Apoptosis

, Volume 13, Issue 12, pp 1450–1464

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

  • Chandan Mandal
  • Avijit Dutta
  • Asish Mallick
  • Sarmila Chandra
  • Laxminarain Misra
  • Rajender S. Sangwan
  • Chitra Mandal
Original Paper

DOI: 10.1007/s10495-008-0271-0

Cite this article as:
Mandal, C., Dutta, A., Mallick, A. et al. Apoptosis (2008) 13: 1450. doi:10.1007/s10495-008-0271-0

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.

Keywords

Acute lymphoblastic leukemiaMyeloid leukemiaWithaferin AApoptosisActivation of p38 MAPKMitochondrial death cascadeTranscription factors

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

Supplementary material

10495_2008_271_MOESM1_ESM.doc (58 kb)
[INSERT CAPTION HERE] (DOC 57 kb)

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Chandan Mandal
    • 1
  • Avijit Dutta
    • 1
    • 2
  • Asish Mallick
    • 1
  • Sarmila Chandra
    • 3
  • Laxminarain Misra
    • 4
  • Rajender S. Sangwan
    • 4
  • Chitra Mandal
    • 1
  1. 1.Department of Infectious diseases and ImmunologyIndian Institute of Chemical BiologyKolkataIndia
  2. 2.Division of Infectious Disease, Department of MedicineChang Gung University School of Medicine and HospitalTaoyuanTaiwan
  3. 3.Kothari Medical CenterKolkataIndia
  4. 4.Central Institute of Medicinal and Aromatic Plants P.O. C.I.M.A.PLucknowIndia