Lu Hang-Qing, Zheng Jie
Institute of Molecular Pathology, Department of Pathology and Pathophysiology, School of Basic Medical Science, Southeast University, Nanjing, Jiangsu, 210009, P. R. China.
Ai Zheng. 2006 Dec;25(12):1470-6.
BACKGROUND & 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.
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.
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.
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.
全反式维甲酸（ATRA）和1,25 - 二羟基维生素D3 [1,25(OH)2D3]可抑制肿瘤细胞增殖并诱导其分化。它们已被用于治疗白血病，但其对实体瘤的作用仍不清楚。本研究旨在探讨ATRA和1,25(OH)2D3对人肝癌细胞系HepG2生长和细胞周期的影响，并探索其分子机制。
用ATRA、1,25(OH)2D3或二者联合处理HepG2细胞后，采用MTT法评估细胞存活率，在光学显微镜下观察形态学变化，通过AnnexinV/PI双染流式细胞术（FCM）检测细胞周期和凋亡情况，并用逆转录 - 聚合酶链反应（RT-PCR）和FCM检测p21(WAF/CIP1)和p27(KIP1) mRNA及蛋白的表达。
ATRA和1,25(OH)2D3单独或联合使用均能以时间和剂量依赖的方式抑制HepG2细胞生长。联合组的抑制作用比ATRA组和1,25(OH)2D3组更明显（P<0.05）。FCM分析表明，10 nmol/L 1,25(OH)2D3单独或与ATRA联合处理72小时，均能强烈诱导HepG2细胞G1期阻滞[分别为(54.27±3.69)%和(65.64±5.65)%，对照为(40.40±1.91)%，P<0.05]，但1 μmol/L ATRA未显示明显作用。它们均诱导细胞凋亡（P<0.05）。用10 nmol/L 1,25(OH)2D3或1,25(OH)2D3与ATRA联合处理24小时的细胞中，p21(WAF/CIP1)的mRNA水平比对照分别提高了35%和56%。与单独使用1,25(OH)2D3相比，1,25(OH)2D3与ATRA联合显著提高了p21(WAF/CIP1)和p27(KIP1)的蛋白水平，而1 μmol/L ATRA未提高HepG2细胞中p21(WAF/CIP1)和p27(KIP1)的蛋白水平。
ATRA和1,
