Acyclic retinoid induces partial differentiation, down-regulates telomerase reverse transcriptase mRNA expression and telomerase activity, and induces apoptosis in human hepatoma-derived cell lines

Background/Aims: 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 α-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 μM 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.