Acyclic retinoid induces partial differentiation, down-regulates telomerase reverse transcriptase mRNA expression and telomerase activity, and induces apoptosis in human hepatoma-derived cell lines.
OBJECTIVE
Acyclic retinoid (AR; all trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid) prevented hepatocarcinogenesis in animal models and in a randomized clinical trial by eradicating premalignant and latent malignant clones of transformed cells from the liver. We investigated the possible mechanism of this clonal deletion at the cellular level.


METHODS
Human hepatoma-derived cell lines, PLC/PRF/5, HuH-7, and JHH-7, were treated in vitro with AR. Secretion of albumin and that of lectin-reactive isoform of alpha-fetoprotein (AFP-L3) were measured as markers of differentiation and dedifferentiation of the cells, respectively. Telomerase reverse transcriptase (TERT) mRNA expression and telomerase activity were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and stretch PCR assay, respectively. Caspase activities were measured by colorimetric protease assay. Mitochondrial membrane permeability transition was examined by Rhodamine staining.


RESULTS
Production of albumin was recovered while that of AFP-L3 was reduced after exposure of the cells to 10 microM AR for 2 days. This differentiation was maintained for another 2 days without retinoid. In parallel, both TERT mRNA expression and telomerase activity were down-regulated. The cells subsequently died due to apoptosis after 4-6 experimental days. Serial increases in mitochondrial membrane permeability and caspase-9 and -3 activities induced apoptosis.


CONCLUSIONS
AR first induces differentiation and reduces telomerase activity. Subsequent apoptosis may contribute to the eradication of the clone.
Yasuda
Ichiro
I
First Department of Internal Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan.
Shiratori
Yoshimune
Y
Adachi
Seiji
S
Obora
Akihiro
A
Takemura
Masao
M
Okuno
Masataka
M
Shidoji
Yoshihiro
Y
Seishima
Mitsuru
M
Muto
Yasutoshi
Y
Moriwaki
Hisataka
H
D000077556:Alitretinoin; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D004268:DNA-Binding Proteins; D015536:Down-Regulation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms; D008928:Mitochondria / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism*; D014212:Tretinoin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D000077556:Alitretinoin; D014212:Tretinoin; C034534:3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid; C509186:TERT protein, human; D019098:Telomerase; D020169:Caspases