title: Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during Withanolide D-induced apoptosis.
abstract: 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.
authors.affiliation: Cancer and Cell Biology Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal, India.
authors.forename: Susmita
authors.initials: S
authors.lastname: Mondal
authors.forename: Kaushik
authors.initials: K
authors.lastname: Bhattacharya
authors.forename: Asish
authors.initials: A
authors.lastname: Mallick
authors.forename: Rajender
authors.initials: R
authors.lastname: Sangwan
authors.forename: Chitra
authors.initials: C
authors.lastname: Mandal
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020014:K562 Cells; D051379:Mice; D008819:Mice, Nude; D008928:Mitochondria / Q000378:metabolism*; D009368:Neoplasm Transplantation; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D054358:Withanolides / Q000494:pharmacology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000502:physiology*
chemical_list: D000970:Antineoplastic Agents; C494772:BAK1 protein, human; C494826:BAX protein, human; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D054358:Withanolides; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C010376:withanolide D; D045304:Cytochromes c