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Phloretin Alleviates Arsenic Trioxide-Induced Apoptosis of H9c2 Cardiomyoblasts via Downregulation in Ca2+/Calcineurin/NFATc Pathway and Inflammatory Cytokine Release

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Abstract

Arsenic trioxide (ATO) is among the first-line chemotherapeutic drugs for treating acute promyelocytic leukemia patients, but its clinical use is hampered due to cardiotoxicity. The present investigation unveils the mechanism underlying ATO-induced oxidative stress that promotes calcineurin (a ubiquitous Ca2+/calmodulin-dependent serine/threonine phosphatase expressed only during sustained Ca2+ elevation) expression, inflammatory cytokine release and apoptosis in H9c2 cardiomyoblasts, and its possible modulation with phloretin (PHL, an antioxidant polyphenol present in apple peel). ATO caused Ca2+ overload resulting in elevated expression of calcineurin and its downstream transcriptional effector NFATc causing the release of cytokines such as IL-2, IL-6, MCP-1, IFN-γ, and TNF-α in H9c2 cardiomyoblast. There was a visible increase in the nuclear fraction of NF-κB and ROS-mediated apoptotic cell death. The expression levels of cardiac-specific genes (troponin, desmin, and caveolin-3) and genes of the apoptotic signaling pathway (BCL-2, BAX, IGF1, AKT, ERK1, -2, RAF1, and JNK) in response to ATO and PHL were studied. The putative binding mode and the potential ligand–target interactions of PHL with calcineurin using docking software (Autodock and iGEMDOCKv2) showed the high binding affinity of PHL to calcineurin. PHL co-treatment significantly reduced Ca2+ influx and normalized the expression of calcineurin, NFATc, NF-κB, and other cytokines. PHL co-treatment resulted in activation of BCL-2, IGF1, AKT, RAF1, ERK1, and ERK2 and inhibition of BAX and JNK. Overall, these results revealed that PHL has a protective effect against ATO-induced apoptosis and we propose calcineurin as a druggable target for the interaction of PHL in ATO cardiotoxicity in H9c2 cells.

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Acknowledgements

We thank the Director, CSIR- NIIST for providing all the necessary facilities for conducting the experiments. We specially thank the Computational Modeling and Simulation Unit, CSIR-NIIST, for providing the necessary help with the docking study.

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  1. Agroprocessing and Technology Division, National Institute for Interdisciplinary Science and Technology (CSIR - NIIST), Thiruvananthapuram, Kerala, 695019, India

    Vineetha Vadavanath Prabhakaran & Raghu Kozhiparambil Gopalan

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12012_2021_9655_MOESM1_ESM.pdf

The effect of ATO-PHL co-treatment was checked with various cancer cell lines such as pancreatic cancer cell line (BxPC3), breast cancer cell line (MCF7), and colon cancer cell line (SW480) to confirm that the anticancer property of ATO was not compromised by PHL. The cancer cell lines were co-treated with ATO and PHL and were evaluated for any alterations in cell morphology (Fig. S.1). Cytotoxicity was evaluated using MTT (Fig. S.2.a) and lactate dehydrogenase (LDH) release (Fig. S.2.b) assays and the level of the cell protectant glutathione (Fig. S.3) was also assessed with ATO-PHL co-treatment. The toxic effect of ATO on cancer cells was potentiated by the co-treatment with PHL. Supplementary file1 (PDF 520 kb)

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Vadavanath Prabhakaran, V., Kozhiparambil Gopalan, R. Phloretin Alleviates Arsenic Trioxide-Induced Apoptosis of H9c2 Cardiomyoblasts via Downregulation in Ca2+/Calcineurin/NFATc Pathway and Inflammatory Cytokine Release. Cardiovasc Toxicol 21, 642–654 (2021). https://doi.org/10.1007/s12012-021-09655-0

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