RGS7 balances acetylation/de-acetylation of p65 to control chemotherapy-dependent cardiac inflammation.

Cardiotoxicity remains a major limitation in the clinical utility of anthracycline chemotherapeutics. Regulator of G-protein Signaling 7 (RGS7) and inflammatory markers are up-regulated in the hearts of patients with a history of chemotherapy particularly those with reduced left-ventricular function. RGS7 knockdown in either the murine myocardium or isolated murine ventricular cardiac myocytes (VCM) or cultured human VCM provided marked protection against doxorubicin-dependent oxidative stress, NF-κB activation, inflammatory cytokine production, and cell death. In exploring possible mechanisms causally linking RGS7 to pro-inflammatory signaling cascades, we found that RGS7 forms a complex with acetylase Tip60 and deacetylase sirtuin 1 (SIRT1) and controls the acetylation status of the p65 subunit of NF-κB. In VCM, the detrimental impact of RGS7 could be mitigated by inhibiting Tip60 or activating SIRT1, indicating that the ability of RGS7 to modulate cellular acetylation capacity is critical for its pro-inflammatory actions. Further, RGS7-driven, Tip60/SIRT1-dependent cytokines released from ventricular cardiac myocytes and transplanted onto cardiac fibroblasts increased oxidative stress, markers of transdifferentiation, and activity of extracellular matrix remodelers emphasizing the importance of the RGS7-Tip60-SIRT1 complex in paracrine signaling in the myocardium. Importantly, while RGS7 overexpression in heart resulted in sterile inflammation, fibrotic remodeling, and compromised left-ventricular function, activation of SIRT1 counteracted the detrimental impact of RGS7 in heart confirming that RGS7 increases acetylation of SIRT1 substrates and thereby drives cardiac dysfunction. Together, our data identify RGS7 as an amplifier of inflammatory signaling in heart and possible therapeutic target in chemotherapeutic drug-induced cardiotoxicity.

Cardiac RGS7 and RGS11 drive TGFß1-dependent liver damage following chemotherapy exposure.

Off target damage to vital organ systems is an unfortunate side effect of cancer chemotherapy and remains a major limitation to the use of these essential drugs in the clinic. Despite decades of research, the mechanisms conferring susceptibility to chemotherapy driven cardiotoxicity and hepatotoxicity remain unclear. In the livers of patients with a history of chemotherapy, we observed a twofold increase in expression of G protein regulator RGS7 and a corresponding decrease in fellow R7 family member RGS11. Knockdown of RGS7 via introduction of RGS7 shRNA via tail vein injection decreased doxorubicin-induced hepatic collagen and lipid deposition, glycogen accumulation, and elevations in ALT, AST, and triglycerides by approximately 50%. Surprisingly, a similar result could be achieved via introduction of RGS7 shRNA directly to the myocardium without impacting RGS7 levels in the liver directly. Indeed, doxorubicin-treated cardiomyocytes secrete the endocrine factors transforming growth factor ß1 (TGFß1) and TGFß superfamily binding protein follistatin-related protein 1 (FSTL1). Importantly, RGS7 overexpression in the heart was sufficient to recapitulate the impacts of doxorubicin on the liver and inhibition of TGFß1 signaling with the receptor blocker GW788388 ameliorated the effect of cardiac RGS7 overexpression on hepatic fibrosis, steatosis, oxidative stress, and cell death as well as the resultant elevation in liver enzymes. Together these data demonstrate that RGS7 controls both the release of TGFß1 from the heart and the profibrotic and pro-oxidant actions of TGFß1 in the liver and emphasize the functional significance of endocrine cardiokine signaling in the pathogenesis of chemotherapy drive multiorgan damage.

Vinpocetine mitigates DMH-induce pre-neoplastic colon damage in rats through inhibition of pro-inflammatory cytokines.

Colorectal cancer (CRC) is currently recognized as the third most prevalent cancer worldwide. Vinpocetine is a synthetic derivative of the vinca alkaloid vincamine. It has been found effective in ameliorating the growth and progression of cancerous cells. However, its pharmacological effect on colon damage remains elusive. Hence, in this study, we have shown the role of vinpocetine in DMH-induced colon carcinogenesis. At first, male albino Wistar rats were administered with DMH consistently for four weeks to induce pre-neoplastic colon damage. Afterward, animals were treated with vinpocetine (4.2 and 8.4 mg/kg/day p.o.) for 15 days. Serum samples were collected to assess the physiological parameters, including ELISA and NMR metabolomics. Colon from all the groups was collected and processed separately for histopathology and western blot analysis. Vinpocetine attenuated the altered plasma parameters; lipid profile and showed anti-proliferative action as evidenced by suppressed COX-2 stimulation and decreased levels of IL-1ß, IL-2, IL-6, and IL-10. Vinpocetine is significantly effective in preventing CRC which may be associated with its anti-inflammatory and antioxidant potential. Accordingly, vinpocetine could serve as a potential anticancer agent for CRC treatment and thus be considered for future clinical and therapeutic research.

RGS7-ATF3-Tip60 Complex Promotes Hepatic Steatosis and Fibrosis by Directly Inducing TNFα.

Aims: The pathophysiological mechanism(s) underlying non-alcoholic fatty liver disease (NAFLD) have yet to be fully delineated and only a single drug, peroxisome proliferator-activated receptor (PPAR) α/γ agonist saroglitazar, has been approved. Here, we sought to investigate the role of Regulator of G Protein Signaling 7 (RGS7) in hyperlipidemia-dependent hepatic dysfunction. Results: RGS7 is elevated in the livers of NAFLD patients, particularly those with severe hepatic damage, pronounced insulin resistance, and high inflammation. In the liver, RGS7 forms a unique complex with transcription factor ATF3 and histone acetyltransferase Tip60, which is implicated in NAFLD. The removal of domains is necessary for ATF3/Tip60 binding compromises RGS7-dependent reactive oxygen species generation and cell death. Hepatic RGS7 knockdown (KD) prevented ATF3/Tip60 induction, and it provided protection against fibrotic remodeling and inflammation in high-fat diet-fed mice translating to improvements in liver function. Hyperlipidemia-dependent oxidative stress and metabolic dysfunction were largely reversed in RGS7 KD mice. Interestingly, saroglitazar failed to prevent RGS7/ATF3 upregulation but it did partially restore Tip60 levels. RGS7 drives the release of particularly tumor necrosis factor α (TNFα) from isolated hepatocytes, stellate cells and its depletion reverses steatosis, oxidative stress by direct TNFα exposure. Conversely, RGS7 overexpression in the liver is sufficient to trigger oxidative stress in hepatocytes that can be mitigated via TNFα inhibition. Innovation: We discovered a novel non-canonical function for an R7RGS protein, which usually functions to regulate G protein coupled receptor (GPCR) signaling. This is the first demonstration for a functional role of RGS7 outside the retina and central nervous system. Conclusion: RGS7 represents a potential novel target for the amelioration of NAFLD. Antioxid. Redox Signal. 38, 137-159.

A RGS7-CaMKII complex drives myocyte-intrinsic and myocyte-extrinsic mechanisms of chemotherapy-induced cardiotoxicity.

Dose-limiting cardiotoxicity remains a major limitation in the clinical use of cancer chemotherapeutics. Here, we describe a role for Regulator of G protein Signaling 7 (RGS7) in chemotherapy-dependent heart damage, the demonstration for a functional role of RGS7 outside of the nervous system and retina. Though expressed at low levels basally, we observed robust up-regulation of RGS7 in the human and murine myocardium following chemotherapy exposure. In ventricular cardiomyocytes (VCM), RGS7 forms a complex with Ca2+/calmodulin-dependent protein kinase (CaMKII) supported by key residues (K412 and P391) in the RGS domain of RGS7. In VCM treated with chemotherapeutic drugs, RGS7 facilitates CaMKII oxidation and phosphorylation and CaMKII-dependent oxidative stress, mitochondrial dysfunction, and apoptosis. Cardiac-specific RGS7 knockdown protected the heart against chemotherapy-dependent oxidative stress, fibrosis, and myocyte loss and improved left ventricular function in mice treated with doxorubicin. Conversely, RGS7 overexpression induced fibrosis, reactive oxygen species generation, and cell death in the murine myocardium that were mitigated following CaMKII inhibition. RGS7 also drives production and release of the cardiokine neuregulin-1, which facilitates paracrine communication between VCM and neighboring vascular endothelial cells (EC), a maladaptive mechanism contributing to VCM dysfunction in the failing heart. Importantly, while RGS7 was both necessary and sufficient to facilitate chemotherapy-dependent cytotoxicity in VCM, RGS7 is dispensable for the cancer-killing actions of these same drugs. These selective myocyte-intrinsic and myocyte-extrinsic actions of RGS7 in heart identify RGS7 as an attractive therapeutic target in the mitigation of chemotherapy-driven cardiotoxicity.

RGS11-CaMKII complex mediated redox control attenuates chemotherapy-induced cardiac fibrosis.

Dose limiting cardiotoxicity remains a major limiting factor in the clinical use of several cancer chemotherapeutics including anthracyclines and the antimetabolite 5-fluorouracil (5-FU). Prior work has demonstrated that chemotherapeutics increase expression of R7 family regulator of G protein signaling (RGS) protein-binding partner Gß5, which drives myocyte cytotoxicity. However, though several R7 family members are expressed in heart, the exact role of each protein in chemotherapy driven heart damage remains unclear. Here, we demonstrate that RGS11, downregulated in the human heart following chemotherapy exposure, possesses potent anti-apoptotic actions, in direct opposition to the actions of fellow R7 family member RGS6. RGS11 forms a direct complex with the apoptotic kinase CaMKII and stress responsive transcription factor ATF3 and acts to counterbalance the ability of CaMKII and ATF3 to trigger oxidative stress, mitochondrial dysfunction, cell death, and release of the cardiokine neuregulin-1 (NRG1), which mediates pathological intercommunication between myocytes and endothelial cells. Doxorubicin triggers RGS11 depletion in the murine myocardium, and cardiac-specific OE of RGS11 decreases doxorubicin-induced fibrosis, myocyte hypertrophy, apoptosis, oxidative stress, and cell loss and aids in the maintenance of left ventricular function. Conversely, RGS11 knockdown in heart promotes cardiac fibrosis associated with CaMKII activation and ATF3/NRG1 induction. Indeed, inhibition of CaMKII largely prevents the fibrotic remodeling resulting from cardiac RGS11 depletion underscoring the functional importance of the RGS11-CaMKII interaction in the pathogenesis of cardiac fibrosis. These data describe an entirely new role for RGS11 in heart and identify RGS11 as a potential new target for amelioration of chemotherapy-induced cardiotoxicity.

Self-assembled dipeptide based fluorescent nanoparticles as a platform for developing cellular imaging probes and targeted drug delivery chaperones.

Self-assembled peptide-based nanostructures, comprised of naturally occurring amino acids, display excellent biocompatibility, biodegradability, flexible responsiveness, and synthetic feasibility and can be customized for various biomedical applications. However, the lack of inherent optical properties of peptide-based nanoparticles is a limitation on their use as imaging probes or drug delivery vehicles. To overcome this impediment, we generated Boc protected tyrosine-tryptophan dipeptide-based nanoparticles (DPNPs) with structure rigidification by Zn(ii), which shifted the peptide's intrinsic fluorescent properties from the ultraviolet to the visible range. These DPNPs are photostable, biocompatible and have visible fluorescence signals that allow for real-time monitoring of their entry into cells. We further show that two DPNPs (PS1-Zn and PS2-Zn) can encapsulate the chemotherapeutic drug doxorubicin (Dox) and facilitate intracellular drug delivery resulting in cancer cell killing actions comparable to the unencapsulated drug. Finally, we chemically modified our DPNPs with an aptamer directed toward the epithelial cell surface marker EPCAM, which improved Dox delivery to the lung cancer epithelial cell line A549. In contrast, the aptamer conjugated DPNPs failed to deliver Dox into the cardiomyocyte cell line AC16. Theoretically, this strategy could be employed in vivo to specifically deliver Dox to cancer cells while sparing the myocardium, a major source of dose-limiting adverse events in the clinic. Our work represents an important proof-of-concept exercise demonstrating that ultra-short peptide-based fluorescent nanostructures have great promise for the development of new imaging probes and targeted drug delivery vehicles.

Eriodictyol mediated selective targeting of the TNFR1/FADD/TRADD axis in cancer cells induce apoptosis and inhibit tumor progression and metastasis.

While the anti-inflammatory activities of Eriodictyol, a plant-derived flavonoid is well-known, reports on its anti-cancer efficacy and selective cytotoxicity in cancer cells are still emerging. However, little is known regarding its mechanism of selective anti-cancer activities. Here, we show the mechanism of selective cytotoxicity of Eriodictyol towards cancer cells compared to normal cells. Investigation reveals that Eriodictyol significantly upregulates TNFR1 expression in tumor cells (HeLa and SK-RC-45) while sparing the normal cells (HEK, NKE and WI-38), which display negligible TNFR1 expression, irrespective of the absence or presence of Eriodictyol. Further investigation of the molecular events reveal that Eriodictyol induces apoptosis through expression of the pro-apoptotic DISC components leading to activation of the caspase cascade. In addition, CRISPR-Cas9 mediated knockout of TNFR1 completely blocks apoptosis in HeLa cells in response to Eriodictyol, confirming that Eriodictyol induced cancer cell apoptosis is indeed TNFR1-dependent. Finally, in vivo data demonstrates that Eriodictyol not only impedes tumor growth and progression, but also inhibits metastasis in mice implanted with 4T1 breast cancer cells. Thus, our study has identified Eriodictyol as a compound with high selectivity towards cancer cells through TNFR1 and suggests that it can be further explored for its prospect in cancer therapeutics.

Ameliorative effect of fluvoxamine against colon carcinogenesis via COX-2 blockade with oxidative and metabolic stress reduction at the cellular, molecular and metabolic levels.

Fluvoxamine's (FLX's) anticancer potential was investigated in pre-clinical research utilizing a DMH-induced colorectal cancer (CRC) rat model. qRT-PCR and immunoblotting validated the mechanistic investigation. The CRC condition was induced in response to COX-2 and IL-6, however, following FLX therapy, the condition returned to normal. FLX's anti-CRC potential may be attributable to COX-2 inhibition since this molecular activity was more apparent for COX-2 than IL-6. FLX repaired the altered metabolites linked to CRC rats, according to 1H-NMR analysis. FLX was shown to be similar to 5-FU in terms of tumor protection, which may be useful in future medication development.

Mechanistic exploration of the activities of poly(lactic-co-glycolic acid)-loaded nanoparticles of betulinic acid against hepatocellular carcinoma at cellular and molecular levels.

The effectiveness of betulinic acid (B) and PLGA loaded nanoparticles of B (Bnp) against hepatocellular carcinoma (HCC) was established and reported earlier. In continuation of our previous report, the present study described the molecular mechanisms of their antineoplastic responses. In this context, the antineoplastic properties of both B and Bnp were evaluated on DEN-induced HCC rat model. The quantitative real-time polymerase chain reaction and western blot analyses revealed that HCC was developed through lower expressions of e-NOS, BAX, BAD, Cyt C and higher expressions of i-NOS, Bcl-xl, Bcl-2. B and Bnp normalised the expressions of these apoptogenic markers. Particularly, both activated i-NOS and e-NOS mediated Bcl-2 family proteins→CytC→Caspase 3 and 9 signalling cascades. The 1H-NMR-based metabolomics study also demonstrated that the perturbed metabolites in DEN-induced rat serum restored to the normal level following both treatments. Moreover, the antineoplastic potential of Bnp was found to be comparable with the marketed product, 5-flurouracil.

Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death.

The pathophysiological mechanism(s) driving non-alcoholic fatty liver disease, the most prevalent chronic liver disease globally, have yet to be fully elucidated. Here, we identify regulator of G protein signaling 6 (RGS6), up-regulated in the livers of NAFLD patients, as a critical mediator of hepatic steatosis, fibrosis, inflammation, and cell death. Human patients with high hepatic RGS6 expression exhibited a corresponding high inflammatory burden, pronounced insulin resistance, and poor liver function. In mice, liver-specific RGS6 knockdown largely ameliorated high fat diet (HFD)-driven oxidative stress, fibrotic remodeling, inflammation, lipid deposition and cell death. RGS6 depletion allowed for maintenance of mitochondrial integrity restoring redox balance, improving fatty acid oxidation, and preventing loss of insulin receptor sensitivity in hepatocytes. RGS6 is both induced by ROS and increases ROS generation acting as a key amplification node to exacerbate oxidative stress. In liver, RGS6 forms a direct complex with ATM kinase supported by key aspartate residues in the RGS domain and is both necessary and sufficient to drive hyperlipidemia-dependent ATM phosphorylation. pATM and markers of DNA damage (γH2AX) were also elevated in livers from NAFLD patients particularly in samples with high RGS6 protein content. Unsurprisingly, RGS6 knockdown prevented ATM phosphorylation in livers from HFD-fed mice. Further, RGS6 mutants lacking the capacity for ATM binding fail to facilitate palmitic acid-dependent hepatocyte apoptosis underscoring the importance of the RGS6-ATM complex in hyperlipidemia-dependent cell death. Inhibition of RGS6, then, may provide a viable means to prevent or reverse liver damage by mitigating oxidative liver damage.

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.

Supramolecular Structures Generated via Self-Assembly of a Cell Penetrating Tetrapeptide Facilitate Intracellular Delivery of a Pro-apoptotic Chemotherapeutic Drug.

Development of drug carriers, which can chaperone xenobiotics directly to their site of action, is an essential step for the advancement of precision medicine. Cationic nanoparticles can be used as a drug delivery platform for various agents including chemotherapeutics, oligonucleotides, and antibodies. Self-assembly of short peptides facilitates the formation of well-defined nanostructures suitable for drug delivery, and varying the polarity of the self-assembly medium changes the nature of noncovalent interactions in such a way as to generate numerous unique nanostructures. Here, we have synthesized an ultrashort cell-penetrating tetrapeptide (sequence Lys-Val-Ala-Val), with Lys as a cationic amino acid, and studied the self-assembly property of the BOC-protected (L1) and -deprotected (L2) analogues. Spherical assemblies obtained from L1/L2 in a 1:1 aqueous ethanol system have the ability to encapsulate small molecules and successfully enter into cells, thus representing them as potential candidates for intracellular drug delivery. To verify the efficacy of these peptides in the facilitation of drug efficacy, we generated encapsulated versions of the chemotherapeutic drug doxorubicin (Dox). L1- and L2-encapsulated Dox (Dox-L1 and Dox-L2), similar to the unencapsulated drug, induced upregulation of regulator of G protein signaling 6 (RGS6) and Gß5, the critical mediators of ATM/p53-dependent apoptosis in Dox-treated cancer cells. Further, Dox-L1/L2 damaged DNA, triggered oxidative stress and mitochondrial dysfunction, compromised cell viability, and induced apoptosis. The ability of Dox-L1 to mediate cell death could be ameliorated via knockdown of either RGS6 or Gß5, comparable to the results obtained with the unencapsulated drug. These data provide an important proof of principle, identifying L1/L2 as drug delivery matrices.

Preclinical Evaluation of Dimethyl Itaconate Against Hepatocellular Carcinoma via Activation of the e/iNOS-Mediated NF-κB-Dependent Apoptotic Pathway.

Hepatocellular carcinoma (HCC) is one of the most common tumors affecting a large population worldwide, with the fifth and seventh greatest mortality rates among men and women, respectively, and the third prime cause of mortality among cancer victims. Dimethyl itaconate (DI) has been reported to be efficacious in colorectal cancer by decreasing IL-1ß release from intestinal epithelial cells. In this study, diethylnitrosamine (DEN)-induced HCC in male albino Wistar rats was treated with DI as an anticancer drug. The function and molecular mechanism of DI against HCC in vivo were assessed using histopathology, enzyme-linked immunosorbent assay (ELISA), and Western blot studies. Metabolomics using 1H-NMR was used to investigate metabolic profiles. As per molecular insights, DI has the ability to trigger mitochondrial apoptosis through iNOS- and eNOS-induced activation of the NF-κB/Bcl-2 family of proteins, CytC, caspase-3, and caspase-9 signaling cascade. Serum metabolomics investigations using 1H-NMR revealed that aberrant metabolites in DEN-induced HCC rats were restored to normal following DI therapy. Furthermore, our data revealed that the DI worked as an anti-HCC agent. The anticancer activity of DI was shown to be equivalent to that of the commercial chemotherapeutic drug 5-fluorouracil.

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.