A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells

Abstract

BackgroundBetulinic 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.MethodsMechanism 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.ResultsThe 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.ConclusionsIn 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.

Simultaneous activation of impaired autophagy and the mammalian target of rapamycin pathway in oral squamous cell carcinoma

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ER stress and impaired autophagy flux in neuronal degeneration and brain injury

“…Autophagy flux is defined as the whole process of cargo moving through the autophagy system, from phagophore formation to autophagosome formation, autophagosome-lysosome fusion, and eventually cargo degradation plus recycling. Autophagy is a dynamic, not static process, and TEM is the gold standard method for assessing the presence of autophagic vesicles, like autophagosomes, lysosomes, autolysosomes (Dutta et al, 2016). Autophagosomes are confirmed by transmission electron microscopy (TEM) as double-membrane organelles with diameters of about 0.3–0.9 μm in yeast (Baba et al, 1997) and 0.5–1.5 μm in mammals (Mizushima et al, 2002).…”

Overaccumulation of p53-mediated autophagy protects against betulinic acid-induced apoptotic cell death in colorectal cancer cells

“…Furthermore, BA or BA analog could induce autophagy in HeLa, A549, MCF7, SW480 and HT29 cells, and autophagic salvage counterbalanced BA-induced cell death in HeLa cells. 10 , 37 Although studies observed that BA could induce autophagy, the role of autophagy as a cell death mechanism was not minutely addressed for BA-treated CRC cells.…”