A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells
Authors: Biswajit Chakraborty
Padma Das
Ankita Sarkar
Debasmita Dutta
Chinmay Chowdhury
Journal: BMC Cancer (2016)
Discipline: CLINICAL MEDICINE
Keywords: Betulinic acid; BA analogue; autophagy; proteasomal degradation pathway; colon cancer
Affiliations: Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. ., Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. .

Abstract:  Background: Betulinic acid (BA), a member of pentacyclic triterpenes has shown important biological activities like anti-bacterial, anti-malarial, anti-inflammatory and most interestingly anticancer property. To overcome its poor aqueous solubility and low bioavailability, structural modifications of its functional groups are made to generate novel lead(s) having better efficacy and less toxicity than the parent compound. BA analogue, 2c was found most potent inhibitor of colon cancer cell line, HT-29 cells with IC50 value 14.9 μM which is significantly lower than standard drug 5-fluorouracil as well as parent compound, Betulinic acid. We have studied another mode of PCD, autophagy which is one of the important constituent of cellular catabolic system as well as we also studied proteasomal degradation pathway to investigate whole catabolic pathway after exploration of 2c on HT-29 cells. Methods: Mechanism of autophagic cell death was studied using fluorescent dye like acridine orange (AO) and monodansylcadaverin (MDC) staining by using fluorescence microscopy. Various autophagic protein expression levels were determined by Western Blotting, qRT-PCR and Immunostaining. Confocal Laser Scanning Microscopy (CLSM) was used to study the colocalization of various autophagic proteins. These were accompanied by formation of autophagic vacuoles as revealed by FACS and transmission electron microscopy (TEM). Proteasomal degradation pathway was studied by proteasome-Glo™ assay systems using luminometer. Results: The formation of autophagic vacuoles in HT-29 cells after 2c treatment was determined by fluorescence staining--confirming the occurrence of autophagy. In addition, 2c was found to alter expression levels of different autophagic proteins like Beclin-1, Atg 5, Atg 7, Atg 5-Atg 12, LC3B and autophagic adapter protein, p62. Furthermore we found the formation of autophagolysosome by colocalization of LAMP-1 with LC3B, LC3B with Lysosome, p62 with lysosome. Finally, as proteasomal degradation pathway downregulated after 2c treatment colocalization of ubiquitin with lysosome and LC3B with p62 was studied to confirm that protein degradation in autophagy induced HT-29 cells follows autolysosomal pathway. Conclusions: In summary, betulinic acid analogue, 2c was able to induce autophagy in HT-29 cells and as proteasomal degradation pathway downregulated after 2c treatment so protein degradation in autophagy induced HT-29 cells follows autolysosomal pathway. 

Retracted: No

PubPeer comments:
 ## Were the MIND descriptors described in this publication conceived independently of Heinrich et al. s MIND descriptors (not referenced in this publication)? The following excerpt shows the MIND (modality independent descriptors) as described in this publication: ![file] The reference cited for these descriptors ([16]) is [Illumination invariant optical flow using neighborhood descriptors by Ali et al. in CVIU, 2016] . In the referenced CVIU publication, these descriptors are called NND (normalized neighborhood descriptors). A detailed comparison of NND with MIND from [MIND: Modality Independent Neighbourhood Descriptor for Multi-Modal Deformable Registration, by Mattias P. Heinrich et al. in Medical Image Analysis (2012)](http: dx.doi.org 10.1016 j.media.2012.05.008) is given in this post on the PubPeer page of the concerned CVIU publication: https: pubpeer.com publications A09D9A3BCBAEF3F84FD4572344DC56#9 These two descriptors (NND MIND by Ali et al. and MIND by Heinrich et al.) are practically the same (except for a trivial difference: an unexplained lack of normalization in NND). Could the authors explain how this extreme similarity came to be? Any particular reason for naming them differently (MIND instead of NND) in this reproduction? Some other observations along these lines in some publications involving Sharib Ali: https: pubpeer.com publications BD8300AE697D7574A34F74DEC40371#2 https: pubpeer.com publications BD8300AE697D7574A34F74DEC40371#6 https: pubpeer.com publications A09D9A3BCBAEF3F84FD4572344DC56#10 **Some concerns about such observations in Sharib Ali s source code files are raised at the end of this post:** https: pubpeer.com publications A4AA4DAC550DE014EC874D3EAC95F0#11 After these concerns were raised, Sharib Ali has added some sources for at least some of the codes that he had published online with claims of his exclusive authorship and has put **the following [disclaimer in the Readme file] of a relatively recent work, seemingly carried out at his current institute (University of Oxford):**  Some parts of code may have been used from external sources. I have tried to reference it but in case not then please notify me and I will add it here. Why should the readers be responsible for finding out the sources used in others work? This is in sharp contrast with Sharib Ali s [emphatic demands of the acknowledgment of his work] for not only using it but also for making its copy. An example of Sharib Ali s apparent practice of claiming authorship of others source code can be seen in the following excerpt of a comparison that juxtaposes [Ali s version of a file] (on the left) with [its source] (on the right): ![file] In a [later version of this file] , Sharib Ali added the source, but still maintained his claim of the authorship of this file and only acknowledged partial borrowing even though this file seems to be reproduced without any modification in the code. This later version was published after concerns about Ali s such practices were made public at the end of [this post] (these practices seem to involve a large number of code files from various sources, including the implementation of MIND descriptors, originally authored by M. P. Heinrich). A statement from Sharib Ali indicates that he does not believe in acknowledging the use of others work and considers even just mentioning them to be optional ( You do not have to acknowledge but you can cite them ): https: pubpeer.com publications A4AA4DAC550DE014EC874D3EAC95F0#12. This statement from him came in an email exchange (reproduced in [this post] ), where his lack of acknowledgment of Heinrich et al. s work involving MIND descriptors is pointed out. **Note:** It is interesting that MIND by Heinrich et al. was developed at IBME, University of Oxford well before Sharib Ali joined this institute or presented it as NND in [[16] ]. ## Some other observations: The reference [16] ([Illumination invariant optical flow using neighborhood descriptors by Ali et al. in CVIU, 2016] ) is also referenced for the regularization used in this publication. However, [16] seems to reproduce this part from already existing literature. In fact, the method described in it seems to be obtained with a single operation that involves substituting an existing transform (MIND) in an existing minimization framework through switching a function call, as pointed out in these posts:  https: pubpeer.com publications A09D9A3BCBAEF3F84FD4572344DC56#10 https: pubpeer.com publications A09D9A3BCBAEF3F84FD4572344DC56#11. Did the authors develop their minimization framework solution from scratch and independently of the already existing framework of [Drulea and Nedevschi] (not cited in this publication)? The authors state to have used Chambolle s 2004 work to solve the minimization problem at hand, but the notations and formulations (e.g. the use of a linear operator ***K***) closely resemble those of Drulea and Nedevshi s and are reminiscent of [the comparison done here] . Concerns about Sharib Ali s lack of acknowledgment of Drulea and Nedevshi s work (and the replacement of Drulea s authorship headers by Ali s in the former s source code files) in his previous work are also raised [here] . The correlation transform used in the formulation of the data term in Drulea and Nedevschi s work seems surprisingly similar to the one used in Gupta et al s formulation (compare Eq. (7) in the former with Eq. (5) in the latter; the only difference appears to be enforcing some boundaries in the denominator term in the latter s formulation). Given so many observed similarities with Drulea and Nedevschi s work makes one wonder if Gupta et al. were aware of this work and if the development of their work was in any way inspired by this work. **See the following post for highlights of other curious observations in several related publications involving Sharib Ali as an author:** https: pubpeer.com publications DEDEDC04CC598FCF47DCC618BD9A3D#1 ,