Comparative analysis of apoptosis-inducing activity of codeine and codeinone.
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.
Hitosugi
Naoko
N
Department of Anesthesiology, Saitama Medical School, Japan. nao-hito@rc4.so-net.ne.jp
Hatsukari
Ikusuke
I
Ohno
Rie
R
Hashimoto
Ken
K
Mihara
Saori
S
Mizukami
Satoshi
S
Nakamura
Shinichi
S
Sakagami
Hiroshi
H
Nagasaka
Hiroshi
H
Matsumoto
Isao
I
Kawase
Masami
M
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
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