title: In vitro anti-breast cancer activity of ethanolic extract of Wrightia tomentosa: role of pro-apoptotic effects of oleanolic acid and urosolic acid.
abstract: BACKGROUND
  Wrightia tomentosa Roem. & Schult. (Apocynaceae) is known in the traditional medicine for anti-cancer activity along with other broad indications like snake and scorpion bites, renal complications, menstrual disorders etc. However, the anti-cancer activity of this plant or its constituents has never been studied systematically in any cancer types so far.
  
  
  OBJECTIVE
  To evaluate the anti-cancer activities of the ethanolic extract of W. tomentosa and identified constituent active molecule(s) against breast cancer.
  
  
  METHODS
  Powdered leaves of W. tomentosa were extracted with ethanol. The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa were tested for its anti-proliferative and pro-apoptotic effects in breast cancer cells MCF-7 and MDA-MB-231.
  
  
  RESULTS
  The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa inhibited the proliferation of human breast cancer cell lines, MCF-7 and MDA-MB-231. The fraction F-4 obtained from hexane fraction inhibited proliferation of MCF-7 and MDA-MB-231 cells in concentration and time dependent manner with IC₅₀ of 50 μg/ml and 30 μg/ml for 24 h, 28 μg/ml and 22 μg/ml for 48 h and 25 μg/ml and 20 μg/ml for 72 h respectively. The fraction F-4 induced G1 cell cycle arrest, reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and subsequent apoptosis. Apoptosis is indicated in terms of increased Bax/Bcl-2 ratio, enhanced Annexin-V positivity, caspase 8 activation and DNA fragmentation. The active molecule isolated from fraction F-4, oleanolic acid and urosolic acid inhibited cell proliferation of MCF-7 and MDA-MB-231 cells at IC₅₀ value of 7.5 μM and 7.0 μM respectively, whereas there is devoid of significant cell inhibiting activity in non-cancer originated cells, HEK-293. In both MCF-7 and MDA-MB-231, oleanolic acid and urosolic acid induced cell cycle arrest and apoptosis as indicated by significant increase in Annexin-V positive apoptotic cell counts.
  
  
  CONCLUSIONS
  Our results suggest that W. tomentosa extracts has significant anti-cancer activity against breast cancer cells due to induction of apoptosis pathway. Olenolic and urosolic acid are important constituent molecules in the extract responsible for anti-cancer activity of W. tomentosa.
authors.affiliation: Division of Endocrinology, CSIR-Central Drug Research Institute-CDRI, U.P., Lucknow 226001, India.
authors.forename: Bandana
authors.initials: B
authors.lastname: Chakravarti
authors.forename: Ranjani
authors.initials: R
authors.lastname: Maurya
authors.forename: Jawed Akhtar
authors.initials: JA
authors.lastname: Siddiqui
authors.forename: Hemant Kumar
authors.initials: HK
authors.lastname: Bid
authors.forename: S M
authors.initials: SM
authors.lastname: Rajendran
authors.forename: Prem P
authors.initials: PP
authors.lastname: Yadav
authors.forename: Rituraj
authors.initials: R
authors.lastname: Konwar
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D029062:Apocynaceae; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000431:Ethanol / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009828:Oleanolic Acid / Q000494:pharmacology*; D010936:Plant Extracts / Q000494:pharmacology*; D018515:Plant Leaves; D012997:Solvents / Q000737:chemistry; D014315:Triterpenes / Q000494:pharmacology*; D000097245:Ursolic Acid
chemical_list: D000970:Antineoplastic Agents; D000431:Ethanol; D007770:L-Lactate Dehydrogenase; D009828:Oleanolic Acid; D010936:Plant Extracts; D012997:Solvents; D014315:Triterpenes; D000097245:Ursolic Acid