TBK1 and GABARAP family members suppress Coxsackievirus B infection by limiting viral production and promoting autophagic degradation of viral extracellular vesicles.

Host-pathogen dynamics are constantly at play during enteroviral infection. Coxsackievirus B (CVB) is a common juvenile enterovirus that infects multiple organs and drives inflammatory diseases including acute pancreatitis and myocarditis. Much like other enteroviruses, CVB is capable of manipulating host machinery to hijack and subvert autophagy for its benefit. We have previously reported that CVB triggers the release of infectious extracellular vesicles (EVs) which originate from autophagosomes. These EVs facilitate efficient dissemination of infectious virus. Here, we report that TBK1 (Tank-binding kinase 1) suppresses release of CVB-induced EVs. TBK1 is a multimeric kinase that directly activates autophagy adaptors for efficient cargo recruitment and induces type-1 interferons during viral-mediated STING recruitment. Positioning itself at the nexus of pathogen elimination, we hypothesized that loss of TBK1 could exacerbate CVB infection due to its specific role in autophagosome trafficking. Here we report that infection with CVB during genetic TBK1 knockdown significantly increases viral load and potentiates the bulk release of viral EVs. Similarly, suppressing TBK1 with small interfering RNA (siRNA) caused a marked increase in intracellular virus and EV release, while treatment in vivo with the TBK1-inhibitor Amlexanox exacerbated viral pancreatitis and EV spread. We further demonstrated that viral EV release is mediated by the autophagy modifier proteins GABARAPL1 and GABARAPL2 which facilitate autophagic flux. We observe that CVB infection stimulates autophagy and increases the release of GABARAPL1/2-positive EVs. We conclude that TBK1 plays additional antiviral roles by inducing autophagic flux during CVB infection independent of interferon signaling, and the loss of TBK1 better allows CVB-laden autophagosomes to circumvent lysosomal degradation, increasing the release of virus-laden EVs. This discovery sheds new light on the mechanisms involved in viral spread and EV propagation during acute enteroviral infection and highlights novel intracellular trafficking protein targets for antiviral therapy.

Asporin, an extracellular matrix protein, is a beneficial regulator of cardiac remodeling.

Heart failure is accompanied by adverse cardiac remodeling involving extracellular matrix (ECM). Cardiac ECM acts as a major reservoir for many proteins including growth factors, cytokines, collagens, and proteoglycans. Activated fibroblasts during cardiac injury can alter the composition and activity of these ECM proteins. Through unbiased analysis of a microarray dataset of human heart tissue comparing normal hearts (n = 135) to hearts with ischemic cardiomyopathy (n = 94), we identified Asporin (ASPN) as the top differentially regulated gene (DEG) in ischemic cardiomyopathy; its gene-ontology terms relate closely to fibrosis and cell death. ASPN is a Class I small leucine repeat protein member implicated in cancer, osteoarthritis, and periodontal ligament mineralization. However, its role in cardiac remodeling is still unknown. Here, we initially confirmed our big dataset analysis through cells, mice, and clinical atrial biopsy samples to demonstrate increased Aspn expression after pressure overload or cardiac ischemia/reperfusion injury. We tested the hypothesis that Aspn, being a TGFß1 inhibitor, can attenuate fibrosis in mouse models of cardiac injury. We found that Aspn is released by cardiac fibroblasts and attenuates TGFß signaling. Moreover, Aspn-/- mice displayed increased fibrosis and decreased cardiac function after pressure overload by transverse aortic constriction (TAC) in mice. In addition, Aspn protected cardiomyocytes from hypoxia/reoxygenation-induced cell death and regulated mitochondrial bioenergetics in cardiomyocytes. Increased infarct size after ischemia/reperfusion injury in Aspn-/- mice confirmed Aspn's contribution to cardiomyocyte viability. Echocardiography revealed greater reduction in left ventricular systolic function post-I/R in the Aspn-/- animals compared to wild type. Furthermore, we developed an ASPN-mimic peptide using molecular modeling and docking which when administered to mice prevented TAC-induced fibrosis and preserved heart function. The peptide also reduced infarct size after I/R in mice, demonstrating the translational potential of ASPN-based therapy. Thus, we establish the role of ASPN as a critical ECM molecule that regulates cardiac remodeling to preserve heart function.

Proteomics of Mouse Heart Ventricles Reveals Mitochondria and Metabolism as Major Targets of a Post-Infarction Short-Acting GLP1Ra-Therapy.

Cardiovascular disease is the main cause of death worldwide, making it crucial to search for new therapies to mitigate major adverse cardiac events (MACEs) after a cardiac ischemic episode. Drugs in the class of the glucagon-like peptide-1 receptor agonists (GLP1Ra) have demonstrated benefits for heart function and reduced the incidence of MACE in patients with diabetes. Previously, we demonstrated that a short-acting GLP1Ra known as DMB (2-quinoxalinamine, 6,7-dichloro-N-[1,1-dimethylethyl]-3-[methylsulfonyl]-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline or compound 2, Sigma) also mitigates adverse postinfarction left ventricular remodeling and cardiac dysfunction in lean mice through activation of parkin-mediated mitophagy following infarction. Here, we combined proteomics with in silico analysis to characterize the range of effects of DMB in vivo throughout the course of early postinfarction remodeling. We demonstrate that the mitochondrion is a key target of DMB and mitochondrial respiration, oxidative phosphorylation and metabolic processes such as glycolysis and fatty acid beta-oxidation are the main biological processes being regulated by this compound in the heart. Moreover, the overexpression of proteins with hub properties identified by protein-protein interaction networks, such as Atp2a2, may also be important to the mechanism of action of DMB. Data are available via ProteomeXchange with identifier PXD027867.

At the heart of mitochondrial quality control: many roads to the top.

Mitochondrial quality control depends upon selective elimination of damaged mitochondria, replacement by mitochondrial biogenesis, redistribution of mitochondrial components across the network by fusion, and segregation of damaged mitochondria by fission prior to mitophagy. In this review, we focus on mitochondrial dynamics (fusion/fission), mitophagy, and other mechanisms supporting mitochondrial quality control including maintenance of mtDNA and the mitochondrial unfolded protein response, particularly in the context of the heart.

Pancreas-specific deletion of protein kinase D attenuates inflammation, necrosis, and severity of acute pancreatitis.

BACKGROUND: Protein kinase D (PKD) family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple cellular functions and human diseases. We recently reported that pharmacologic inhibition of PKD ameliorated the pathologic responses and severity of pancreatitis. However, to further investigate the importance of PKD family members in pancreatitis, it is necessary to explore the effects of pancreas-specific genetic inhibition of PKD isoform on pathology of pancreatitis. METHODS: We generated a mouse model (referred as PKD3Δpanc mice) with pancreas-specific deletion of PKD3, the predominant PKD isoform in mouse pancreatic acinar cells, by crossing Pkd3flox/flox mice with Pdx1-Cre transgenic mice which express Cre recombinase under the control of the mouse Pdx1 promoter. Pancreas-specific deletion of the PKD3 gene and PKD3 protein was confirmed by PCR and Western blot analysis. Experimental pancreatitis was induced in PKD3Δpanc and Pkd3flox/flox (control mice) littermates by intraperitoneal injections of cerulein or L-arginine. RESULTS: Compared to the control mice, PKD3Δpanc mice displayed significant attenuation in inflammation, necrosis, and severity of pancreatitis in both experimental models. PKD3Δpanc mice had markedly decreased NF-κB and trypsinogen activation, pancreatic mRNA expression of multiple inflammatory molecules, and the receptor-interacting protein kinase 1 (RIP1) activation in pancreatitis. PKD3Δpanc mice also had less pancreatic ATP depletion, increased pro-survival Bcl-2 family protein expression, and autophagy promotion. CONCLUSION: With PKD3Δpanc mouse model, we further demonstrated that PKD plays a critical role in pathobiological process of pancreatitis and PKD constitutes a novel therapeutic target to treat this disorder.

Intermittent Use of a Short-Course Glucagon-like Peptide-1 Receptor Agonist Therapy Limits Adverse Cardiac Remodeling via Parkin-dependent Mitochondrial Turnover.

Given that adverse remodeling is the leading cause of heart failure and death in the USA, there is an urgent unmet need to develop new methods in dealing with this devastating disease. Here we evaluated the efficacy of a short-course glucagon-like peptide-1 receptor agonist therapy-specifically 2-quinoxalinamine, 6,7-dichloro-N-(1,1-dimethylethyl)-3-(methylsulfonyl)-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB; aka Compound 2) - in attenuating adverse LV remodeling. We also examined the role, if any, of mitochondrial turnover in this process. Wild-type, Parkin knockout and MitoTimer-expressing mice were subjected to permanent coronary artery ligation, then treated briefly with DMB. LV remodeling and cardiac function were assessed by histology and echocardiography. Autophagy and mitophagy markers were examined by western blot and mitochondrial biogenesis was inferred from MitoTimer protein fluorescence and qPCR. We found that DMB given post-infarction significantly reduced adverse LV remodeling and the decline of cardiac function. This paralleled an increase in autophagy, mitophagy and mitochondrial biogenesis. The salutary effects of the drug were lost in Parkin knockout mice, implicating Parkin-mediated mitophagy as part of its mechanism of action. Our findings suggest that enhancing Parkin-associated mitophagy and mitochondrial biogenesis after infarction is a viable target for therapeutic mitigation of adverse remodeling.

Antiviral Effects of Menthol on Coxsackievirus B.

Coxsackievirus B (CVB) is a common human enterovirus that causes systemic infection but specifically replicates to high titers in the pancreas. It was reported that certain viruses induce mitochondrial fission to support infection. We documented that CVB triggers mitochondrial fission and blocking mitochondrial fission limits infection. The transient receptor potential channels have been implicated in regulating mitochondrial dynamics; namely, the heat and capsaicin receptor transient receptor potential cation channel subfamily V member 1 (TRPV1) contributes to mitochondrial depolarization and fission. When we transiently warmed HeLa cells to 39 °C prior to CVB exposure, infection was heightened, whereas cooling cells to 25 °C reduced infection. Inducing "cold" by stimulating transient receptor potential cation channel subfamily M member 8 (TRPM8) with menthol led to reduced infection and also resulted in lower levels of mitochondrial fission during infection. Additionally, menthol stabilized levels of mitochondrial antiviral signaling (MAVS) which is known to be tied to mitochondrial dynamics. Taken together, this highlights a novel pathway wherein CVB relies on TRPV1 to initiate proviral mitochondrial fission, which may contribute to the disruption of antiviral immunity. TRPM8 has been shown to antagonize TRPV1, and thus we hypothesize that stimulating TRPM8 blocks TRPV1-mediated mitochondrial fragmentation following CVB exposure and attenuates infection.

Imatinib modulates pro-inflammatory microenvironment with angiostatic effects in experimental lung carcinogenesis.

Lung cancer has second highest rate of incidence and mortality around the world. Smoking cigarettes is the main stream cause of lung carcinogenesis along with other factors such as spontaneous mutations, inactivation of tumor suppressor genes. The present study was aimed to identify the mechanistic role of Imatinib in the chemoprevention of experimental lung carcinogenesis in rat model. Gross morphological observations for tumor formation, histological examinations, RT-PCR, Western blotting, fluorescence spectroscopy and molecular docking studies were performed to elucidate the chemopreventive effects of Imatinib and support our hypothesis by various experiments. It is evident that immuno-compromised microenvironment inside solid tumors is responsible for tumor progression and drug resistance. Therefore, it is inevitable to modulate the pro-inflammatory signaling inside solid tumors to restrict neoangiogenesis. In the present study, we observed that Imatinib could downregulate the inflammatory signaling and also attributed angiostatic effects. Moreover, Imatinib also altered the biophysical properties of BAL cells such as plasma membrane potential, fluidity and microviscosity to restrict their infiltration and thereby accumulation to mount immuno-compromised environment inside the solid tumors during angiogenesis. Our molecular docking studies suggest that immunomodulatory and angiostatic properties of Imatinib could be either independent of each other or just a case of synergistic pleiotropy. Imatinib was observed to activate the intrinsic or mitochondrial pathway of apoptosis to achieve desired effects in cancer cell killings. Interestingly, binding of Imatinib inside the catalytic domain of PARP-1 also suggests that it has caspase-independent properties in promoting cancer cell deaths.

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis.

BACKGROUND: During pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence. METHODS: We examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis. RESULTS: Pancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin. CONCLUSION: Our findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.

Centromere protein F (CENPF), a microtubule binding protein, modulates cancer metabolism by regulating pyruvate kinase M2 phosphorylation signaling.

Prostate cancer (PC) is the most commonly diagnosed cancer in men and is the second leading cause of male cancer-related death in North America. Metabolic adaptations in malignant PC cells play a key role in fueling the growth and progression of the disease. Unfortunately, little is known regarding these changes in cellular metabolism. Here, we demonstrate that centromere protein F (CENPF), a protein associated with the centromere-kinetochore complex and chromosomal segregation during mitosis, is mechanically linked to altered metabolism and progression in PC. Using the CRISPR-Cas9 system, we silenced the gene for CENPF in human PC3 cells. These cells were found to have reduced levels of epithelial-mesenchymal transition markers and inhibited cell proliferation, migration, and invasion. Silencing of CENPF also simultaneously improved sensitivity to anoikis-induced apoptosis. Mass spectrometry analysis of tyrosine phosphorylated proteins from CENPF knockout (CENPFKO) and control cells revealed that CENPF silencing increased inactive forms of pyruvate kinase M2, a rate limiting enzyme needed for an irreversible reaction in glycolysis. Furthermore, CENPFKO cells had reduced global bio-energetic capacity, acetyl-CoA production, histone acetylation, and lipid metabolism, suggesting that CENPF is a critical regulator of cancer metabolism, potentially through its effects on mitochondrial functioning. Additional quantitative immunohistochemistry and imaging analyzes on a series of PC tumor microarrays demonstrated that CENPF expression is significantly increased in higher-risk PC patients. Based on these findings, we suggest the CENPF may be an important regulator of PC metabolism through its role in the mitochondria.

Ethanol Induced Disordering of Pancreatic Acinar Cell Endoplasmic Reticulum: An ER Stress/Defective Unfolded Protein Response Model.

BACKGROUND & AIMS: Heavy alcohol drinking is associated with pancreatitis, whereas moderate intake lowers the risk. Mice fed ethanol long term show no pancreas damage unless adaptive/protective responses mediating proteostasis are disrupted. Pancreatic acini synthesize digestive enzymes (largely serine hydrolases) in the endoplasmic reticulum (ER), where perturbations (eg, alcohol consumption) activate adaptive unfolded protein responses orchestrated by spliced X-box binding protein 1 (XBP1). Here, we examined ethanol-induced early structural changes in pancreatic ER proteins. METHODS: Wild-type and Xbp1+/- mice were fed control and ethanol diets, then tissues were homogenized and fractionated. ER proteins were labeled with a cysteine-reactive probe, isotope-coded affinity tag to obtain a novel pancreatic redox ER proteome. Specific labeling of active serine hydrolases in ER with fluorophosphonate desthiobiotin also was characterized proteomically. Protein structural perturbation by redox changes was evaluated further in molecular dynamic simulations. RESULTS: Ethanol feeding and Xbp1 genetic inhibition altered ER redox balance and destabilized key proteins. Proteomic data and molecular dynamic simulations of Carboxyl ester lipase (Cel), a unique serine hydrolase active within ER, showed an uncoupled disulfide bond involving Cel Cys266, Cel dimerization, ER retention, and complex formation in ethanol-fed, XBP1-deficient mice. CONCLUSIONS: Results documented in ethanol-fed mice lacking sufficient spliced XBP1 illustrate consequences of ER stress extended by preventing unfolded protein response from fully restoring pancreatic acinar cell proteostasis during ethanol-induced redox challenge. In this model, orderly protein folding and transport to the secretory pathway were disrupted, and abundant molecules including Cel with perturbed structures were retained in ER, promoting ER stress-related pancreas pathology.

Transient Receptor Potential Ankyrin 1 Activation within the Cardiac Myocyte Limits Ischemia-reperfusion Injury in Rodents.

BACKGROUND: Recent evidence suggests that cross talk exists between cellular pathways important for pain signaling and ischemia-reperfusion injury. Here, the authors address whether the transient receptor potential ankyrin 1 (TRPA1) channel, important in pain signaling, is present in cardiac myocytes and regulates cardiac ischemia-reperfusion injury. METHODS: For biochemical analysis of TRPA1, techniques including quantitative polymerase chain reaction, Western blot, and immunofluorescence were used. To determine how TRPA1 mediates cellular injury, the authors used an in vivo model of rat cardiac ischemia-reperfusion injury and adult rat-isolated cardiac myocytes subjected to hypoxia-reoxygenation. RESULTS: The authors' biochemical analysis indicates that TRPA1 is within the cardiac myocytes. Further, using a rat in vivo model of cardiac injury, the TRPA1 activators ASP 7663 and optovin reduce myocardial injury (45 ± 5%* and 44 ± 8%,* respectively, vs. control, 66 ± 6% infarct size/area at risk; n = 6 per group; mean ± SD; *P < 0.001). TRPA1 inhibition also blocked the infarct size-sparing effects of morphine. In isolated cardiac myocytes, the TRPA1 activators ASP 7663 and optovin reduce cardiac myocyte cell death when given during reoxygenation (20 ± 3%* and 22 ± 4%* vs. 36 ± 3%; percentage of dead cells per field, n = 6 per group; mean ± SD; *P < 0.05). For a rat in vivo model of cardiac injury, the infarct size-sparing effect of TRPA1 activators also occurs during reperfusion. CONCLUSIONS: The authors' data suggest that TRPA1 is present within the cardiac myocytes and is important in regulating myocardial reperfusion injury. The presence of TRPA1 within the cardiac myocytes may potentially explain why certain pain relievers that can block TRPA1 activation, such as cyclooxygenase-2 inhibitors or some nonsteroidal antiinflammatory drugs, could be associated with cardiovascular risk.

Transient Receptor Potential Vanilloid 1 Regulates Mitochondrial Membrane Potential and Myocardial Reperfusion Injury.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) mediates cellular responses to pain, heat, or noxious stimuli by calcium influx; however, the cellular localization and function of TRPV1 in the cardiomyocyte is largely unknown. We studied whether myocardial injury is regulated by TRPV1 and whether we could mitigate reperfusion injury by limiting the calcineurin interaction with TRPV1. METHODS AND RESULTS: In primary cardiomyocytes, confocal and electron microscopy demonstrates that TRPV1 is localized to the mitochondria. Capsaicin, the specific TRPV1 agonist, dose-dependently reduced mitochondrial membrane potential and was blocked by the TRPV1 antagonist capsazepine or the calcineurin inhibitor cyclosporine. Using in silico analysis, we discovered an interaction site for TRPV1 with calcineurin. We synthesized a peptide, V1-cal, to inhibit the interaction between TRPV1 and calcineurin. In an in vivo rat myocardial infarction model, V1-cal given just prior to reperfusion substantially mitigated myocardial infarct size compared with vehicle, capsaicin, or cyclosporine (24±3% versus 61±2%, 45±1%, and 49±2%, respectively; n=6 per group; P<0.01 versus all groups). Infarct size reduction by V1-cal was also not seen in TRPV1 knockout rats. CONCLUSIONS: TRPV1 is localized at the mitochondria in cardiomyocytes and regulates mitochondrial membrane potential through an interaction with calcineurin. We developed a novel therapeutic, V1-cal, that substantially reduces reperfusion injury by inhibiting the interaction of calcineurin with TRPV1. These data suggest that TRPV1 is an end-effector of cardioprotection and that modulating the TRPV1 protein interaction with calcineurin limits reperfusion injury.

Downregulation of telomerase activity by diclofenac and curcumin is associated with cell cycle arrest and induction of apoptosis in colon cancer.

Uncontrolled cell proliferation is the hallmark of cancer, and cancer cells have typically acquired damage to genes that directly regulate their cell cycles. The synthesis of DNA onto the end of chromosome during the replicative phase of cell cycle by telomerase may be necessary for unlimited proliferation of cells. Telomerase, a ribonucleoprotein enzyme is considered as a universal therapeutic target of cancer because of its preferential expression in cancer cells and its presence in 90 % of tumors. We studied the regulation of telomerase and telomerase reverse transcriptase catalytic subunit (TERT) by diclofenac and curcumin, alone and also in combination, in 1, 2-dimethylhydrazine dihydrochloride-induced colorectal cancer in rats. The relationship of telomerase activity with tumors suppressor proteins (p51, Rb, p21), cell cycle machinery, and apoptosis was also studied. Telomerase is highly expressed in DMH group and its high activity is associated with increased TERT expression. However, telomerase is absent or is present at lower levels in normal tissue. CDK4, CDK2, cyclin D1, and cyclin E are highly expressed in DMH as assessed by RT-PCR, qRT-PCR, Western blot, and immunofluorescence analysis. Diclofenac and curcumin overcome these carcinogenic effects by downregulating telomerase activity, diminishing the expression of TERT, CDK4, CDK2, cyclin D1, and cyclin E. The anticarcinogenic effects shown after the inhibition of telomerase activity by diclofenac and curcumin may be associated with upregulation of tumor suppressor proteins p51, Rb, and p21, whose activation induces the cells cycle arrest and apoptosis.

Downregulation of PI3-K/Akt/PTEN pathway and activation of mitochondrial intrinsic apoptosis by Diclofenac and Curcumin in colon cancer.

Phosphatidylinositol 3-kinase (PI3-K)/PTEN/Akt signaling is over activated in various tumors including colon cancer. Activation of this pathway regulates multiple biological processes such as apoptosis, metabolism, cell proliferation, and cell growth that underlie the biology of a cancer cell. In the present study, the chemopreventive effects have been observed of Diclofenac, a preferential COX-2 inhibitory non-steroidal anti-inflammatory drugs, and Curcumin, a natural anti-inflammatory agent, in the early stage of colorectal carcinogenesis induced by 1,2-dimethylhydrazine dihydrochloride in rats. The tumor-promoting role of PI3-K/Akt/PTEN signal transduction pathway and its association with anti-apoptotic family of proteins are also observed. Both Diclofenac and Curcumin downregulated the PI3-K and Akt expression while promoting the apoptotic mechanism. Diclofenac and Curcumin administration significantly increased the expression of pro-apoptotic Bcl-2 family members (Bad and Bax) while decreasing the anti-apoptotic Bcl-2 protein. An up-regulation of cysteine protease family apoptosis executioner, such as caspase-3 and -9, is seen. Diclofenac and Curcumin inhibited the Bcl-2 protein by directly interacting at the active site by multiple hydrogen bonding, as also evident by negative glide score of Bcl-2. These drugs stimulated apoptosis by increasing reactive oxygen species (ROS) generation and simultaneously decreasing the mitochondrial membrane potential (ΔΨ M). Diclofenac and Curcumin showed anti-neoplastic effects by downregulating PI3-K/Akt/PTEN pathway, inducing apoptosis, increasing ROS generation, and decreasing ΔΨ M. The anti-neoplastic and apoptotic effects were found enhanced when both Diclofenac and Curcumin were administered together, rather than individually.

Sulindac and Celecoxib regulate cell cycle progression by p53/p21 up regulation to induce apoptosis during initial stages of experimental colorectal cancer.

In the present study we have elaborated the putative mechanisms could be followed by the non-steroidal anti-inflammatory drugs (NSAIDs) viz. Sulindac and Celecoxib in the regulation of cell cycle checkpoints along with tumor suppressor proteins to achieve their chemopreventive effects in the initial stages of experimental colorectal cancer. Male Sprague-Dawley rats were administered with 1,2-dimethylhydrazine dihydrochloride (DMH) to produce early stages of colorectal carcinogenesis. The mRNA expression profiles of various target genes were analyzed by RT-PCR and validated by quantitative real-time PCR, whereas protein expression was analyzed by Western blotting. Nuclear localization of transcription factors or other nuclear proteins was analyzed by electrophoretic mobility shift assay and immunofluorescence. Flowcytometry was performed to analyze the differential apoptotic events and cell cycle regulation. Molecular docking studies with different target proteins were also performed to deduce the various putative mechanisms of action followed by Sulindac and Celecoxib. We observed that DMH administration has abruptly increased the proliferation of colonic cells which is macroscopically visible in the form of multiple plaque lesions and co-relates with the disturbed molecular mechanisms of cell cycle regulation. However, co-administration of NSAIDs has shown regulatory effects on cell cycle checkpoints via induction of various tumor suppressor proteins. We may conclude that Sulindac and Celecoxib could possibly follow p53/p21 mediated regulation of cell proliferation, where down regulation of NF-κB signaling and activation of PPARγ might serve as important additional events in vivo.

Dolastatin, along with Celecoxib, stimulates apoptosis by a mechanism involving oxidative stress, membrane potential change and PI3-K/AKT pathway down regulation.

BACKGROUND: Phosphoinositide 3-kinase (PI3-K) is an important regulator of oncogenesis and apoptosis in various types of cancers including colon cancer. A combinatorial strategy of using Cyclooxygenase-2 inhibitor, Celecoxib and Dolastatin, a linear peptide from marine mollusks of Indian Ocean origin has shown anti-neoplastic effects in colon cancer in a rat model. METHODS: The signal transduction pathway of PI3-K/AKT and the downstream signaling proteins had been studied in an early stage of colon carcinogenesis (DMH induced) by gene and protein expression, apoptotic studies by colonocyte apoptotic bleb assay, intracellular calcium level by fluorescence spectrometry, mitochondrial membrane potential by Rhodamine 123 flow cytometry and Reactive oxygen species measurement. Molecular docking analysis was employed to study the interaction of oncogenic proteins and the ligand, Celecoxib and Dolastatin. RESULTS: Apoptotic cell index was lowered with DMH while both the drugs increased it and inhibited PI3-K and AKT expression. Docking studies revealed both the proteins targeted by the drugs via an ATP binding site. An increased expression of GSK-3ß, pro-apoptotic protein Bad, transcription factor Egr-1, tumor suppressor protein PTEN while a downregulation of G1-associated cell cycle protein, Cyclin D1 and increased intracellular calcium as well as reactive oxygen species were observed. Also, the number of cells having a higher mitochondrial membrane potential was lowered. CONCLUSION: Celecoxib and Dolastatin inhibited the tumor development through regulation of the PI3-K/AKT pathway which can act as a novel target for these drugs. GENERAL SIGNIFICANCE: The anti-cancer properties of Dolastatin, a peptide isolated from marine mollusks in colorectal cancer is shown.

NSAIDs may regulate EGR-1-mediated induction of reactive oxygen species and non-steroidal anti-inflammatory drug-induced gene (NAG)-1 to initiate intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer.

This study aims to investigate the unclear molecular relationship involved in the activation of intrinsic pathway of apoptosis and NSAID-activated gene-1 (NAG-1) induction as a putative target in NSAIDs-mediated chemoprevention of colorectal cancer. Male Sprague-Dawley rats were administered with a colon-specific pro-carcinogen, 1,2-dimethylhydrazine dihydrochloride to achieve the early stages of colorectal cancer. Histopathological examination was performed for the analysis of neoplastic lesions while flow cytometry was performed for the relative quantification of intracellular reactive oxygen species (ROS), differential mitochondrial membrane potential (MMP or ΔΨ(M)), and apoptotic events. Various target biomolecules were analyzed either for their mRNA or protein expression profiles via RT-PCR and quantitative Real-Time PCR, or Western blotting and immunofluorescence, respectively. Enhanced gene as well as protein expression of pro-apoptotic agents was observed with the daily oral administration of two NSAIDs viz. Sulindac (cyclooxygenase (COX)-non-specific) and Celecoxib (a selective COX-2 inhibitor). A significant increase in early growth response-1 (EGR-1) protein expression and nuclear localization in NSAIDs co-administered animals may have positively regulated the expression of NAG-1 with a significant enhancement of intracellular ROS in turn decreasing the ΔΨ(M) to initiate apoptosis. In silico molecular docking analysis also showed that Sulindac and Celecoxib can block the active site pocket of B-cell lymphoma-extra large (Bcl-xL, anti-apoptotic transmembrane mitochondrial protein) which could be a putative mechanism followed by these NSAIDs to overcome anti-apoptotic properties of the molecule. NSAIDs-mediated up-regulation of EGR-1 and thereby NAG-1 along with implication of higher ROS load may positively regulate the intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer.

Up-regulation of p53 and mitochondrial signaling pathway in apoptosis by a combination of COX-2 inhibitor, Celecoxib and Dolastatin 15, a marine mollusk linear peptide in experimental colon carcinogenesis.

Programmed cell death, also known as apoptosis, is an active process occurring in eukaryotic cells and it depends on various sets of pro and anti-apoptotic proteins. Chemoprevention of colorectal cancer can be achieved by inducing apoptosis using synthetic compound, Celecoxib and natural peptide, Dolastatin 15 in an effective manner. But the apoptotic signaling by these two drugs remain unclear. The present study was thus focused on the role of Bcl2 family of proteins and their interplay with p53 in rats during the chemoprevention by these two drugs. After treatment for 6 wk with 1, 2-dimethylhydrazine (DMH), animals showed a marked occurrence of multiple plaque lesions. However, a simultaneous treatment with Celecoxib and Dolastatin 15 decreases such number to a significant level. DMH treatment also decreases the number of apoptotic cells in the colonic enterocytes which were corrected to the normal level by Celecoxib and Dolastatin 15. An increased expression of Bcl2 while other proteins like Bax, Apaf-1, cyt c, and caspases in the apoptotic pathway, and the tumor suppressor proteins, p53 and p21 get down-regulated after DMH treatment which were reverted back to normal with Celecoxib and Dolastatin 15. Also, cells having high mitochondrial membrane potential had been seen to increase to significant levels which were reduced after the administration of these anti-inflammatory drugs. In silico molecular docking studies also showed that Dolastatin 15 and Celecoxib may bind to the active site pocket of Bcl2 , thus revealing the direct target of Dolastatin 15 and Celecoxib apart from binding to COX-2.

Angiostatic properties of sulindac and celecoxib in the experimentally induced inflammatory colorectal cancer.

Initiation of various cancers has been observed to be regulated via a prolonged inflammatory state in the tissues. However, molecular role of such a localized inflammation is not clear in the advanced stages of colorectal cancer. In this study, we have elaborated the role of various pro- and anti-inflammatory cytokines, transcription, and angiogenic factors in the progression of the 1,2-dimethylhydrazine dihydrochloride (DMH)-induced late phage colorectal cancer and also observed the chemopreventive role of the two non-steroidal anti-inflammatory drugs (NSAIDs), viz., Sulindac and Celecoxib. Carcinogenic changes were observed with morphological and histopathological studies, whereas mRNA and protein regulations of various biomolecules were identified via RT- or qRT-PCR, western blot and immunofluorescence analysis, respectively. Activity of inducible nitric oxide (NO) and cyclooxygenase-2 enzymes were analyzed using standard NO assay and prostaglandin E2 immunoassay, whereas activities of matrix metalloproteinases (MMP-2 and-9) were identified by gelatin zymography. Flowcytometry was performed for the relative quantification of the apoptotic events. Molecular docking studies of Sulindac and Celecoxib were also performed with different target proteins to observe their putative mechanisms of action. As a result, we found that DMH-treated animals were having over-expression of various pro-inflammatory cytokines (IL-1ß, IL-2, and IFNγ), aberrant nuclear localization of activated cell survival transcription factors (NF-κB and Stat3) along with the increased incidence of activated angiogenic factors (MMP-2 and MMP-9) suggesting a marked role of inflammation in the tumor progression. However, NSAIDs co-administration has significantly reduced the angiogenic potential of the growing neoplasm.