Apigenin-induced apoptosis in A375 and A549 cells through selective action and dysfunction of mitochondria
Authors: Jayeeta Das
Naoual Boujedaini
Anisur Rahman Khuda-Bukhsh
Sreemanti Das
Asmita Samadder
Journal: Experimental Biology and Medicine (2012)
Discipline: CLINICAL MEDICINE
Keywords: apigenin; anticancer potential; apoptosis induction; DNA interaction; mitochondrial dysfunction
Affiliations: Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India, Boiron Laboratory, 20 rue de la Libération - 69110 Sainte Foy-lès, Lyon, France

Abstract:  We isolated apigenin (5,7,4'-trihydroxy flavone) from ethanolic extract of Lycopodium clavatum (LC) used as a homeopathic mother tincture for treatment of various diseases. We assessed the anticancer potentials of the compound using human malignant melanoma cell line A375 and a lung carcinoma cell line A549 and focussed on its putative molecular mechanism of action on apoptosis induction. We examined the cytotoxicity of apigenin in both cancer cells and normal peripheral blood mononuclear cells (PBMC). A375 cells were more prone to apigenin-induced apoptosis, as compared with A549 cells after 24 h of treatment, while PBMC showed little or no cytotoxicity to apigenin. We also evaluated the effects of apigenin on interaction with DNA by comparative analysis of circular dichroism spectral data and melting temperature profiles (Tm) of calf thymus DNA (CT-DNA) treated with or without apigenin. Reactive oxygen species (ROS) accumulation in mitochondria, super-oxide dismutase and total thiol group (GSH) activities were also analyzed. The apoptotic process involved mitochondrial pathway associated with apigenin-DNA interaction, DNA fragmentation, ROS accumulation, cytochrome c (cyt c) release and mitochondrial transmembrane potential depolarization, Bax, caspase 3, 9, PARP, up-regulation, Bcl-2 down-regulation and down-regulation of cyt c in the mitochondrial fraction. Results of mitochondrial inner membrane swelling measurements, intracellular ADP/ATP ratio and ATPase activity showed that in A549 cells, apigenin did not appear to directly target the mitochondrial oxidative phosphorylation system but rather acted at an upstream step to activate the mitochondrial apoptotic pathway. However, apigenin could directly target and impair mitochondrial function in A375 cells by breaking down their oxidative phosphorylation system. Collectively, these results suggest that apigenin exhibits anticancer potential in A375 and A549 cells that may be mediated through DNA interaction, damage and mitochondrial dysfunction either by direct or indirect action on mitochondrial oxidative phosphorylation system. 

Retracted: No

PubPeer comments:
 Concern regarding the data published by Sreemanti das et al 2012 Identical images have been used as a result of different treatments. Significant portions (dots events) of one histogram plot is matching with other histogram plots. Below is one such example (plot C and plot F). ![file] ![file] When combined, several events overlapped (click on the image for gif visualization) ![file] , 
 Fig 7A. In panel C, cells are much bigger than in rest of the panel. For better view contrast enhanced. ![file] , 
 Similar concern in Figure 8B. When roughly 20% from one blot was cut off, stretched vertically the blot looks similar to another blot . See below for detail. Similar looking areas of the two blots (in square box) marked with pink arrows. First original them. Below highlighted. ![file] ![file] , 
 **Overlapping areas encircled** ![file] , 
 Concern with another figure presented at figure 8A. Look carefully to see the similarity. **The bands of G3PDH protein from A375 cells is unusually identical with G3PDH protein bands of A549 cells when flipped horizontally** ![file] , 
 #1 Thanks for your attention on our paper published in 2012. Though I retired in 2013 and away fully from active research since 2015 (after completion of my emeritus fellowship), I still looked into your observation again and also consulted my coauthors, particularly the first author, and now respond to your queries one by one. 1.tThe FACS data are instrument generated. As the no. of events were same and also same gating was used, hence the FACS data in quadrant 1 only shows some similarities at some points. But if observed and compared closely and properly, these are definitely not similar images. In case of immunofluorescence data, this data was repeated thrice. It was observed that with this specific drug concentration, every time the cells were getting elongated and with bipolar shape. We could not understand it clearly till we got a similar reference (Endothelin-induced Apoptosis of A375 Human Melanoma Cells. The Journal of Biological Chemistry, 1998, 12584-12592) when we could finally interpret the data. This reference suggests that induction of pro apoptotic activity after 24hrs may cause drastic morphological changes of the surviving cells from epitheloid to an elongated bipolar shape. You can also see a current reference in support of this contention: (Effect of ABT-888 on the apoptosis, motility and invasiveness of BRAFi-resistant melanoma cells. Int J Oncol, 2018, 1149-1159). , 
 2.tThe blots were definitely different. If they are observed minutely it can be clearly understood that they are not at all similar. If there are similar areas, that may happen by chance which is sometimes unavoidable in some cases. 3.tThe FACS data are instrument generated. The four quartets should be observed. As the same gating and same no. of events have been used, this may be possible. There is no question of overlap. Also cell no. was counted before doing FACS. 4.tThe PCR images as observed in the zoomed image, are significantly different in most of the areas. If one band portion is similar to another minute part of another band portion then that does not validate that the data has been copied. Even band intensities are totally different which have been normalized. , 
 #6 1. Firstly, thank you professor Khuda-Bukhsh for taking the time to read the comments and reply. We understand that all FACS data are instrument generated. In such experiments the counted events and gating should always have to be exactly same, otherwise the data won t make any sense. But it doesn t mean same settings would generate dots in identical positions in the plot in several experiments (I just observed similar thing in another figure, that may appear here someday). We understand there would be some overlapped area, but the  nature of dots clearly showing those have been replicated. With due respect I would like to inform you that, we had a very close look in big screen, and trust me these are unreal . 2. For the immunofluorescence image, I was thinking, that might have happened due to higher magnification (set mistakenly) while acquiring the image. 3. Again, I very much understand your point. But it s the kind of similarities, which is completely unrealistic. It simply can t match (read overlap) each other completely. 4. In FACS experiments, how many quadrants should be considered that entirely depends on the question, why the assay is being carried out. There is absolutely no need to counts the cells for FACS assays. The instrument itself counts the cells quite accurately and stop counting after completion of set number of events . As you know, depending on your settings, these events are the counts. You only need to count cells prior FACS acquisition just to make sure that high concentration of cells beads don t clog the channel. Again, I repeat, these similarities are unexpected. 5. I completely disagree that the G3PDH bands are different. Although you mentioned that  the band intensities are totally different but all I see that, they are all stated as value 1 (for G3PDH protein bands from two different cell lines). , 
 Thanks for your clarifying note and your further comments. Please allow me to respond to your specific points one by one again as per our understanding. Your points #1,3 4. Answer: We did not have any facility to amplify the figures in the way you perhaps could, and that may be the reason why the learned Peer-Reviewers of the paper also possibly were not in a position to compare so closely. In order to avoid such unforeseen problem in comparing the FACS results we did not depend solely on a single protocol and compared results of several other protocols parameters (e.g., DAPI staining, DNA Fragmentation Assay, Cytochrome-c release from mitochondria, depolarization of mitichondrial membrane potential, AnnexinV-FITC, most importantly ROS generation, etc) to arrive at a specific inference (in this case anticancer activity and the possible pathways). In biological systems, particularly in case of cancer cells , there may be some subtle variations in some cases and in rather lower magnification, some observation may also suffer from human error or variation. Therefore, what may look like subtly dissimilar at this magnification, even when compared, with amplified figures, to us (we noted some degree of dissimilarlity between the Fig. C and Fig. F), which you term as the  same and  unreal . However, the number of learned reviewers of the journals were either convinced about our observation, or might not have also enlarged with other gadgets to question this issue. They quite agreed with our observation. #2 . We hope that you agree with our observation on the immunofluorescence data in which higher magnification would have given much larger morphological features of the cells and not like the ones shown in these figures. #5. Sorry, I mistakenly referred to G3PDH band in the Western Blots to be different in my earlier response, which is actually similar, as this is the representative House-keeping gene expression. Though we used the same band as representative of house-keeping gene, and the the learned reviewers also did not object to this, we possibly could have used another band of the same gene from another cell line, which could look like very similar, but not exactly the same. , 
 2020 expression of concern. https: journals.sagepub.com doi full 10.1177 1535370219899001 SAGE and the Editor of Experimental Biology and Medicine hereby issue an expression of concern for the following article: Das S, Das J, Samadder A, Boujedaini N, Khuda-Bukhsh AR. Apigenin-induced apoptosis in A375 and A549 cells through selective action and dysfunction of mitochondria. Experimental Biology and Medicine. 2012;237(12):1433--48 The Editor and SAGE were alerted to manipulation of images used in the paper. The institutions of the authors of this article have been contacted and asked to undertake an investigation. The Editor and SAGE strive to uphold the very highest standards of publication ethics and are committed to supporting the high standards of integrity of Experimental Biology and Medicine. Authors, reviewers, editors and interested readers should consult the ethics section of the SAGE website for guidelines on publication ethics. , 
 #10 It would be appreciated if Dr. Khuda-Bukhsh could also respond to some of the other papers with questions on PubPeer: https: pubpeer.com search?q=Anisur+Rahman+Khuda-Bukhsh , 
 #1  When combined, several events overlapped (click on the image for gif visualization) The GIF is nice. Here is my own visualisation, colour-reversing panel (F) and superimposing it on (C) so that identical details cancel out. It is clear that the discrepancies between the two panels are confined to small rectangular areas. ![file] Regions of (C) are more similar than one would expect [black rectangles]. ,