Mahalingam S. Sundaram

Novel Synthetic Biscoumarins Target Tumor Necrosis Factor-α in Hepatocellular Carcinoma In Vitro and In Vivo

Background: TNF-α-induced NF-κB pathway is associated with the progression of several cancers and abrogation of TNF signaling a potential target for cancer treatment. Results: Novel biscoumarin inhibits TNF signaling in vitro and in vivo in IBD model. Conclusion: The lead compound interrupts the trimeric structure of TNF to achieve this effect. Significance: This study introduces a novel TNF inhibitor with the potential to target pro-inflammatory diseases. TNF is a pleotropic cytokine known to be involved in the progression of several pro-inflammatory disorders. Many therapeutic agents have been designed to counteract the effect of TNF in rheumatoid arthritis as well as a number of cancers. In the present study we have synthesized and evaluated the anti-cancer activity of novel biscoumarins in vitro and in vivo. Among new compounds, BIHC was found to be the most cytotoxic agent against the HepG2 cell line while exhibiting less toxicity toward normal hepatocytes. Furthermore, BIHC inhibited the proliferation of various hepatocellular carcinoma (HCC) cells in a dose- and time-dependent manner. Subsequently, using in silico target prediction, BIHC was predicted as a TNF blocker. Experimental validation was able to confirm this hypothesis, where BIHC could significantly inhibit the recombinant mouse TNF-α binding to its antibody with an IC50 of 16.5 μm. Furthermore, in silico docking suggested a binding mode of BIHC similar to a ligand known to disrupt the native, trimeric structure of TNF, and also validated with molecular dynamics simulations. Moreover, we have demonstrated the down-regulation of p65 phosphorylation and other NF-κB-regulated gene products upon BIHC treatment, and on the phenotypic level the compound shows inhibition of CXCL12-induced invasion of HepG2 cells. Also, we demonstrate that BIHC inhibits infiltration of macrophages to the peritoneal cavity and suppresses the activity of TNF-α in vivo in mice primed with thioglycollate broth and lipopolysaccharide. We comprehensively validated the TNF-α inhibitory efficacy of BIHC in an inflammatory bowel disease mice model.

Melatonin elevates apoptosis in human platelets via ROS mediated mitochondrial damage

Melatonin is a pineal hormone that regulates circadian and seasonal rhythms. The chronobiotic role of melatonin corresponds with a repertoire of pharmacological properties. Besides, it has a wide range of therapeutic applications. However, recent studies have demonstrated its direct interaction with platelets: at physiological concentration it promotes platelet aggregation; on the other hand, at pharmacological doses it raises intracellular Ca(2+) leading to platelet activation, thrombus formation and cardiovascular disorders. In order to further probe its effects on platelets, the current study targeted platelet apoptosis and melatonin was found to stimulate apoptosis. The mitochondrial pathway of apoptosis was mainly investigated because of its susceptibility to oxidative stress-inducing factors including therapeutic and dietary elements. Melatonin significantly increased the generation of intracellular ROS and Ca(2+), facilitating mitochondrial membrane depolarization, cytochrome c release, caspase activation, protein phosphorylation and phosphatidylserine externalization. Further, the overall toxicity of melatonin on platelets was confirmed by MTT and lactate dehydrogenase assays. The elevated rate of platelet apoptosis has far reaching consequences including thrombocytopenia. Besides, platelets undergoing apoptosis release microparticles, which fuel thrombus formation and play a significant role in the pathophysiology of a number of diseases. In many parts of the world melatonin is an over-the-counter dietary supplement and alternative medicine. Since, melatonin displays platelet proapoptotic effect at a concentration attainable through therapeutic dosage, the present study sends a warning signal to the chronic use of melatonin as a therapeutic drug and questions its availability without a medical prescription.

Unconjugated bilirubin exerts pro-apoptotic effect on platelets via p38-MAPK activation

Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation.

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide.

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.

A New Ibuprofen Derivative Inhibits Platelet Aggregation and ROS Mediated Platelet Apoptosis

Thrombocytopenia is a serious issue connected with the pathogenesis of several human diseases including chronic inflammation, arthritis, Alzheimers disease, cardiovascular diseases (CVDs) and other oxidative stress-associated pathologies. The indiscriminate use of antibiotics and other biological drugs are reported to result in thrombocytopenia, which is often neglected during the treatment regime. In addition, augmented oxidative stress induced by drugs and pathological conditions has also been shown to induce thrombocytopenia, which seems to be the most obvious consequence of elevated rate of platelet apoptosis. Thus, blocking oxidative stress-induced platelet apoptosis would be of prime importance in order to negotiate thrombocytopenia and associated human pathologies. The current study presents the synthesis and platelet protective nature of novel ibuprofen derivatives. The potent anti-oxidant ibuprofen derivative 4f was selected for the study and the platelet protective efficacy and platelet aggregation inhibitory property has been demonstrated. The compound 4f dose dependently mitigates the oxidative stress-induced platelet apoptosis in both platelet rich plasma and washed platelets. The platelet protective nature of compound 4f was determined by assessing various apoptotic markers such as ROS generation, cytosolic Ca2+ levels, PS externalization, cytochrome C translocation, Caspase activation, mitochondrial membrane depolarization, cytotoxicity, LDH leakage and tyrosine phosphorylation of cytosolic proteins. Furthermore, compound 4f dose dependently ameliorated agonist induced platelet aggregation. Therefore, compound 4f can be estimated as a potential candidate in the treatment regime of pathological disorders associated with platelet activation and apoptosis. In addition, compound 4f can be used as an auxiliary therapeutic agent in pathologies associated with thrombocytopenia.

NETosis and lack of DNase activity are key factors in Echis carinatus venom-induced tissue destruction

Indian Echis carinatus bite causes sustained tissue destruction at the bite site. Neutrophils, the major leukocytes in the early defence process, accumulate at the bite site. Here we show that E. carinatus venom induces neutrophil extracellular trap (NET) formation. The NETs block the blood vessels and entrap the venom toxins at the injection site, promoting tissue destruction. The stability of NETs is attributed to the lack of NETs-degrading DNase activity in E. carinatus venom. In a mouse tail model, mice co-injected with venom and DNase 1, and neutropenic mice injected with the venom, do not develop NETs, venom accumulation and tissue destruction at the injected site. Strikingly, venom-induced mice tail tissue destruction is also prevented by the subsequent injection of DNase 1. Thus, our study suggests that DNase 1 treatment may have a therapeutic potential for preventing the tissue destruction caused by snake venom.

Novel apigenin based small molecule that targets snake venom metalloproteases

The classical antivenom therapy has appreciably reduced snakebite mortality rate and thus is the only savior drug available. Unfortunately, it considerably fails to shield the viper bite complications like hemorrhage, local tissue degradation and necrosis responsible for severe morbidity. Moreover, the therapy is also tagged with limitations including anaphylaxis, serum sickness and poor availability. Over the last decade, snake venom metalloproteases (SVMPs) are reported to be the primary component responsible for hemorrhage and tissue degradation at bitten site. Thus, antivenom inability to offset viper venom-induced local toxicity has been a basis for an insistent search for SVMP inhibitors. Here we report the inhibitory effect of compound 5d, an apigenin based molecule against SVMPs both in silico and in vivo. Several apigenin analogues are synthesized using multicomponent Ugi reactions. Among them, compound 5d effectively abrogated Echis carinatus (EC) venom-induced local hemorrhage, tissue necrosis and myotoxicity in a dose dependant fashion. The histopathological study further conferred effective inhibition of basement membrane degradation, and accumulation of inflammatory leucocytes at the site of EC venom inoculation. The compound also protected EC venom-induced fibrin and fibrinogen degradation. The molecular docking of compound 5d and bothropasin demonstrated the direct interaction of hydroxyl group of compound with Glu146 present in hydrophobic pocket of active site and does not chelate Zn2+. Hence, it is concluded that compound 5d could be a potent agent in viper bite management.

Oxidative stress-induced methemoglobinemia is the silent killer during snakebite: a novel and strategic neutralization by melatonin.

Oxidative stress‐induced methemoglobinemia remained an untouched area in venom pharmacology till date. This study for the first time explored the potential of animal venoms to oxidize hemoglobin to methemoglobin. In in vitro whole‐blood assay, methemoglobin forming ability of venoms varied as Naja naja > Ophiophagus hannah > Echis carinatus > Daboia russellii > Apis mellifera > Mesobuthus tamulus > Hippasa partita. Being highly potential, N. naja venom was further studied to observe methemoglobin formation in RBCs and in combinations with PMNs and PBMCs, where maximum effect was observed in RBCs + PMNs combination. Naja naja venom/externally added methemoglobin‐induced methemoglobin formation was in parallel with ROS generation in whole blood/RBCs/RBCs + PMNs/RBCs + PBMCs. In in vivo studies, the lethal dose (1 mg/kg body weight, i.p.) of N. naja venom readily induced methemoglobin formation, ROS generation, expression of inflammatory markers, and hypoxia‐inducible factor‐3α. Although the mice administered with three effective doses of antivenom recorded zero mortality; the methemoglobin and ROS levels remained high. However, one effective dose of antivenom when administered along with melatonin (1:50; venom/melatonin, w/w), not only offered 100% survival of experimental mice, but also significantly reduced methemoglobin level, and oxidative stress markers including hypoxia‐inducible factor‐3α. This study provides strong drive that, complementing melatonin would not only reduce the antivenom load, but for sure greatly increase the success rate of antivenom therapy and drastically minimize the global incidence of snakebite deaths. However, further detailed investigations are needed before translating the combined therapy towards the bed side.

Tamarind Seed (Tamarindus indica) Extract Ameliorates Adjuvant-Induced Arthritis via Regulating the Mediators of Cartilage/Bone Degeneration, Inflammation and Oxidative Stress

Medicinal plants are employed in the treatment of human ailments from time immemorial. Several studies have validated the use of medicinal plant products in arthritis treatment. Arthritis is a joint disorder affecting subchondral bone and cartilage. Degradation of cartilage is principally mediated by enzymes like matrix metalloproteinases (MMPs), hyaluronidases (HAase), aggrecanases and exoglycosidases. These enzymes act upon collagen, hyaluronan and aggrecan of cartilage respectively, which would in turn activate bone deteriorating enzymes like cathepsins and tartrate resistant acid phosphatases (TRAP). Besides, the incessant action of reactive oxygen species and the inflammatory mediators is reported to cause further damage by immunological activation. The present study demonstrated the anti-arthritic efficacy of tamarind seed extract (TSE). TSE exhibited cartilage and bone protecting nature by inhibiting the elevated activities of MMPs, HAase, exoglycosidases, cathepsins and TRAP. It also mitigated the augmented levels of inflammatory mediators like interleukin (IL)-1β, tumor necrosis factor-α, IL-6, IL-23 and cyclooxygenase-2. Further, TSE administration alleviated increased levels of ROS and hydroperoxides and sustained the endogenous antioxidant homeostasis by balancing altered levels of endogenous antioxidant markers. Overall, TSE was observed as a potent agent abrogating arthritis-mediated cartilage/bone degradation, inflammation and associated stress in vivo demanding further attention.

Novel Synthetic Oxazines Target NF-κB in Colon Cancer In Vitro and Inflammatory Bowel Disease In Vivo

Aberrant activation of nuclear factor kappa B (NF-κB) has been linked with the pathogenesis of several proinflammatory diseases including number of cancers and inflammatory bowel diseases. In the present work, we evaluated the anticancer activity of 1,2-oxazines derivatives against colorectal cancer cell lines and identified 2-((2-acetyl-6,6-dimethyl-4-phenyl-5,6-dihydro-2H-1,2-oxazin-3-yl)methyl)isoindoline-1,3-dione (API) as the lead anticancer agent among the tested compounds. The apoptosis inducing effect of API was demonstrated using flow cytometry analysis and measuring the caspase 3/7 activity in API treated cells. Based on the literature on inhibition of NF-κB by oxazines, we evaluated the effect of 1,2-oxazines against the ability of NF-κB binding to DNA, NF-κB-dependent luciferase expression and IκBα phosphorylation. We found that, API abrogate constitutive activation of NF-κB and inhibits IκBα phosphorylation in HCT116 cells. Our in silico analysis revealed the binding of oxazines to the hydrophobic cavity that present between the interface of p65 and IκBα. Given the relevance with aberrant activation of NF-κB in inflammation bowel disease (IBD), we evaluated the effect of API on dextran sulphate sodium-induced IBD mice model. The treatment of IBD induced mice with API decreased the myeloperoxidase activity in colonic extract, modulated the colon length and serum levels of pro- and anti-inflammatory cytokines such as TNF-α, IFN-γ, IL-6, IL-1β and IL-10. Furthermore, the histological analysis revealed the restoration of the distorted cryptic epithelial structure of colon in the API treated animals. In conclusion, we comprehensively validated the NF-κB inhibitory efficacy of API that targets NF-κB in in vitro colon cancer and an in vivo inflammatory bowel disease model.