Department Of Biochemistry And Microbial Sciences

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    Anti-cancer drug doxorubicin induced cardiotoxicity: Understanding the mechanisms involved in ros generation resulting in mitochondrial dysfunction
    (Rasayan Journal of Chemistry, c/o Dr. Pratima Sharma, 2020) Upadhayay, S; Sharma, N; Mantha, A.K; Dhiman, M.
    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.
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    Assessment of Extract of Syzygium cumini Against Doxorubicin Induced Cardiotoxicity
    (Central University of Punjab, 2018) Chayan, Mukherjee; Monisha Dhiman
    For the past four decades, doxorubicin (DOX) has been used to treat cancer, mainly solid tumours and haematological malignancies. However, clinical community is greatly concerned regarding the administration of this as DOX treatment is commonly associated with dose-dependent cardiotoxicity. Attempts at alleviating drug generated cardiac damage using an extract from different parts of plants with radical scavenging property are a promising area of research. Hydroalcoholic extract derived from fruit pulp of Syzygiumcumini which has a significant antiradical scavenging effect. This study aims to assess the effect of parallel administration of SC fruit pulp extract (SC) on mitigating or preventing DOX induced cardiotoxicity in vitro using H9c2 cardiomyoblast cell lines. Addition of SC fruit pulp extract and DOX were performed for both treatment and control sets on H9c2 cells. SC fruit pulp extract showed strong ABTS cation radical scavenging activity in a dose dependent manner. MTT assay was used to study the cytotoxic effect of SC fruit pulp extract and DOX. ROS levels were estimated using NBT assay and DHE assay. The results showed that DOX has significant cytotoxic effect in a dose dependent manner while SC fruit pulp extract did not display any significant cytotoxicity on H9c2 cells. The DOX induced ROS production was found to be significantly reduced in SC fruit pulp extract treated cells. Results of the current study also suggest that the treatment of SC fruit pulp extract along with DOX, displayed cardioprotective potential in H9c2 cells by: 1) reducing lipid peroxidation; 2) decreasing extracellular nitric oxide (NO); 3) decreasing the expression of the protein p47phox and iNOS/NOS-2. These results clearly suggest that treatment of SC fruit pulp extract along with DOX reduces the DOX induced toxicity and hence can be a promising therapeutic intervention in managing DOX mediated cardiotoxicity.