Bak Compensated for Bax in p53-null Cells to Release Cytochrome c for the Initiation of Mitochondrial Signaling during Withanolide D-Induced Apoptosis

The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD), a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53−/− cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53−/− over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53−/− cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax−Bak−) reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax−Bak+/HCT116Bax+Bak−) was only marginally effective after WithaD treatment. In HCT116p53−/− cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells

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

Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growthinhibitory 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