The Effects Of A Novel All-trans Retinoid Acid Derivative 4-Amino-2-trifluoromethyl-phenyl Retinate On The Proliferation Of HepG2 Cell Line And The Probable Molecular Mechanisms

Objective The present study was conducted to elucidate the influence of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) on the proliferation in human hepatocellular carcinoma (HCC) HepG2 cells and to preliminarily investigate the underlying mechanisms.Methods HepG2 cells were served as the study model and exposed to all-trans retinoic acid (ATRA) and ATPR respectively. MTT assay was used to measure the cells growth viability. Plate colony formation experiment was performed to assess the cloning efficiency by counting clone number. To verify the influence on proliferation, the distribution of cell cycle was investigated by flow cytometry. The apoptosis was observed by Hoechst staining. To explore the underlying molecular mechanisms, immunofluorescence was applied to observe the distribution of tumour suppressor p53. The transcription and translation levels of p53 were analyzed by real-time quantitative RT-PCR (qRT-PCR) and Western blot. The expression of murine double minute 2 (MDM2), apoptosis stimulating proteins of p53 (ASPP), cycle-and apoptosis-associated proteins were determined by Western blot. Finally, Western blot was used to detect retinoic acid receptors (RARs) and retinoid x receptors (RXRs) expression.Results After HepG2 cells were treated with ATRA and ATPR, HepG2 cells growth was inhibited in a dose-and time-dependent manner and the inhibition rate of ATPR was much higher than that of ATRA at the same dose for the same indicated time. Further, ATPR significantly inhibited HepG2 cells colony formation and arrested cells at G0/G1 phase, while ATRA had no the inhibitory effects. An apoptotic mode of cell death was shown by Hoechst staining. Moreover, the fluorescent density of p53 was higher in the nuclei following ATPR treatment than that in ATRA group. Similarly, HepG2 cells exposed to ATPR revealed the more elevated mRNA and protein levels of p53 than ATRA-treated cells. Western blot showed that ATPR increased ASPP1, p21, Bax and caspase-3 expression and decreased MDM2, iASPP, cyclinD, cyclinE, cyclin-dependent kinase 6 (CDK6) and Bcl-2 expression, while CDK4 and ASPP2 expression were constant in each group. For RARs and RXRs expression, ATRA and ATPR upregulated RARa and downregulated RXRβ,RXRγ levels in HepG2 cells and the effect was more significant in ATPR groups. However, RARγ and RXRα expression has no obvious changes in each group. Besides, the decreased expression of RARβ was observed in HepG2 cells exposed to ATRA while no significant difference was observed in ATPR groups.Conclusion ATPR could better inhibit the proliferation of HCC HepG2 cell line than ATRA at the same concentration and the same time. The mechanisms might be partly through upregulating expression of p53 and ASPP1 and downregulating iASPP via binding to various RARs or RXRs, thereby resulting in G0/G1 cell cycle arrest and apoptosis.