title: A potent betulinic acid analogue ascertains an antagonistic mechanism between autophagy and proteasomal degradation pathway in HT-29 cells.
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.
authors.affiliation: Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. debasmita.biochem@gmail.com.
authors.forename: Debasmita
authors.initials: D
authors.lastname: Dutta
authors.affiliation: Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. biswajitmom@gmail.com.
authors.forename: Biswajit
authors.initials: B
authors.lastname: Chakraborty
authors.affiliation: Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. ankita9sarkar@gmail.com.
authors.forename: Ankita
authors.initials: A
authors.lastname: Sarkar
authors.affiliation: Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. chinmay@iicb.res.in.
authors.forename: Chinmay
authors.initials: C
authors.lastname: Chowdhury
authors.affiliation: Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. padmadas2005@yahoo.co.in.
authors.forename: Padma
authors.initials: P
authors.lastname: Das
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D019073:HT29 Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000096:biosynthesis; D053978:Pentacyclic Triterpenes; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects* / Q000235:genetics; D059748:Proteolysis / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D014315:Triterpenes / Q000008:administration & dosage* / Q000737:chemistry; D014617:Vacuoles / Q000187:drug effects / Q000473:pathology; D000094062:Betulinic Acid
chemical_list: D009363:Neoplasm Proteins; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; D046988:Proteasome Endopeptidase Complex; D000094062:Betulinic Acid