Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during Withanolide D-induced apoptosis
Authors: Rajender Sangwan
Susmita Mondal
Kaushik Bhattacharya
Asish Mallick
Chitra Mandal
Journal: PloS one (2012)
Discipline: Multidisciplinary
Keywords: cancer chemotherapy; apoptosis; Withanolide D; mitochondria; p53
Affiliations: Cancer and Cell Biology Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal, India.

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. 

Retracted: Yes

PubPeer comments:
null, 
 Concern about Figure 6D,  spleen panels. There appears to be an overlap in the 40x panels between the untreated and treated panels. Shown with brown boxes. There appears to be many similarities within the 20x  untreated panel. Several of those areas appear to also be visible in the  withad panel. The similarities suggest that the  untreated panel looks sort of like a Frankenstein monster built out of pieces taken from the  withad panel. ![file] , 
 2020 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0228839 After this article [1] was published, concerns were raised about results reported in Figs 1, 2, and 6. Specifically: The same u03b2-actin data are reported for each cell line in Fig 1C and 1D, and similarly the same u03b2-actin data are reported for each cell line in Fig 2C and 2D. The authors explained that panels C and D of each of these figures report results of experiments using the same lysates from the same experiments. Equal amounts of the same protein samples were run on parallel gels to obtain the data reported in panels C and D, as such, the same u03b2-actin controls applied. In Fig 6D, for Spleen data, it appears as though the right half of the 40X untreated panel overlaps with the left half of the 40X WithaD panel. The authors noted that this was due to an error in preparing the WithaD panel and provided a replacement image. In Fig 6D, there are several regions which appear to be repeated within the 20X Spleen Untreated panel, and between the 20X Spleen Untreated and 20X Spleen WithaD panels. The authors commented that animal experiments and subsequent histology were conducted at two different institutes and were unable to clarify the reason(s) for or origins of the similarities. The authors offered replacement data which they noted were obtained by staining additional sections from the original paraffin embedded tissues for this experiment. In follow-up discussions, the authors provided body weight and tumor weight data to support results statements referencing u201cdata not shownu201d, and they noted that all data supporting the results in the article are available except for the original flow cytometry output files. The unresolved concerns about the 20X Spleen data reported in Fig 6D call into question the integrity of the results reported in this figure. In light of this issue, the PLOS ONE Editors retract this article. CM notified the journal that all authors disagree with the retraction. SM and KB confirmed their disagreement. The other authors either could not be reached or did not reply directly. Reference 1.Mondal S, Bhattacharya K, Mallick A, Sangwan R, Mandal C (2012) Bak Compensated for Bax in p53-null Cells to Release Cytochrome c for the Initiation of Mitochondrial Signaling during Withanolide D-Induced Apoptosis. PLoS ONE 7(3): e34277. https: doi.org 10.1371 journal.pone.0034277 ,