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Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade

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Abstract

Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growth-inhibitory effect on several human leukemic cell lines and on primary cells from patients with lymphoblastic and myeloid leukemia in a dose-dependent manner, showing no toxicity on normal human lymphocytes and primitive hematopoietic progenitor cells. WA-mediated decrease in cell viability was observed through apoptosis as demonstrated by externalization of phosphatidylserine, a time-dependent increase in Bax/Bcl-2 ratio; loss of mitochondrial transmembrane potential, cytochrome c release, caspases 9 and 3 activation; and accumulation of cells in sub-G0 region based on DNA fragmentation. A search for the downstream pathway further reveals that WA-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2 and HSP27 in leukemic cells. The RNA interference of p38MAPK protected these cells from WA-induced apoptosis. The RNAi knockdown of p38MAPK inhibited active phosphorylation of p38MAPK, Bax expression, activation of caspase 3 and increase in Annexin V positivity. Altogether, these findings suggest that p38MAPK in leukemic cells promotes WA-induced apoptosis. WA caused increased levels of Bax in response to MAPK signaling, which resulted in the initiation of mitochondrial death cascade, and therefore it holds promise as a new, alternative, inexpensive chemotherapeutic agent for the treatment of patients with leukemia of both lymphoid and myeloid origin.

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Abbreviations

ALL:

Acute lymphoblastic leukemia

DMSO:

Dimethyl sulphoxide

DTT:

Dithiothreitol

dUTP:

2′-Deoxyuridine 5′-triphosphate

TUNEL:

Terminal deoxyneuleotidyltransferase enzyme-mediated dUTP end labeling

FITC:

Fluorescein isothiocyanate

JC-1:

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide

Δψm :

Mitochondrial trans-membrane potential

MS:

Mass spectroscopy

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

2D NMR:

Two-dimensional nuclear magnetic resonance

1HNMR:

1H (proton) Nuclear magnetic resonance

PBMC:

Peripheral blood mononuclear cells

PI:

Propidium iodide

WA:

Withaferrin A

Z-VAD-fmk:

Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl keton

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Acknowledgments

Chandan Mandal and Avijit Dutta are Senior Research Fellowship from the University Grant Commission and Indian Council of Medical Research respectively. This work received financial assistance from the Council of Scientific and Industrial Research, I.I.C.B. and Department of Biotechnology, Govt. of India. Suchandra Chowdhury and Susmita Mondal are acknowledged for their help in processing patient samples. The information provided in this report is protected by pending Indian patent application.

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Correspondence to Chitra Mandal.

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Mandal, C., Dutta, A., Mallick, A. et al. Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade. Apoptosis 13, 1450–1464 (2008). https://doi.org/10.1007/s10495-008-0271-0

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Keywords

  • Acute lymphoblastic leukemia
  • Myeloid leukemia
  • Withaferin A
  • Apoptosis
  • Activation of p38 MAPK
  • Mitochondrial death cascade
  • Transcription factors