Department Of Biochemistry And Microbial Sciences

Doxorubicin (DOX), despite being an effective anti-cancer drug has offsite targets that affect the vital organs such as heart, brain and kidney. DOX-induced cardiotoxicity is reported as a multi-factorial process that interferes with mitochondrial bioenergetics. These responses increase the threshold of oxidant-mediated injury and redox-mediated apoptosis in the cardiomyocytes. Oxidative stress particularly mitochondrial dysfunction in cardiomyocytes associated with cardiovascular diseases. In the present study we examined the effect of DOX on H9c2 cardiomyocyte where cells were treated with 5 μM DOX. To rule out the source of reactive oxygen species (ROS) during DOX-induced toxicity, the DOX-treated cardiomyocytes were incubated with 100 ?M diphenyleneiodonium (DPI), 50 μM salicyl hydroxamic acid (SHX), 20 μM Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), which are specific inhibitors of NADPH oxidase (NOX), Myeloperoxidase (MPO), and mitochondrial oxidative phosphorylation respectively and 10 μM N-acetyl cysteine (NAC, free radical scavenger) was also used to perceive the role of ROS. H2O2 (100 ?M) treated H9c2 cardiomyocytes were used as positive controls. The cell viability, reactive oxygen species (ROS) level and oxidative stress were determined using MTT assay, NBT assay/Flow-cytometry and Western blotting based assays. The effect of DOX on mitochondria was evaluated using Amplex Red assay; fluorescent probes such as MitoSOX and MitoTracker were used to examine the DOX-induced ROS production from the mitochondrial matrix. The mitochondrial membrane potential was evaluated using JC-1 dye. Western blotting was performed for cytochrome c release and apoptosis was examined with Annexin V-FITC assay. DOX was found to reduce cell viability, increase ROS level followed by enhanced oxidative stress in the form of protein carbonyls. DOX also showed a reduction in the mitochondrial membrane potential and allowed the release of cytochrome c which further leads to apoptosis and cell death. Further to rule out the pathway/mechanism(s) of DOX-mediated cardiac pathologies, the treatment with inhibitors of the classical ROS sources such as NADPH oxidase, Myeloperoxidase, mitochondria and general ROS scavenger (NAC) suggested that ROS via NOX and MPO during DOX-induced toxicity plays a crucial role in cardiomyocytes. The mitochondrial integrity was conserved when the cells were treated with NOX and MPO inhibitors, the cytochrome C release and apoptosis reduced in presence of these inhibitors. Taken together, these results demonstrate that DOX leads to ROS production and oxidative stress in cardiomyocytes which ultimately affects the mitochondrial integrity and functions, most importantly the ROS released via NOX and MPO is critical during DOX-induced cardiotoxicity. - RAS?YAN. All rights reserved.

Tumor micro-environment has potential to customize the behavior of the immune cell according to their need. In immune-eliminating phase, immune cells eliminate transformed cells but after tumor establishment innate and adaptive immune cells synergistically provide shelter as well as fulfill their requirement that helps in progression. In between eliminating and establishment phase, equilibrium and escaping phase regulate the immune cells response. During immune-escaping, (1) the antigenic response generated is either inadequate, or focused entirely on tolerance, and (2) immune response generated is specific and effective, but the tumor skips immune recognition. In this review, we are discussing the critical role of immune cells and their cytokines before and after the establishment of tumor which might play a critical role during immunotherapy. ? 2018 Wiley Periodicals, Inc.

Department Of Biochemistry And Microbial Sciences

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