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Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by N-acetyl cysteine

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Abstract

Induction of apoptosis in cancer cells has become the major focus of anti-cancer therapeutics development. WithaferinA, a major chemical constituent of Withania somnifera, reportedly shows cytotoxicity in a variety of tumor cell lines while its molecular mechanisms of action are not fully understood. We observed that withaferinA primarily induces oxidative stress in human leukemia HL-60 cells and in several other cancer cell lines. The withanolide induced early ROS generation and mitochondrial membrane potential (Δψmt) loss, which preceded release of cytochrome c, translocation of Bax to mitochondria and apoptosis inducing factor to cell nuclei. These events paralleled activation of caspases −9, −3 and PARP cleavage. WA also activated extrinsic pathway significantly as evidenced by time dependent increase in caspase-8 activity vis-à-vis TNFR-1 over expression. WA mediated decreased expression of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Furthermore, withaferinA inhibited DNA binding of NF-κB and caused nuclear cleavage of p65/Rel by activated caspase-3. N-acetyl-cysteine rescued all these events suggesting thereby a pro-oxidant effect of withaferinA. The results of our studies demonstrate that withaferinA induced early ROS generation and mitochondrial dysfunction in cancer cells trigger events responsible for mitochondrial -dependent and -independent apoptosis pathways.

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Abbreviations

AIF:

Apoptosis inducing factor

DCFH-DA:

Dichlorofluorescein diacetate

EMSA:

Electrophoretic mobility shift assay

HPLC:

High Performance Liquid Chromatography

IR:

Infra red

NAC:

N-acetyl-cysteine

NF-κB:

Nuclear factor κB

PI:

Propidium iodide

ROS:

Reactive oxygen species

TNFR:

Tumor necrosis factor receptor

WA:

Withaferin A

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Acknowledgments

We are highly grateful to Dr. M.S.Majumdar of Institute of Microbial Technology, Chandigarh, India for providing facilities to perform EMSA. Thanks are also to the Council of Scientific and Industrial Research, India, for financial support for senior research fellowoships to Fayaz Malik and Ajay Kumar.

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Authors and Affiliations

  1. Division of Pharmacology, Indian Institute of Integrative Medicine, Council of Scientific and Industrial Research, Canal Road, Jammu-Tawi, 180001, India

    Fayaz Malik, Ajay Kumar, Shashi Bhushan, Sheema Khan, Krishan Avtar Suri, Ghulam Nabi Qazi & Jaswant Singh

  2. Department of Biotechnology, Punjabi University, Patiala, Punjab, 147002, India

    Aruna Bhatia

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  1. Fayaz Malik
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Correspondence to Jaswant Singh.

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Malik, F., Kumar, A., Bhushan, S. et al. Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by N-acetyl cysteine. Apoptosis 12, 2115–2133 (2007). https://doi.org/10.1007/s10495-007-0129-x

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