title: A novel all-trans retinoic acid derivative 4-amino‑2‑trifluoromethyl-phenyl retinate inhibits the proliferation of human hepatocellular carcinoma HepG2 cells by inducing G0/G1 cell cycle arrest and apoptosis via upregulation of p53 and ASPP1 and downregulation of iASPP.
abstract: 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, was reported to function as a tumor inhibitor in various types of cancer cells in vitro. However, little is known concerning its antitumor effect on human hepatocellular carcinoma (HCC) HepG2 cells. The aims of the present study were to investigate the effects of ATPR on the proliferation of HepG2 cells and to explore the probable mechanisms. A series of experiments were performed following the treatment of HepG2 cells with ATRA and ATPR. MTT and plate colony formation assays were used to measure the cell viability. To confirm the influence on proliferation, flow cytometry was used to detect the distribution of the cell cycle. Apoptosis was observed by Hoechst staining and flow cytometry. In addition, to characterize the underlying molecular mechanisms, immunofluorescence was applied to observe the distribution of p53. The transcription and translation levels of p53 were analyzed by real-time quantitative RT-PCR (qRT-PCR) and western blotting. The expression levels of murine double minute 2 (MDM2), apoptosis stimulating proteins of p53 (ASPP), cell cycle- and apoptosis-associated proteins were detected by western blotting. After HepG2 cells were incubated with ATRA and ATPR, the viability of the HepG2 cells was inhibited in a dose- and time-dependent manner. As well, ATPR significantly suppressed HepG2 cell colony formation and arrested cells at the G0/G1 phase, while ATRA had no obvious effects. Both Hoechst staining and flow cytometry unveiled the apoptosis of HepG2 cells. Moreover, the fluorescent density of p53 was higher in the nuclei after exposure to ATPR than that in the ATRA group. HepG2 cells treated with ATPR showed elevated mRNA and protein levels of p53 when compared with these levels in the ATRA-treated cells. Western blotting showed that ATPR increased ASPP1, p21 and Bax expression and decreased MDM2, iASPP, cyclin D and E, cyclin-dependent kinase 6 (CDK6) and Bcl-2 expression, while CDK4 and ASPP2 expression were scarcely altered. Consequently, ATPR exerted a better inhibitory effect on the proliferation of HepG2 cells than ATRA through increased expression of p53 and ASPP1 and downregulation of iASPP, thereby resulting in G0/G1 cell cycle arrest and apoptosis.
authors.affiliation: Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Hefei, Anhui 230032, P.R. China.
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.affiliation: College of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, P.R. China.
authors.forename: Feihu
authors.initials: F
authors.lastname: Chen
authors.affiliation: Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Hefei, Anhui 230032, P.R. China.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Hefei, Anhui 230032, P.R. China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Gene Research of Anhui Province, Anhui Medical University, Hefei, Anhui 230032, P.R. China.
authors.forename: Shuyu
authors.initials: S
authors.lastname: Gui
authors.affiliation: Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Hefei, Anhui 230032, P.R. China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D015536:Down-Regulation / Q000187:drug effects; D016193:G1 Phase / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D016192:Resting Phase, Cell Cycle / Q000187:drug effects; D012176:Retinoids / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects; D014212:Tretinoin / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C585745:4-amino-2-trifluoromethyl-phenyl retinate; D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; C498406:PPP1R13B protein, human; C119612:PPP1R13L protein, human; D012333:RNA, Messenger; D012097:Repressor Proteins; D012176:Retinoids; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D014212:Tretinoin; D051736:Proto-Oncogene Proteins c-mdm2