[Synergistic inhibitory effect of all-trans retinoic acid and 1,25-dihydroxy vitamin D3 on growth of human hepatoma cell line HepG2].
OBJECTIVE
All-trans retinoic acid (ATRA) and 1,25-dihydroxy vitamin D3 [1,25(OH)2D3] can inhibit the proliferation of tumor cells and induce their differentiation. They have been used to treat leukemia, but their effects on solid tumors remain unclear. This study was to investigate the effects of ATRA and 1,25(OH)2D3 on growth and cell cycle of human hepatoma cell line HepG2, and explore the molecular mechanism.


METHODS
After HepG2 cells were treated with ATRA, 1,25(OH)2D3, or the combination of both chemicals, cell survival was assessed by MTT assay, morphologic changes were observed under light microscope, cell cycle and apoptosis were determined by flow cytometry (FCM) with dual staining of AnnexinV/PI, and the expression of p21(WAF/CIP1) and p27(KIP1) mRNA and protein were determined by reverse transcription-polymerase chain reaction (RT-PCR) and FCM.


RESULTS
ATRA and 1,25(OH)2D3, used alone or in combination, inhibited the growth of HepG2 cells in a time- and dose-dependent manner. The inhibition was more obvious in the combination group than in ATRA group and 1,25(OH)2D3 group (P<0.05). FCM analysis indicated that 10 nmol/L 1,25(OH)2D3, used alone or in combination with ATRA for 72 h, strongly induced G1 phase arrest of HepG2 cells [(54.27+/-3.69)% and (65.64+/-5.65)% vs. (40.40+/-1.91)% of the control, P<0.05], but 1 micromol/L ATRA did not show obvious effect. All of them induced apoptosis (P<0.05). The mRNA level of p21(WAF/CIP1) was enhanced by 35% and 56% of control in the cells treated with 10 nmol/L 1,25(OH)2D3 or 1,25(OH)2D3 combined with ATRA for 24 h. The combination of 1,25(OH)2D3 and ATRA markedly enhanced the protein levels of p21(WAF/CIP1) and p27(KIP1) as compared with 1,25(OH)2D3 alone, and 1 micromol/L ATRA did not enhance the protein levels of p21(WAF/CIP1) and p27(KIP1) in HepG2 cells.


CONCLUSIONS
ATRA and 1,25(OH)2D3 could inhibit growth and induce apoptosis of HepG2 cells, and the molecular basis of the cell cycle blockade by 1,25(OH)2D3 may be associated with the up-regulation of CDK inhibitors p21(WAF/CIP1) and p27(KIP1), which mediate G1 arrest. Furthermore, the combination of ATRA and 1,25(OH)2D3 can exert synergistic inhibitory effect on the growth of HepG2 cells.
Lu
Hang-Qing
HQ
Institute of Molecular Pathology, Department of Pathology and Pathophysiology, School of Basic Medical Science, Southeast University, Nanjing, Jiangsu, 210009, P. R. China.
Zheng
Jie
J
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004357:Drug Synergism; D016193:G1 Phase / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*; D015854:Up-Regulation; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
D000970:Antineoplastic Agents; C493956:CDKN1A protein, human; C436291:CDKN1B protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D050760:Cyclin-Dependent Kinase Inhibitor p27; D014212:Tretinoin