A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
View Archive Info| Field | Value | |
| Title |
A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation
pathway in HT-29 cells
|
|
| Creator |
Dutta, Debasmita
Chakraborty, Biswajit Sarkar, Ankita Chowdhury, Chinmay Das, Padma |
|
| Subject |
Cancer Biology and Inflammatory Disorder Division
Chemistry |
|
| Description |
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.
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.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. 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
|
|
| Publisher |
BioMed Central
|
|
| Date |
2016
|
|
| Type |
Article
PeerReviewed |
|
| Format |
application/pdf
|
|
| Identifier |
http://www.eprints.iicb.res.in/2446/1/BMC_CANCER__Volume__16______Article_Number__23;2016.pdf
Dutta, Debasmita and Chakraborty, Biswajit and Sarkar, Ankita and Chowdhury, Chinmay and Das, Padma (2016) A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells. BMC Cancer, 16 (23). 01p.-19p.. |
|
| Relation |
http://dx.doi.org/10.1186/s12885-016-2055-1
http://www.eprints.iicb.res.in/2446/ |
|