RGS6 drives cardiomyocyte death following nucleolar stress by suppressing Nucleolin/miRNA-21.

BACKGROUND: Prior evidence demonstrated that Regulator of G protein Signaling 6 (RGS6) translocates to the nucleolus in response to cytotoxic stress though the functional significance of this phenomenon remains unknown. METHODS: Utilizing in vivo gene manipulations in mice, primary murine cardiac cells, human cell lines and human patient samples we dissect the participation of a RGS6-nucleolin complex in chemotherapy-dependent cardiotoxicity. RESULTS: Here we demonstrate that RGS6 binds to a key nucleolar protein, Nucleolin, and controls its expression and activity in cardiomyocytes. In the human myocyte AC-16 cell line, induced pluripotent stem cell derived cardiomyocytes, primary murine cardiomyocytes, and the intact murine myocardium tuning RGS6 levels via overexpression or knockdown resulted in diametrically opposed impacts on Nucleolin mRNA, protein, and phosphorylation.RGS6 depletion provided marked protection against nucleolar stress-mediated cell death in vitro, and, conversely, RGS6 overexpression suppressed ribosomal RNA production, a key output of the nucleolus, and triggered death of myocytes. Importantly, overexpression of either Nucleolin or Nucleolin effector miRNA-21 counteracted the pro-apoptotic effects of RGS6. In both human and murine heart tissue, exposure to the genotoxic stressor doxorubicin was associated with an increase in the ratio of RGS6/Nucleolin. Preventing RGS6 induction via introduction of RGS6-directed shRNA via intracardiac injection proved cardioprotective in mice and was accompanied by restored Nucleolin/miRNA-21 expression, decreased nucleolar stress, and decreased expression of pro-apoptotic, hypertrophy, and oxidative stress markers in heart. CONCLUSION: Together, these data implicate RGS6 as a driver of nucleolar stress-dependent cell death in cardiomyocytes via its ability to modulate Nucleolin. This work represents the first demonstration of a functional role for an RGS protein in the nucleolus and identifies the RGS6/Nucleolin interaction as a possible new therapeutic target in the prevention of cardiotoxicity.

A conserved nutrient responsive axis mediates autophagic degradation of miRNA-mRNA hybrids in blood cell progenitors.

In animals, microRNAs are amongst the primary non-coding RNAs involved in regulating the gene expression of a cell. Most mRNAs in a cell are targeted by one or many miRNAs. Although several mechanisms can be attributed to the degradation of miRNA and mRNA within a cell, but the involvement of autophagy in the clearance of miRNA and its target mRNA is not known. We discover a leucine-responsive axis in blood cell progenitors that can mediate an autophagy-directed degradation of miRNA-bound mRNA in Drosophila melanogaster and Homo sapiens. This previously unknown miRNA clearance axis is activated upon amino acid deprivation that can traffic miRNA-mRNA-loaded Argonaute for autophagic degradation in a p62-dependent manner. Thus, our research not only reports a novel axis that can address the turnover of a catalytically active miRISC but also elucidates a slicer-independent mechanism through which autophagy can selectively initiate the clearance of target mRNA.

G protein ß5-ATM complexes drive acetaminophen-induced hepatotoxicity.

Excessive ingestion of the common analgesic acetaminophen (APAP) leads to severe hepatotoxicity. Here we identify G protein ß5 (Gß5), elevated in livers from APAP overdose patients, as a critical regulator of cell death pathways and autophagic signaling in APAP-exposed liver. Liver-specific knockdown of Gß5 in mice protected the liver from APAP-dependent fibrosis, cell loss, oxidative stress, and inflammation following either acute or chronic APAP administration. Conversely, overexpression of Gß5 in liver was sufficient to drive hepatocyte dysfunction and loss. In hepatocytes, Gß5 depletion ameliorated mitochondrial dysfunction, allowed for maintenance of ATP generation and mitigated APAP-induced cell death. Further, Gß5 knockdown also reversed impacts of APAP on kinase cascades (e.g. ATM/AMPK) signaling to mammalian target of rapamycin (mTOR), a master regulator of autophagy and, as a result, interrupted autophagic flux. Though canonically relegated to nuclear DNA repair pathways, ATM also functions in the cytoplasm to control cell death and autophagy. Indeed, we now show that Gß5 forms a direct, stable complex with the FAT domain of ATM, important for autophosphorylation-dependent kinase activation. These data provide a viable explanation for these novel, G protein-independent actions of Gß5 in liver. Thus, Gß5 sits at a critical nexus in multiple pathological sequelae driving APAP-dependent liver damage.

Biphasic changes in TGF-ß1 signaling drive NSAID-induced multi-organ damage.

The clinical utility of non-steroidal anti-inflammatory drugs (NSAIDs), used extensively worldwide, is limited by adverse cardiac events resulting from chronic drug exposure. Here, we provide evidence identifying transforming growth factor ß (TGF-ß1), released from multiple tissues, as a critical driver of NSAID-induced multi-organ damage. Biphasic changes in TGF-ß1 levels in liver and heart were accompanied by ROS generation, cell death, fibrotic remodeling, compromised cardiac contractility and elevated liver enzymes. Pharmacological inhibition of TGF-ßRI signaling markedly improved heart and liver function and increased overall survival of animals exposed to multiple NSAIDs, effects likely mediated by reductions in NOX-dependent ROS generation. Notably, the beneficial impact of TGF-ßRI blockade was confined to a critical window wherein consecutive, but not concurrent, inhibitor administration improved cardiac and hepatic endpoints. Remarkably, in addition to ameliorating indomethacin-mediated myofilament disruptions, cardiac TGF-ßRI knockdown lead to drastic reductions in TGF-ß1 production accompanied by lessening in intestinal lesioning underscoring the importance of endocrine TGF-ß1 signaling in NSAID-driven tissue injury. Indeed, gastric ulceration was associated with a higher incidence of cardiac complications in a human cohort underscoring the critical importance of circulation-facilitated peripheral organ system interconnectedness in efforts seeking to mitigate the toxic side effects of chronic NSAID use.

Malabaricone C Attenuates Nonsteroidal Anti-Inflammatory Drug-Induced Gastric Ulceration by Decreasing Oxidative/Nitrative Stress and Inflammation and Promoting Angiogenic Autohealing.

Aims: Nonsteroidal anti-inflammatory drugs (NSAIDs), among the most commonly used drugs worldwide, are associated with gastrointestinal (GI) complications that severely limit the clinical utility of this essential class of pain medications. Here, we mechanistically dissect the protective impact of a natural product, malabaricone C (Mal C), on NSAID-induced gastropathy. Results: Mal C dose dependently diminished erosion of the stomach lining and inflammation in mice treated with NSAIDs with the protective impact translating to improvement in survival. By decreasing oxidative and nitrative stress, Mal C treatment prevented NSAID-induced mitochondrial dysfunction and cell death; nuclear factor κ-light-chain enhancer of activated B cell induction, release of proinflammatory cytokines and neutrophil infiltration; and disruptions in the vascular endothelial growth factor/endostatin balance that contributes to mucosal autohealing. Importantly, Mal C failed to impact the therapeutic anti-inflammatory properties of multiple NSAIDs in a model of acute inflammation. In all assays tested, Mal C proved as or more efficacious than the current first-line therapy for NSAID-dependent GI complications, the proton pump inhibitor omeprazole. Innovation: Given that omeprazole-mediated prophylaxis is, itself, associated with a shift in NSAID-driven GI complications from the upper GI to the lower GI system, there is a clear and present need for novel therapeutics aimed at ameliorating NSAID-induced gastropathy. Mal C provided significant protection against NSAID-induced gastric ulcerations impacting multiple critical signaling cascades contributing to inflammation, cell loss, extracellular matrix degradation, and angiogenic autohealing. Conclusion: Thus, Mal C represents a viable lead compound for the development of novel gastroprotective agents.

Antineoplastic properties of zafirlukast against hepatocellular carcinoma via activation of mitochondrial mediated apoptosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwideand haslimited treatment options. In view of this, zafirlukast (ZAF) was administered orally to DEN-induced HCC rats to evaluate its antineoplastic properties. ELISA, qRT-PCR and Western blot were used to determine the molecular mechanism associated with ZAF therapy for HCC. We found that HCC developed as a result of lower expression of caspases 3 and 9, but their levels returned to normal when the expression of eNOS, BAX, BAD, and Cyt C was decreased and when the expression of iNOS, Bcl-xl, and Bcl-2 was increased. Again, ZAF (80 mg/kg dose) treatment normalized the expression of caspase-mediated apoptotic factors, i.e. BAX and Bcl-2 proteins, as established through Western blot analysis. Later, 1H NMR-based serum metabolomics study revealed that levels of perturbed metabolites in DEN-induced rat serum returned to normal after ZAF administration. Altogether, the antineoplastic potential of ZAF was found to be comparable, and to some degree better, than the marketed chemotherapeutic 5-flurouracil, which may be beneficial for anti-HCC treatment from a future drug design perspective.

Zolmitriptan attenuates hepatocellular carcinoma via activation of caspase mediated apoptosis.

A preclinical study using DEN-induced HCC rat model was attempted to evaluate the antitumor potential of zolmitriptan (ZOL). The molecular insights were investigated using ELISA, qRT-PCR and Western blot techniques. The result confirmed that the HCC condition was developed in response to lower expressions of caspase 3 and 9 which, in turn, was due to the upstream regulation of iNOS, Bcl-xl and Bcl-2, and downstream regulation of eNOS, BAX, BAD and Cyt C. The treatment with ZOL caused the significant activation of caspase mediated apoptotic signals that could be responsible for its anti-HCC potential. Later, 1H NMR based serum metabolomics study confirmed that ZOL restored the perturbed metabolites associated with DEN-induced HCC. The antineoplastic potential of ZOL was found comparable or to some degree better than the marketed chemotherapeutics, 5-flurouracil.

Atypical G Protein ß5 Promotes Cardiac Oxidative Stress, Apoptosis, and Fibrotic Remodeling in Response to Multiple Cancer Chemotherapeutics.

The clinical use of multiple classes of cancer chemotherapeutics is limited by irreversible, dose-dependent, and sometimes life-threatening cardiotoxicity. Though distinct in their mechanisms of action, doxorubicin, paclitaxel, and 5-FU all induce rapid and robust upregulation of atypical G protein Gß5 in the myocardium correlating with oxidative stress, myocyte apoptosis, and the accumulation of proinflammatory and profibrotic cytokines. In ventricular cardiac myocytes (VCM), Gß5 deficiency provided substantial protection against the cytotoxic actions of chemotherapeutics, including reductions in oxidative stress and simultaneous attenuation of ROS-dependent activation of the ATM and CaMKII proapoptotic signaling cascades. In addition, Gß5 loss allowed for maintenance of Δψm, basal mitochondrial calcium uniporter expression, and mitochondrial Ca2+ levels, effects likely to preserve functional myocyte excitation-contraction coupling. The deleterious effects of Gß5 are not restricted to VCM, however, as Gß5 knockdown also reduces chemotherapy-induced release of proinflammatory cytokines (e.g., TNFα), hypertrophic factors (e.g., ANP), and profibrotic factors (e.g., TGFß1) from both VCM and ventricular cardiac fibroblasts, with the most dramatic reduction occurring in cocultured cells. Our experiments suggest that Gß5 facilitates the myofibroblast transition, the persistence of which contributes to pathologic remodeling and heart failure. The convergence of Gß5-mediated, ROS-dependent signaling pathways in both cell types represents a critical etiological factor in the pathogenesis of chemotherapy-induced cardiotoxicity. Indeed, intracardiac injection of Gß5-targeted shRNA allowed for heart-specific protection against the damaging impact of chronic chemotherapy. Together, our results suggest that inhibition of Gß5 might represent a novel means to circumvent cardiotoxicity in cancer patients whose treatment regimens include anthracyclines, taxanes, or fluoropyrimidines.Significance: These findings suggest that inhibiting an atypical G-protein might provide a strategy to limit the cardiotoxicity in cancer patients treated with anthracyclines, taxanes, or fluoropyrimidines. Cancer Res; 78(2); 528-41. ©2017 AACR.