ROS- and HIF1α-dependent IGFBP3 upregulation blocks IGF1 survival signaling and thereby mediates high-glucose-induced cardiomyocyte apoptosis.

The prevalence of chronic hyperglycemia and its complications, imposing a critical burden on the worldwide economy and the global healthcare system, is a pressing issue. Mounting evidence indicates that oxidative stress and hypoxia, two noticeable features of hyperglycemia, play a joint crucial role in mediating cellular apoptosis. However, the underlying detailed molecular mechanism remains elusive. Triggered by the observation that insulin-like growth factor (IGF1)-binding protein 3 (IGFBP3) can mediate, in renal cells, high-glucose-induced apoptosis by elevating oxidative stress, we wish to, in this study, know whether or not the similar scenario holds in cardiac cells and, if so, to find its relevant molecular key players, thereby dissecting the underlying molecular pathway. Specifically, we used a combination of three different cellular sources (H9c2 cells, diabetic rats, and neonatal rat ventricular cardiomyocytes) as our model systems of study. We made use of Co-IP assay and western blot analysis in conjunction with loss-of-function reasoning, gain-of-function logic, and inhibitor treatment as our main analytical tools. As a result, briefly, our main findings are that hyperglycemia can induce cardiac IGFBP3 overexpression and secretion, that high levels of IGFBP3 can sequester IGF1 from IGF1 survival pathway, leading to apoptosis, and that IGFBP3 gene upregulation is hypoxia-inducible factor (HIF)1α-dependent and reactive oxygen species dependent. Piecing these findings together allows us to propose the improved molecular regulatory mechanism. In conclusion, we have established the molecular roles of IGFBP3, HIF1, and prolyl hydroxylase domain in connecting oxidative stress with hypoxia and in cellular apoptosis under hyperglycemia.

Diallyl trisulfide suppresses doxorubicin-induced cardiomyocyte apoptosis by inhibiting MAPK/NF-κB signaling through attenuation of ROS generation.

BACKGROUND: Doxorubicin (Dox) is an effective anticancer agent. However, its effectiveness is limited by its cardiotoxic effects. It has also been reported that the mitogen-activated protein kinase family and NF-κB can be activated by Dox treatment. DATS has been shown to be a potent antioxidant with cardioprotective effects. We investigate whether Dox induces cardiac apoptosis through JNK- and ERK-dependent NF-κB upregulation that can be reduced by DATS treatment. METHODS AND MATERIAL: H9c2 cells were treated with 0.5-1.5 µM Dox for 24 hours. Dox promoted apoptosis and ROS generation and inhibited viability in a dose-dependent manner. Then, the phosphorylation levels of JNK, ERK, and NF-κB evaluated by western blot were elevated. We used inhibitors of JNK, ERK, and NF-κB to determine which of these proteins were involved in Dox-induced apoptosis. Furthermore, Dox-exposed cells were treated with DATS at doses of 1, 5, and 10 µM, and the data demonstrated that ROS generation and apoptotic proteins were decreased and that ERK and NF-κB were downregulated in a dose-dependent manner. Additionally, six-week-old rats were divided into three groups (n = 6 per group) designed as an eight-week study. Normal, Dox (at dose 3.75 mg/kg by ip) administered with or without DATS (at dose 40 mg/kg by gavage) treatment groups. The results indicate that cardiac dysfunction, apoptosis, and JNK, ERK, and NF-κB activation by Dox were reversed by treatment with DATS. CONCLUSION: DATS appears to suppress Dox-induced cardiomyocyte apoptosis by inhibiting NADPH oxidase-related ROS production and the downstream JNK/ERK/NF-κB signaling pathway; DATS may possess clinical therapeutic potential by blocking Dox-induced cardiotoxicity.

Nrf2 Activation as a Protective Feedback to Limit Cell Death in High Glucose-Exposed Cardiomyocytes.

Hyperglycemia leads to excess reactive oxygen species (ROS) generation, which causes many diabetic complications, such as cardiomyopathy. Nuclear factor erythroid 2-related factor 2 (Nrf2), a redox-sensing transcription factor, can up-regulate its downstream antioxidant gene expressions in response to oxidative stress. However, the regulatory signal pathway in which high glucose (HG) induces Nrf2 activation is still unclear. Our results demonstrated that HG (33 mM) can indeed stimulate Nrf2 protein expression and translocation into the nucleus in cardiomyocytes, enhancing the downstream antioxidant protein levels. Using siRNAs, p38, JNK, PKCα, and PKCδ, as well as ROS scavengers, it was observed that the dependence of PKCα/PKCδ on ROS production to enhance JNK and p38 phosphorylation mediated HG-induced cardiac Nrf2 expression and activation. Knockdown of Nrf2 by siRNA transfection increased cleaved-caspase3, reduced Bcl2 in the cellular protein level and further exacerbated HG-induced apoptosis. In addition, all of these proteins induced by HG in vitro were also increased in STZ-induced diabetic rat ventricles in vivo. Our study demonstrated that HG-induced cardiac Nrf2 activation occurs through PKCα/PKCδ-ROS-JNK/p38 signaling. These findings may provide a therapeutic target to counteract the oxidative stress associated with diabetic cardiomyopathy. J. Cell. Biochem. 118: 1659-1669, 2017. © 2016 Wiley Periodicals, Inc.

Lactate dehydrogenase downregulation mediates the inhibitory effect of diallyl trisulfide on proliferation, metastasis, and invasion in triple-negative breast cancer.

The Warburg effect plays a critical role in tumorigenesis, suggesting that specific agents targeting Warburg effect key proteins may be a promising strategy for cancer therapy. Previous studies have shown that diallyl trisulfide (DATS) inhibits proliferation of breast cancer cells by inducing apoptosis in vitro and in vivo. However, whether the Warburg effect is involved with the apoptosis-promoting action of DATS is unclear. Here, we show that the action of DATS is associated with downregulation of lactate dehydrogenase A (LDHA), an essential protein of the Warburg effect whose upregulation is closely related to tumorigenesis. Interestingly, inhibition of the Warburg effect by DATS in breast cancer cells did not greatly affect normal cells. Furthermore, DATS inhibited growth of breast cancer cells, particularly in MDA-MB-231, a triple-negative breast cancer (TNBC) cell, and reduced proliferation and migration; invasion was reversed by over-expression of LDHA. These data suggest that DATS inhibits breast cancer growth and aggressiveness through a novel pathway targeting the key enzyme of the Warburg effect. Our study shows that LDHA downregulation is involved in the apoptotic effect of DATS on TNBC. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1390-1398, 2017.

Pkcδ Activation is Involved in ROS-Mediated Mitochondrial Dysfunction and Apoptosis in Cardiomyocytes Exposed to Advanced Glycation End Products (Ages).

Diabetic patients exhibit serum AGE accumulation, which is associated with reactive oxygen species (ROS) production and diabetic cardiomyopathy. ROS-induced PKCδ activation is linked to mitochondrial dysfunction in human cells. However, the role of PKCδ in cardiac and mitochondrial dysfunction caused by AGE in diabetes is still unclear. AGE-BSA-treated cardiac cells showed dose- and time-dependent cell apoptosis, ROS generation, and selective PKCδ activation, which were reversed by NAC and rotenone. Similar tendency was also observed in diabetic and obese animal hearts. Furthermore, enhanced apoptosis and reduced survival signaling by AGE-BSA or PKCδ-WT transfection were reversed by kinase-deficient (KD) of PKCδ transfection or PKCδ inhibitor, respectively, indicating that AGE-BSA-induced cardiomyocyte death is PKCδ-dependent. Increased levels of mitochondrial mass as well as mitochondrial fission by AGE-BSA or PKCδ activator were reduced by rottlerin, siPKCδ or KD transfection, indicating that the AGE-BSA-induced mitochondrial damage is PKCδ-dependent. Using super-resolution microscopy, we confirmed that PKCδ colocalized with mitochondria. Interestingly, the mitochondrial functional analysis by Seahorse XF-24 flux analyzer showed similar results. Our findings indicated that cardiac PKCδ activation mediates AGE-BSA-induced cardiomyocyte apoptosis via ROS production and may play a key role in the development of cardiac mitochondrial dysfunction in rats with diabetes and obesity.

Diallyl trisulfide protects against high glucose-induced cardiac apoptosis by stimulating the production of cystathionine gamma-lyase-derived hydrogen sulfide.

BACKGROUND: Cystathionine-γ-lyase (CSE)-derived hydrogen sulfide (H2S) is a potent cardioprotective agent. We investigated the effects of diallyl trisulfide (DATS) on CSE expression and H2S generation in myocardium and examined whether DATS-mediated H2S generation effectively protects rat heart from diabetes-induced cardiac damage. METHODS: The correlations between the effects of hyperglycemia and diabetes on CSE expression and the effects of DATS and H2S on hyperglycemia and diabetes were examined in vitro in the cardiomyocyte cell line H9c2 and in vivo in hearts from rats with streptozotocin-induced diabetes mellitus (DM). RESULTS: Expression of CSE, a catalyst of H2S production, was suppressed in H9c2 cells treated with high glucose (33 mM) and in DM rat hearts. CSE suppression also correlated with a decrease in the activation of the pro-survival protein kinase Akt. Treatment of H9c2 cells with DATS resulted in increased CSE expression and a reduction in apoptosis via a mechanism involving IGF1R/pAkt signaling and by modulating the expression of reactive oxygen species-related enzymes. The role CSE plays in the cardioprotective effects of DATS was further confirmed by CSE inhibition assays including inhibitors and siRNA. CONCLUSION: DATS produces H2S as efficiently as NaSH and DATS-derived H2S provides effective cardioprotection. Further, our data indicate that H2S plays a major role in the protective effect of DATS against apoptosis of cardiomyocytes.