A distinctive feature of the early stages of apoptosis is the disruption of active mitochondria, including changes in membrane potential and alterations to redox potential. In healthy live cells, jc - 1 selectively accumulates in mitochondria and forms j-aggregates that exhibit red fluorescence whereas in apoptotic cells with reduced mitochondria membrane potential, jc-1 exists as a monomer resulting in green fluorescence.
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Pinostrobin (PN) is a naturally occurring dietary bioflavonoid, found in various medicinal herbs/plants. Though anti-cancer potential of many such similar constituents has been demonstrated, critical biochemical targets and exact mechanism for their apoptosis-inducing actions have not been fully elucidated. The present study was aimed to investigate if PN induced apoptosis in cervical cancer cells (HeLa) of human origin. It is demonstrated that PN at increasing dose effectivity reduced the cell viability as well as GSH and NO2- levels. Condensed nuclei with fragmented chromatin and changes in mitochondrial matrix morphology clearly indicated the role of mitochondria in PN induced apoptosis. A marked reduction in mitochondrial membrane potential and increased ROS production after PN treatment showed involvement of free radicals, which in turn further augment ROS levels. PN treatment resulted in DNA damage, which could have been triggered by an increase in ROS levels. Decrease in apoptotic cells in the presence of caspase 3 inhibitor in PN-treated cells suggested that PN induced apoptosis via caspase dependent pathways. Additionally, a significant increase in the expression of proteins of extrinsic (TRAIL R1/DR4, TRAIL R2/DR5, TNF RI/TNFRSF1A, FADD, Fas/TNFRSF6) and intrinsic pathway (Bad, Bax, HTRA2/Omi, SMAC/Diablo, cytochrome C, Pro-Caspase-3, Cleaved Caspase-3) was observed in the cells exposed to PN. Taken together, these observations suggest that PN efficiently induces apoptosis through ROS mediated extrinsic and intrinsic dependent signaling pathways, as well as ROS mediated mitochondrial damage in HeLa cells.
Citation: Jaudan A, Sharma S, Malek SNA, Dixit A (2018) Induction of apoptosis by pinostrobin in human cervical cancer cells: Possible mechanism of action. PLoS ONE 13(2): e0191523. doi:10.1371/journal.pone.0191523
Editor: Yi-Hsien Hsieh, Institute of Biochemistry and Biotechnology, TAIWAN
Received: September 9, 2017; Accepted: January 5, 2018; Published: February 8, 2018
Copyright: © 2018 Jaudan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The Jawaharlal Nehru University, New Delhi, India is acknowledged for providing financial support. The Department of Science and Technology, New Delhi, India is acknowledged for PURSE grant [SR/PURSE/Phase2/11(C) 2015] to the Jawaharlal Nehru University, New Delhi, India. The Department of Biotechnology, Ministry of Science and Technology, India is acknowledged for providing research fellowship to AJ. Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi is acknowledged for providing the TEM, FACS, Confocal and Live Cell Imaging Facilities.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ΔΨm, Mitochondrial membrane potentia;DCFH-DA, 2, 7-Dichlorodihydrofluoresceindiacetate;JC-1, 5,5,6,6-Tetrachloro-1,1,3,3-tetraethylimidacarbocyanine;HPV, Human papilloma virus;Cyt c, cytochrome c;Smac, Second mitochondria-derived activator of caspases;ApoAF-1, Apoptotic protease activating factor-1;IAP, Inhibitor of apoptosis protein;FITC, Fluorescein isothiocyanate;PI, Propidium iodide;PN, Pinostrobin;DX, Doxorubicin
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The mechanism(s) involved in the clearance of senescent platelets are largely unknown. We have recently demonstrated that platelet aging in vivo is associated with loss of membrane phospholipid asymmetry, a universal phenomenon in cells undergoing apoptosis. Thus, we postulated that senescent platelets may exhibit programmed cell death changes. which may trigger their removal from circulation. Since platelets contain the apoptosis machinery as well as mitochondria, a key organelle in the regulation of apoptosis, we studied the appearance of apoptotic-like changes during platelet aging in vivo. To investigate this, we assessed changes in mitochondrial membrane potential (deltapsi) in circulating canine platelets during decline in platelet count after suppression of thrombopoiesis by estradiol injection, a validated model to obtain circulating platelets of increasing mean age. Phosphatidylserine (PS) exposure was determined by flow cytometry by binding of FITC-labeled annexin V. Mitochondrial deltapsi was studied with the cationic lipophilic dye DIOC6 (3) and the J-aggregate-forming cation JC-1 and analysis by flow cytometry. The proportion of platelets with exposed PS rose significantly with age, from 2.88% before to 6.7%, 8 days after estradiol injection. By flow cytometry it was demonstrated a significant decreased in DIOC6 (3) fluorescence (median fluorescence intensity 791+/-98 vs 567+/-102 day 0 vs day 8 post injection of estradiol, respectively; n: 11; p <0.01), consistent with mitochondrial deltapsi collapse