PMID- 26772983
OWN - NLM
STAT- MEDLINE
DCOM- 20161005
LR  - 20221207
IS  - 1471-2407 (Electronic)
IS  - 1471-2407 (Linking)
VI  - 16
DP  - 2016 Jan 16
TI  - A potent betulinic acid analogue ascertains an antagonistic mechanism between 
      autophagy and proteasomal degradation pathway in HT-29 cells.
PG  - 23
LID - 10.1186/s12885-016-2055-1 [doi]
LID - 23
AB  - 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 muM 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.
FAU - Dutta, Debasmita
AU  - Dutta D
AD  - 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.
FAU - Chakraborty, Biswajit
AU  - Chakraborty B
AD  - Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical 
      Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. 
      biswajitmom@gmail.com.
FAU - Sarkar, Ankita
AU  - Sarkar A
AD  - Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of 
      Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. 
      ankita9sarkar@gmail.com.
FAU - Chowdhury, Chinmay
AU  - Chowdhury C
AD  - Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical 
      Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. chinmay@iicb.res.in.
FAU - Das, Padma
AU  - Das P
AD  - 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.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20160116
PL  - England
TA  - BMC Cancer
JT  - BMC cancer
JID - 100967800
RN  - 0 (Neoplasm Proteins)
RN  - 0 (Pentacyclic Triterpenes)
RN  - 0 (Triterpenes)
RN  - EC 3.4.25.1 (Proteasome Endopeptidase Complex)
RN  - 4G6A18707N (Betulinic Acid)
SB  - IM
MH  - Autophagy/*drug effects
MH  - Colonic Neoplasms/*drug therapy/genetics/pathology
MH  - HT29 Cells
MH  - Humans
MH  - Neoplasm Proteins/biosynthesis
MH  - Pentacyclic Triterpenes
MH  - Proteasome Endopeptidase Complex/*drug effects/genetics
MH  - Proteolysis/drug effects
MH  - Signal Transduction/drug effects
MH  - Triterpenes/*administration & dosage/chemistry
MH  - Vacuoles/drug effects/pathology
MH  - Betulinic Acid
PMC - PMC4715307
EDAT- 2016/01/17 06:00
MHDA- 2016/10/07 06:00
PMCR- 2016/01/16
CRDT- 2016/01/17 06:00
PHST- 2015/08/07 00:00 [received]
PHST- 2016/01/06 00:00 [accepted]
PHST- 2016/01/17 06:00 [entrez]
PHST- 2016/01/17 06:00 [pubmed]
PHST- 2016/10/07 06:00 [medline]
PHST- 2016/01/16 00:00 [pmc-release]
AID - 10.1186/s12885-016-2055-1 [pii]
AID - 2055 [pii]
AID - 10.1186/s12885-016-2055-1 [doi]
PST - epublish
SO  - BMC Cancer. 2016 Jan 16;16:23. doi: 10.1186/s12885-016-2055-1.