title: Hederagenin from the leaves of ivy (Hedera helix L.) induces apoptosis in human LoVo colon cells through the mitochondrial pathway.
abstract: BACKGROUND
  Colorectal cancer has become one of the leading cause of cancer morbidity and mortality throughout world. Hederagenin, a derivative of oleanolic acid isolated from the leaves of ivy (Hedera helix L.), has been shown to have potential anti-tumor activity. The study was conducted to evaluate whether hederagenin could induce apoptosis of human colon cancer LoVo cells and explore the possible mechanism.
  
  
  METHODS
  MTT assay was used for evaluating cell viability while Annexin V-FITC/PI assay and Hoechst 33342 nuclear stainining were used for the determination of apoptosis and mitochondrial membrane potential. DCFH-DA fluorescence staining and flow cytometry were used to measure ROS generation. Real-time PCR and western blot analysis were performed for apoptosis-related protein expressions.
  
  
  RESULTS
  MTT assay showed that hederagenin could significantly inhibit the viability of LoVo cells in a concentration-dependent and time-dependent manner by IC50 of 1.39 μM at 24 h and 1.17 μM at 48 h. The apoptosis ratio was significantly increased to 32.46% and 81.78% by the induction of hederagenin (1 and 2 μM) in Annexin V-FITC/PI assay. Hederagenin could also induce the nuclear changes characteristic of apoptosis by Hoechst 33342 nuclear stainining under fluorescence microscopy. DCFH-DA fluorescence staining and flow cytometry showed that hederagenin could increase significantly ROS generation in LoVo cells. Real-time PCR showed that hederagenin induced the up-regulation of Bax and down-regulation of Bcl-2, Bcl-xL and Survivin. Western blotting analysis showed that hederagenin decreased the expressions of apoptosis-associated proteins Bcl-2, procaspase-9, procaspase-3, and polyADP- ribosepolymerase (PARP) were increased, while the expressions of Bax, caspase-3, caspase-9 were increased. However, there was no significant change on caspase-8.
  
  
  CONCLUSIONS
  These results indicated that the disruption of mitochondrial membrane potential might contribute to the apoptosis of hederagenin in LoVo cells. Our findings suggested that hederagenin might be a promising therapeutic candidate for human colon cancer.
authors.forename: Bao-Xin-Zi
authors.initials: BX
authors.lastname: Liu
authors.forename: Jin-Yong
authors.initials: JY
authors.lastname: Zhou
authors.forename: Yu
authors.initials: Y
authors.lastname: Li
authors.forename: Xi
authors.initials: X
authors.lastname: Zou
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.forename: Jun-Fei
authors.initials: JF
authors.lastname: Gu
authors.forename: Jia-Rui
authors.initials: JR
authors.lastname: Yuan
authors.forename: Bing-Jie
authors.initials: BJ
authors.lastname: Zhao
authors.affiliation: Department of Oncology, Jiangsu Province Hospital of Traditional Chinese Medicine, The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, P, R, China. wenmoxiushi@163.com.
authors.forename: Liang
authors.initials: L
authors.lastname: Feng
authors.forename: Xiao-Bin
authors.initials: XB
authors.lastname: Jia
authors.forename: Rui-Ping
authors.initials: RP
authors.lastname: Wang
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D035981:Hedera / Q000737:chemistry*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010936:Plant Extracts / Q000494:pharmacology*; D018515:Plant Leaves / Q000737:chemistry; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D010936:Plant Extracts; D051028:bcl-2-Associated X Protein; D009828:Oleanolic Acid; D053148:Caspase 3; D053453:Caspase 9; C025763:hederagenin