Histone deacetylase inhibitors sensitize lung cancer cells to hyperthermia: involvement of Ku70/SirT-1 in thermo-protection
Authors: Zainab Mohamed
Mohamed K Hassan
Yusuke Ohba
Ahmed S Sultan
Hidemichi Watari
Noriaki Sakuragi
Yoichiro Fujioka
Alaa-Eldin Salah-Eldin
Journal: PLoS ONE (2014)
Discipline: Multidisciplinary
Keywords: thermal stress; histone deacetylase inhibitors; Ku70; lung cancer cells; hyperthermia
Affiliations: Department of Obstetrics and Gynecology, Hokkaido University, Graduate School of Medicine, Sapporo, Japan; Biotechnology program, Department of Zoology, Faculty of Science, Port Said University, Port Said, Egypt., Department of Obstetrics and Gynecology, Hokkaido University, Graduate School of Medicine, Sapporo, Japan., Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, United States of America., Department of Cell Physiology, Hokkaido University, Graduate School of Medicine, Sapporo, Japan.

Abstract:  This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5°C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer. 

Retracted: Yes

PubPeer comments:
 Figure 1A and S1: Some panels in Figure 1A (PC-10 cells) appear to look similar or show an overlap with panels in Figure S1 (H1299 cells). In some cases panels appear to have been rotated. ![file] , 
 Figure 2A 2B 4A S4. Some panels or subsets of panels appear to look more similar to each other than expected. Boxes of the same color show lanes that look similar. ![file] , 
 Figure 6A. * Green arrows: In the Actin panel, lanes 1-3 look similar to lanes 4-6 if one of these sets is rotated 180 degrees. A vertical line appears to be visible between lanes 3 and 4. ![file] , 
 Figure S6. * Aqua arrows: sharp horizontal and vertical background transitions appear to be visible. ![file] , 
 Figure 2A and Figure 7B. * In the Ponceau S panel of Figure 2A, lanes 2 and 3 appear to look similar. * Lanes 1, 2, 4, and 5 in Figure 2A s Ponceau S panel look similar to lanes 1-4 in Figure 7A s Ponceau S panel. The two panels appear to show a different experiment (with or without Control siRNA) and the right lanes represent different timepoints. ![file] , 
 Retracted on August 6, 2020. Here is the retraction notice: https: journals.plos.org plosone article?id=10.1371 journal.pone.0237589 From the retraction notice:  *After this article [1] was published, concerns were raised about several of the reported results. (...)* *A corresponding author commented on the concerns and in some cases disagreed with the issues noted. Supporting data from related or replicate experiments were provided in response to some of the concerns, but the author noted that the original data underlying results reported in [1] are not available, except for the microscopy images provided in support of Fig 1A and S1 Fig. Without the original data, the image concerns cannot be clarified.* *In light of the above concerns which call into question the reliability of the reported results, the PLOS ONE Editors retract this article.* *HW, YF, YO and NS agreed with retraction. A-eS-e, ASS, and ZM either could not be reached or did not respond directly. MKH disagreed with the retraction and noted that he stands by the findings reported in the article.* , 
 2020 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0237589 After this article [1] was published, concerns were raised about several of the reported results. Specifically: Similarities were noted between the following panels of Fig 1A and S1 Fig. These two figures report results of experiments using different cell lines. u25cb. Fig 1A No treatment, S1 Fig No treatment, and S1 Fig Hyperthermia only u25cb. Fig 1A NM only and S1 Fig NAM only u25cb. Fig 1A TSA only and S1 Fig TsA only u25cb. Fig 1A TSA+NAM only and S1 Fig NAM+TsA only u25cb. Fig 1A TSA+NAM+hyperthermia and S1 Fig NAM+hyperthermia u25cb. S1 Fig TsA+hyperthermia and S1 Fig NAM+TsA+hyperthermia The authors provided some image data to support these experiments and commented that the PC-10 results had been shown in S1 Fig in error. For several panels, there were discrepancies in data labels provided in the published figures versus in the image data file provided post-publication, and as such, questions about the reported results remain unresolved for both the PC-10 and H1299 experiments. Furthermore, based on the data received, it is unclear whether the results reported for H1299 cells were supported by the data. In Fig 1, no loading control data are reported for the Bax western blot experiment in panel C, and panel D (described in the legend) is missing from the figure. An author commented that some proteins commonly used as loading controls (e.g. actin and tubulin) may be affected by hyperthermia and so they routinely use CBB or Ponceau S staining to visualize protein levels. Loading control data for the Fig 1C experiment are not available. Data were not provided to support the claims made in the article based on the missing Fig 1D panel. Similarities were noted between the following western blot panels: u25cb. Fig 2A Bax lanes 1u20134, which reports a western blot using whole cell extracts as input, and Fig 3A Bax, which reports an immunoprecipitation-western blot experiment. u25cb. Fig 2A BclxL (lanes 2, 3) and S4 Fig BclxL. u25cb. Fig 2A Bcl2, lanes 4 and 5 appear similar and there appears to be a vertical discontinuity near the left side of lane 5. u25cb. Fig 2A Ku70 (lanes 2u20135) and Fig 4A AntiKu70 Ab (W.B.). In response to this, the authors noted that the same data applied in the two experiments. However, different timepoints are indicated for these data in the two figures. u25cb. Fig 2A Ku70 (lanes 4u20135) and S4 Fig Ku70; these data represent results for different cell lines and different time points. u25cb. Fig 2B Bcl2 40 u03bcg panel and S4 Fig Bcl2. u25cb. Fig 2B BclxL panels for 20 and 40 u03bcg. u25cb. In the Fig 4C Ku70 panel, lanes 2 and 3 appear similar, when aspect ratio is adjusted. u25cb. In the Fig 6A Actin panel, lanes 1u20133 appear similar to lanes 4u20136 when rotated 180 degrees. The author commented that this may have been due to an error. In Fig 2A, lanes 2 and 3 of the Ponceau S panel appear similar. A corresponding author commented that these similarities are due to similarities in loading. However, per our editorial assessment, the image data in the indicated lanes are more similar than would be expected for different experimental results. In addition, lanes 1, 2, 4, and 5 of the Fig 2A Ponceau S panel appear similar to lanes 1u20134, respectively, of the Fig 7B Control siRNA Ponceau S panel. These represent different experiments. In Fig 3B, there appears to be a vertical discontinuity suggestive of image splicing after lane 4 of the Ku70 blot, and there is no visible image data in lane 5 of this blot. The author confirmed that there is no image data in lane 5 of the blots in this figure (except for Bax) but did not clarify whether the figure includes negative data for 6-h (IgG) samples, or whether 6-h (IgG) data were not included in the Ku70 and Ku80 panels. In S6 Fig, there appear to be several vertical and or horizontal discontinuities suggestive of image splicing, and in the IP:Bax Ab, Bax panel there appears to be a box overlay in lane 5 that is discontinuous with the rest of the panel image. The authors commented that images were spliced between lanes 4 and 5 in preparing the figures; they did not comment on the other visible discontinuities. The backgrounds of several western blot panels reported in the article appear uniform and or adjusted such that one cannot distinguish features in the background image. A corresponding author commented that the quality of the reported images may have been affected by the imaging technique: according to the author, results were photographed using polaroid cameras and then scanned to obtain digital images. A corresponding author commented on the concerns and in some cases disagreed with the issues noted. Supporting data from related or replicate experiments were provided in response to some of the concerns, but the author noted that the original data underlying results reported in [1] are not available, except for the microscopy images provided in support of Fig 1A and S1 Fig. Without the original data, the image concerns cannot be clarified. In light of the above concerns which call into question the reliability of the reported results, the PLOS ONE Editors retract this article. HW, YF, YO and NS agreed with retraction. A-eS-e, ASS, and ZM either could not be reached or did not respond directly. MKH disagreed with the retraction and noted that he stands by the findings reported in the article. Reference 1.Hassan MK, Watari H, Salah-eldin A-e, Sultan AS, Mohamed Z, Fujioka Y, et al. (2014) Histone Deacetylase Inhibitors Sensitize Lung Cancer Cells to Hyperthermia: Involvement of Ku70 SirT-1 in Thermo-Protection. PLoS ONE 9(4): e94213. https: doi.org 10.1371 journal.pone.0094213 ,