title: Biological evaluation of a halogenated triterpenoid, 2α-bromo-dihydrobelulonic acid as inhibitor of human topoisomerase IIα and HeLa cell proliferation.
abstract: BACKGROUND
  The pentacyclic lupane-type (6-6-6-6-5 type) triterpenoid, Betulinic acid (BA) is a potent inhibitor of topoisomerases and is of immense interest as anticancer drugs. However, the compound being highly lipophilic, has limited in vivo uptake capacity. BA derivatives with halogen substituent at C-2 have improved membrane permeability and cytotoxicity against cancer cells.
  
  
  OBJECTIVE
  The halogenated triterpenoid, 2α-bromo-dihydrobetulonic acid (B1) was synthesized from betulinic acid (BA) isolated from Bischofia javanica. Aim of the study was to determine whether B1 could act as a more efficient inhibitor of Topo IIα activity and HeLa cell proliferation, in comparison to BA.
  
  
  RESULTS
  B1 displayed efficient inhibition of DNA relaxation activity of topoisomerase IIα and the inhibitory effect was markedly improved upon pre-incubation of the compound with enzyme. Topoisomerase IIα inhibition by B1 was relieved in presence of increasing concentrations of DNA suggesting the compound as a reversible catalytic inhibitor. Subsequent UV and fluorescence spectroscopy studies indicated that B1 interacts and intercalates with DNA at concentrations signicantly greater than that required for topoisomerase IIα inhibition. The compound showed cytotoxic activity against HeLa cells with significantly lower IC50value (7.5 μM) as compared to that of BA (30 μM) and had very low damaging/cytotoxic effect on normal cells. Treatment of B1 impaired HeLa cell proliferation by inducing Go-G1 arrest through lowered expression of cyclin D1 and PCNA polypeptides, and enhanced expression of p21. B1 treatment also increased the accumulation of early and late apoptotic cells in a concentration dependent manner as indicated by annexin V-FITC/PI binding assay.
authors.affiliation: Department of Biotechnology, University of North Bengal, Darjeeling, 734 013, West Bengal, India. Electronic address: ghosshilpi@gmail.com.
authors.forename: Shilpi
authors.initials: S
authors.lastname: Ghosh
authors.affiliation: Department of Biotechnology, University of North Bengal, Darjeeling, 734 013, West Bengal, India.
authors.forename: Swagata
authors.initials: S
authors.lastname: Mukhopadhyay
authors.affiliation: Experimental Medicine & Stem Cell Research Laboratory, Department of Physiology, West Bengal State University, Barasat, Kolkata, 700 126, India.
authors.forename: Mrinmoy
authors.initials: M
authors.lastname: Sarkar
authors.affiliation: Natural Products and Polymer Laboratory, Department of Chemistry, University of North Bengal, Darjeeling, 734 013, West Bengal, India; Molecular Complexicity Laboratory, Department of Chemistry, Raiganj University, Raiganj, 733134, West Bengal, India.
authors.forename: Amitava
authors.initials: A
authors.lastname: Mandal
authors.affiliation: Department of Biotechnology, University of North Bengal, Darjeeling, 734 013, West Bengal, India.
authors.forename: Vaskar
authors.initials: V
authors.lastname: Das
authors.affiliation: Department of Biotechnology, University of North Bengal, Darjeeling, 734 013, West Bengal, India.
authors.forename: Anoop
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Experimental Medicine & Stem Cell Research Laboratory, Department of Physiology, West Bengal State University, Barasat, Kolkata, 700 126, India; Department of Physiology, University of Gour Banga, Malda, 732103, West Bengal, India. Electronic address: bgiri.emscrl@gmail.com.
authors.forename: Biplab
authors.initials: B
authors.lastname: Giri
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D054879:Halogenation; D006367:HeLa Cells; D006801:Humans; D007364:Intercalating Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D053978:Pentacyclic Triterpenes; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D057949:Streptophyta / Q000737:chemistry; D059005:Topoisomerase II Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D014315:Triterpenes / Q000737:chemistry / Q000494:pharmacology; D000094062:Betulinic Acid
chemical_list: C000617652:2-bromo-dihydrobetulonic acid; D000951:Antigens, Neoplasm; C530104:CCND1 protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D007364:Intercalating Agents; D053978:Pentacyclic Triterpenes; D018809:Proliferating Cell Nuclear Antigen; D059005:Topoisomerase II Inhibitors; D014315:Triterpenes; D019938:Cyclin D1; D009828:Oleanolic Acid; D004247:DNA; D004250:DNA Topoisomerases, Type II; D000094062:Betulinic Acid
keywords: 2α-bromo-dihydrobelulonic acid; Betulinic acid; Bischofia javanica; HeLa cells; Topoisomerase IIα