title: Comparative analysis of apoptosis-inducing activity of codeine and codeinone.
abstract: BACKGROUND
  There are relatively few studies about the antiproliferative effects of codeine-related compounds on human cancer cell lines, compared with those of morphine-related compounds. The authors previously found that codeinone, an oxidation metabolite of codeine, among 10 opioids, showed the highest cytotoxic activity (DNA fragmentation-inducing activity) against human promyelocytic leukemic cell lines (HL-60). This was counteracted by an antioxidant, N-acetyl-L-cysteine (NAC). These findings prompted us to perform a more detailed study of apoptosis induction after codeinone treatment.
  
  
  METHODS
  Apoptosis was induced by treating HL-60 cells for 1-6 h with codeine or codeinone. DNA fragmentation was assessed by both agarose gel electrophoresis and fluorometric determination of the fragmented DNA after staining with diamidinophenylindole (DAPI). The appearance of apoptotic cells was monitored by microscopic observation after staining with Hoechst (H)-33342, and fluorescence activated cell sorter (FACS) after staining with Annexin. The release of cytochrome c and cytochrome oxidase from mitochondria and activation of caspase 3 were monitored by Western blot analysis. Intracellular caspase 3-like activity was confirmed by FACS, using cell permeable substrate. Mitochondrial manganese-containing superoxide dismutase (MnSOD) activity and mRNA expression were assayed by activity staining after separation on the polyacrylamide gel electrophoresis, and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively.
  
  
  RESULTS
  Codeinone induced internucleosomal DNA fragmentation and production of Annexin-positive apoptotic cells more potently than codeine in HL-60 cells. Codeinone stimulated the release of both cytochrome c and cytochrome oxidase, and cleavage of procaspase 3 without significant changes in both the activity and expression of MnSOD.
  
  
  CONCLUSIONS
  Codeinone was found to possess both apoptosis and necrosis-inducing activity, in addition to the reported antinociceptive activity, further substantiating its antitumor potential.
authors.affiliation: Department of Anesthesiology, Saitama Medical School, Japan. nao-hito@rc4.so-net.ne.jp
authors.forename: Naoko
authors.initials: N
authors.lastname: Hitosugi
authors.forename: Ikusuke
authors.initials: I
authors.lastname: Hatsukari
authors.forename: Rie
authors.initials: R
authors.lastname: Ohno
authors.forename: Ken
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Saori
authors.initials: S
authors.lastname: Mihara
authors.forename: Satoshi
authors.initials: S
authors.lastname: Mizukami
authors.forename: Shinichi
authors.initials: S
authors.lastname: Nakamura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakagami
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagasaka
authors.forename: Isao
authors.initials: I
authors.lastname: Matsumoto
authors.forename: Masami
authors.initials: M
authors.lastname: Kawase
mesh_terms: D000701:Analgesics, Opioid / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003061:Codeine / Q000031:analogs & derivatives* / Q000097:blood / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018922:HL-60 Cells; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism
chemical_list: D000701:Analgesics, Opioid; D003574:Cytochrome c Group; D012333:RNA, Messenger; C046414:6-codeinone; D013482:Superoxide Dismutase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003061:Codeine