumour growth, increases cisplatin efficacy and OS. Mechanistically, silencing MICU1 activates pyruvate dehydrogenase (PDH) by stimulating the PDPhosphatase-phosphoPDH-PDH axis. Forced-expression of MICU1 in normal cells phenocopies the metabolic aberrations of malignant cells. Consistent with the in vitro and in vivo findings we observe a significant correlation between MICU1 and pPDH (inactive form of PDH) expression with poor prognosis. Thus, MICU1 could serve as an important therapeutic target to normalize metabolic aberrations responsible for poor prognosis in ovarian cancer.

Inhibition of BMI1 induces autophagy-mediated necroptosis.

The clonal self-renewal property conferred by BMI1 is instrumental in maintenance of not only normal stem cells but also cancer-initiating cells from several different malignancies that represent a major challenge to chemotherapy. Realizing the immense pathological significance, PTC-209, a small molecule inhibitor of BMI1 transcription has recently been described. While targeting BMI1 in various systems significantly decreases clonal growth, the mechanisms differ, are context-dependent, and somewhat unclear. We report here that genetic or pharmacological inhibition of BMI1 significantly impacts clonal growth without altering CDKN2A/INK4/ARF or CCNG2 and induces autophagy in ovarian cancer (OvCa) cells through ATP depletion. While autophagy can promote survival or induce cell death, targeting BMI1 engages the PINK1-PARK2-dependent mitochondrial pathway and induces a novel mode of nonapoptotic, necroptosis-mediated cell death. In OvCa, necroptosis is potentiated by activation of the RIPK1-RIPK3 complex that phosphorylates its downstream substrate, MLKL. Importantly, genetic or pharmacological inhibitors of autophagy or RIPK3 rescue clonal growth in BMI1 depleted cells. Thus, we have established a novel molecular link between BMI1, clonal growth, autophagy and necroptosis. In chemoresistant OvCa where apoptotic pathways are frequently impaired, necroptotic cell death modalities provide an important alternate strategy that leverage overexpression of BMI1.

Therapeutic evaluation of microRNA-15a and microRNA-16 in ovarian cancer.

Treatment of chemo-resistant ovarian cancer (OvCa) remains clinically challenging and there is a pressing need to identify novel therapeutic strategies. Here we report that multiple mechanisms that promote OvCa progression and chemo-resistance could be inhibited by ectopic expression of miR-15a and miR-16. Significant correlations between low expression of miR-16, high expression of BMI1 and shortened overall survival (OS) were noted in high grade serous (HGS) OvCa patients upon analysis of The Cancer Genome Atlas (TCGA). Targeting BMI1, in vitro with either microRNA reduced clonal growth of OvCa cells. Additionally, epithelial to mesenchymal transition (EMT) as well as expression of the cisplatin transporter ATP7B were inhibited by miR-15a and miR-16 resulting in decreased degradation of the extra-cellular matrix and enhanced sensitization of OvCa cells to cisplatin. Nanoliposomal delivery of the miR-15a and miR-16 combination, in a pre-clinical chemo-resistant orthotopic mouse model of OvCa, demonstrated striking reduction in tumor burden compared to cisplatin alone. Thus, with the advent of miR replacement therapy some of which are in Phase 2 clinical trials, miR-15a and miR-16 represent novel ammunition in the anti-OvCa arsenal.

Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Deficiencies of the human cystathionine ß-synthase (CBS) enzyme are characterized by a plethora of vascular disorders and hyperhomocysteinemia. However, several clinical trials demonstrated that despite reduction in homocysteine levels, disease outcome remained unaffected, thus the mechanism of endothelial dysfunction is poorly defined. Here, we show that the loss of CBS function in endothelial cells (ECs) leads to a significant down-regulation of cellular hydrogen sulfide (H2S) by 50% and of glutathione (GSH) by 40%. Silencing CBS in ECs compromised phenotypic and signaling responses to the VEGF that were potentiated by decreased transcription of VEGF receptor (VEGFR)-2 and neuropilin (NRP)-1, the primary receptors regulating endothelial function. Transcriptional down-regulation of VEGFR-2 and NRP-1 was mediated by a lack in stability of the transcription factor specificity protein 1 (Sp1), which is a sulfhydration target of H2S at residues Cys68 and Cys755. Reinstating H2S but not GSH in CBS-silenced ECs restored Sp1 levels and its binding to the VEGFR-2 promoter and VEGFR-2, NRP-1 expression, VEGF-dependent proliferation, and migration phenotypes. Thus, our study emphasizes the importance of CBS-mediated protein S-sulfhydration in maintaining vascular health and function.-Saha, S., Chakraborty, P. K., Xiong, X., Dwivedi, S. K. D., Mustafi, S. B., Leigh, N. R., Ramchandran, R., Mukherjee, P., Bhattacharya, R. Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Bmi-1: At the crossroads of physiological and pathological biology.

Bmi-1 is a member of the Polycomb Repressor Complex1 that mediates gene silencing by regulating chromatin structure and is indispensable for self-renewal of both normal and cancer stem cells. Despite three decades of research that have elucidated the transcriptional regulation, post-translational modifications and functions of Bmi-1 in regulating the DNA damage response, cellular bioenergetics, and pathologies, the entire potential of a protein with such varied function remains to be realized. This review attempts to synthesize the current knowledge on Bmi-1 with an emphasis on its role in both normal physiology and cancer. Additionally, since cancer stem cells are emerging as a new paradigm for therapy resistance, the role of Bmi-1 in this perspective is also highlighted. The wide spectrum of malignancies that implicate Bmi-1 as a signature for stemness and oncogenesis also make it a suitable candidate for therapy. Nonetheless new approaches are vitally needed to further characterize physiological roles of Bmi-1 with the long-term goal of using Bmi-1 as a prognostic marker and a therapeutic target.

Characterization of the Cardiac Overexpression of HSPB2 Reveals Mitochondrial and Myogenic Roles Supported by a Cardiac HspB2 Interactome.

Small Heat Shock Proteins (sHSPs) are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2), which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait) and a human cardiac library (prey) coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 "cardiac interactome" to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID). A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is tempting to speculate that small molecule activators of HspB2 might be deployed to mitigate mitochondrial related diseases such as cardiomyopathy and neurodegenerative disease.

Role of cystathionine beta synthase in lipid metabolism in ovarian cancer.

Elevated lipid metabolism is implicated in poor survival in ovarian cancer (OC) and other cancers; however, current lipogenesis-targeting strategies lack cancer cell specificity. Here, we identify a novel role of cystathionine beta-synthase (CBS), a sulphur amino acid metabolizing enzyme highly expressed in several ovarian cancer cell lines, in driving deregulated lipid metabolism in OC. We examined the role of CBS in regulation of triglycerides, cholesterol and lipogenic enzymes via the lipogenic transcription factors SREBP1 and SREBP2. CBS silencing attenuated the expression of number of key enzymes involved in lipid synthesis (FASN and ACC1). Additionally CBS abrogates lipid uptake in OC cells. Gene silencing of CBS or SREBPs abrogated cellular migration and invasion in OC, while ectopic expression of SREBPs can rescue phenotypic effects of CBS silencing by restoring cell migration and invasion. Mechanistically, CBS represses SREBP1 and SREBP2 at the transcription levels by modulating the transcription factor Sp1. We further established the roles of both CBS and SREBPs in regulating ovarian tumor growth in vivo. In orthotopic tumor models, CBS or SREBP silencing resulted in reduced tumor cells proliferation, blood vessels formation and lipid content. Hence, cancer-selective disruption of the lipid metabolism pathway is possible by targeting CBS and, at least for OC, promises a profound benefit.

Beta catenin is degraded by both caspase-3 and proteasomal activity during resveratrol-induced apoptosis in HeLa cells in a GSK3ß-independent manner.

Increased activity of ß-catenin, an important transcriptional activator for survival and proliferation-associated genes has been linked with many cancers. We examined whether ß-catenin is a target of resveratrol and whether its degradation contributes to the pro-apoptotic effects of resveratrol. HeLa cells were exposed to 60 µM resveratrol for 48 h. Apoptosis was confirmed by measurement of annexin V externalization, caspase-3 activation and DNA fragmentation. Induction of apoptosis was observed as early as 12 h, when both caspase-3 activation and PARP (poly ADP ribose polymerase) cleavage occurred. Nuclear ß-catenin levels remained unchanged for 48 h during resveratrol exposure. However, extranuclear cell lysate ß-catenin underwent a decrease at a late stage of apoptosis namely at 36-48 h. Alterations in the phosphorylation status of Akt/GSK3ß were not observed during resveratrol-induced apoptosis. Furthermore, inhibition of GSK3ß activity which is. largely responsible for ß-catenin degradation failed to influence ß-catenin stability. However, inhibition of caspase-3 activity prevented the decline in ß-catenin levels at 36-48 h of resveratrol exposure. Lactacystin, a proteosomal inhibitor also prevented the degradation of ß-catenin by resveratrol. In conclusion, resveratrol induced apoptosis in HeLa cells in an Akt/GSK3ß-independent manner and down-regulated ß-catenin levels during apoptosis through action of caspase-3 and proteasomal degradation, independent of GSK3ß-mediated phosphorylation.

Corrigendum to 'Bmi-1: At the crossroads of physiological and pathological biology' [Genes & Diseases 2 (2015) 225-239].

[This corrects the article DOI: 10.1016/j.gendis.2015.04.001.].

Assessment of thermal stress adaptation by monitoring Hsp70 and MnSOD in the freshwater gastropod, Bellamya bengalensis (Lamark 1882).

Expression of the stress biomarkers 70-kDa heat shock proteins (Hsp70) and manganese superoxide dismutase (MnSOD) was measured as the molecular basis of adaptive response against increased experimental temperatures (32-40 °C for a span of 24-72 h) on the fresh water molluscan species, Bellamya bengalensis (Lamark 1882). The experimental snail specimens were collected during summer and winter seasons from two contrasting wetlands: an ecorestored (free from human interference) site (SI) and other experiencing anthropogenic stresses (SII). The mortality rate of the B. bengalensis and the immunoblotting of MnSOD and Hsp70 of their digestive glands were performed at regular intervals during the period of heat stress. The SI provided a lower stress environment based on physicochemical parameters such as pH, dissolved oxygen (DO), biological oxygen demand (BOD), chemical oxygen demand (COD), and alkalinity for the survival of test species, although both sites experienced mortality due to thermal stresses. The parity in protein expressions displayed a uniform mode of adaptive impact to temperature elevations in both field and laboratory exposure. The Hsp70 expression was minimal at lower thermal stress, but increased with a rise in temperature. It is very likely that higher Hsp70 levels are not directly related to survival or adaptation. In contrast, MnSOD levels appeared to be an indicator of adaptive responses vis-a-vis survival of the animals. So, the expression levels of a universal free radical scavenger like MnSOD are recognized as a potential biomarker in a bioindicator species like Bellamya.

Reductive stress linked to small HSPs, G6PD, and Nrf2 pathways in heart disease.

SIGNIFICANCE: Aerobic organisms must exist between the dueling biological metabolic processes for energy and respiration and the obligatory generation of reactive oxygen species (ROS) whose deleterious consequences can reduce survival. Wide fluctuations in harmful ROS generation are circumvented by endogenous countermeasures (i.e., enzymatic and nonenzymatic antioxidants systems) whose capacity decline with aging and are enhanced by disease states. RECENT ADVANCES: Substantial efforts on the cellular and molecular underpinnings of oxidative stress has been complemented recently by the discovery that reductive stress similarly predisposes to inheritable cardiomyopathy, firmly establishing that the biological extremes of the redox spectrum play essential roles in disease pathogenesis. CRITICAL ISSUES: Because antioxidants by nutritional or pharmacological supplement to prevent or mitigate disease states have been largely disappointing, we hypothesize that lack of efficacy of antioxidants might be related to adverse outcomes in responders at the reductive end of the redox spectrum. As emerging concepts, such as reductive, as opposed, oxidative stress are further explored, there is an urgent and critical gap for biochemical phenotyping to guide the targeted clinical applications of therapeutic interventions. FUTURE DIRECTIONS: New approaches are vitally needed for characterizing redox states with the long-term goal to noninvasively assess distinct clinical states (e.g., presymptomatic, end-stage) with the diagnostic accuracy to guide personalized medicine.

HSPB2 is dispensable for the cardiac hypertrophic response but reduces mitochondrial energetics following pressure overload in mice.

BACKGROUND: CryAB (HspB5) and HspB2, two small heat shock genes located adjacently in the vertebrate genome, are hypothesized to play distinct roles. Mice lacking both cryab and hspb2 (DKO) are viable and exhibit adult-onset degeneration of skeletal muscle but confounding results from independent groups were reported for cardiac responses to different stressful conditions (i.e., ischemia/reperfusion or pressure overload). To determine the specific requirements of HSPB2 in heart, we generated cardiac-specific HSPB2 deficient (HSPB2cKO) mice and examined their cardiac function under basal conditions and following cardiac pressure overload. METHODOLOGY/PRINCIPAL FINDINGS: Transverse aortic constriction (TAC) or sham surgery was performed in HSPB2cKO mice and their littermates (HSPB2wt mice). Eight weeks after TAC, we found that expression of several small HSPs (HSPB2, 5, 6) was not markedly modified in HSPB2wt mice. Both cardiac function and the hypertrophic response remained similar in HSPB2cKO and HSPB2wt hearts. In addition, mitochondrial respiration and ATP production assays demonstrated that the absence of HSPB2 did not change mitochondrial metabolism in basal conditions. However, fatty acid supported state 3 respiration rate (ADP stimulated) in TAC operated HSPB2cKO hearts was significantly reduced in compared with TAC operated HSPB2wt mice (10.5±2.2 vs. 12.8±2.5 nmol O(2)/min/mg dry fiber weight, P<0.05), and ATP production in HSPB2cKO hearts was significantly reduced in TAC compared with sham operated mice (29.8±0.2 vs. 21.1±1.8 nmol ATP/min/mg dry fiber weight, P<0.05). Although HSPB2 was not associated with mitochondria under cardiac stress, absence of HSPB2 led to changes in transcript levels of several metabolic and mitochondrial regulator genes. CONCLUSIONS/SIGNIFICANCE: The present study indicates that HSPB2 can be replaced by other members of the multigene small HSP family under basal conditions while HSPB2 is implicated in the regulation of metabolic/mitochondrial function under cardiac stress such pressure overload.

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 microM H(2)O(2) for 4 weeks. Repetitively stressed cells were found to be significantly resistant to apoptosis-inducing 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, NFkappaB p65 became phosphorylated and appreciable nuclear localization of p65 was observed. NFkappaB 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, NFkappaB 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 NFkappaB p65 phosphorylation. However, inhibition of Akt activity through PI3 kinase inhibitor LY294002 did not result in obstruction of p38MAPK phosphorylation by H(2)O(2). Also, Resveratrol was effective as an antioxidant in counteracting a rise in reactive oxygen species (ROS) and p38MAPK activation by H(2)O(2) 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 NFkappaB.