title: Involvement of Smac, p53, and caspase pathways in induction of apoptosis by gossypol in human retinoblastoma cells.
abstract: OBJECTIVE
  Retinoblastoma is a malignant tumor of the retina usually occurring in young children. To date, the conventional treatments for retinoblastoma have been enucleation, cryotherapy, external beam radiotherapy, or chemotherapy. Most of these treatments, however, have possible side effects, including blindness, infections, fever, gastrointestinal toxicity, and neurotoxicity. More effective treatments are therefore imperative. Gossypol has been reported as a potential inhibitor of cell proliferation in various types of cancers, such as prostate cancer, breast cancer, leukemia, and lung cancer. This study investigates the possible antiproliferative effect of gossypol on retinoblastoma.
  
  
  METHODS
  Human retinoblastoma cells were cultured with various concentrations of gossypol and checked for cell viability with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nuclear condensation caused by cell apoptosis was detected by staining retinoblastoma cells with 4',6-diamidino-2-phenylindole (DAPI), counting those with condensed nuclei, and determining the percentage of apoptotic cells. In addition, the stages of apoptosis and phases in cell cycles were examined with flow cytometry. The possible signal transduction pathways involved were examined with a protein array assay and western blot analysis.
  
  
  RESULTS
  After incubation, the cell survival rate was significantly lower after treatment with 5, 10, and 20 µM of gossypol. The maximum antisurvival effect of gossypol was observed at 20 µM, and the number of apoptotic cells was higher in the preparations cultured with 10 and 20 µM of gossypol. The results in flow cytometry indicated that at concentrations of 10 and 20 µM, gossypol increased the proportion of early- and late-apoptotic retinoblastoma cells and induced cell arrest of retinoblastoma cells at the same concentrations. This antiproliferative effect was later confirmed by upregulating the expression of death receptor 5 (DR5), caspase 8, caspase 9, caspase 3, cytochrome C, tumor protein 53 (p53), and second mitochondria-derived activator of caspases (Smac) in the signal transduction pathways.
  
  
  CONCLUSIONS
  We concluded that gossypol has an antiproliferative effect on retinoblastoma cells.
authors.affiliation: School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan.
authors.forename: Wei-Ting
authors.initials: WT
authors.lastname: Hsiao
authors.forename: Ming-Dar
authors.initials: MD
authors.lastname: Tsai
authors.forename: Guey-Mei
authors.initials: GM
authors.lastname: Jow
authors.forename: Lu-Tai
authors.initials: LT
authors.lastname: Tien
authors.forename: Yih-Jing
authors.initials: YJ
authors.lastname: Lee
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006072:Gossypol / Q000494:pharmacology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism; D019572:Retinal Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D012175:Retinoblastoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D016159:Tumor Suppressor Protein p53; D045304:Cytochromes c; D020169:Caspases; D006072:Gossypol