Chen Bin, He Tao, Wu Li, Cao Ting, Zheng Hongmei
Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China, 430060.
J BUON. 2020 Jan-Feb;25(1):421-426.
The main purpose of the current study was to evaluate the anticancer action of 7-Methoxyheptaphylline in Y-79 human retinoblastoma cells along with evaluating its effects on cellular apoptosis, cell cycle phase distribution and Wnt/β-catenin signalling pathway.
The retinoblastoma cell line RbY-79 was used in this study. Cell viability was assessed by WTS-1 assay while apoptotic studies were carried out by DAPI, acridine orange (AO)/ethidium bromide (EB), annexin V + propidium iodide (PI) staining using fluorescence microscopy and flow cytometry. Effects on cell cycle progression were studied using Annexin V/PI staining in combination with flow cytometry. Western blot assay was used to examine the effects on Bax, Bcl-2 and proteins associated with Wnt/β-catenin signalling pathway.
The results indicated that 7-Methoxyheptaphylline suppressed the viability of the Y-79 cells concentration-dependently with IC50 value of 15.5 μM. The percentage of the DAPI-positive cells showed a significant upsurge reminiscent of the apoptosis in the Y-79 retinoblastoma cells. 7-Methoxyheptaphylline also caused considerable nuclear fragmentation of the Y-79 retinoblastoma cells, representative of apoptosis. The apoptosis percentage increased significantly as the dose of the 7-Methoxyheptaphylline increased from 0 to 12, 24 and 48 µM. The molecule caused upregulation of Bax and downregulation of Bcl-2 in Y-79 retinoblastoma cells. 7-Methoxyheptaphylline also caused S-phase cell cycle arrest with concomitant concentration-dependent decline in the expression levels of cyclin A, E and D1. It was also seen that increasing doses of 7-Methoxyheptaphylline led to a dose-dependent decline in the expression levels of wnt-13a and β-catenin.
In conclusion, it is believed that the molecule may prove to be a promising anticancer agent for the treatment of retinoblastoma.
本研究的主要目的是评估7-甲氧基七叶茶碱对Y-79人视网膜母细胞瘤细胞的抗癌作用，并评估其对细胞凋亡、细胞周期阶段分布和Wnt/β-连环蛋白信号通路的影响。
本研究使用视网膜母细胞瘤细胞系RbY-79。通过WTS-1试验评估细胞活力，同时使用荧光显微镜和流式细胞术通过DAPI、吖啶橙（AO）/溴化乙锭（EB）、膜联蛋白V +碘化丙啶（PI）染色进行凋亡研究。使用膜联蛋白V/PI染色结合流式细胞术研究对细胞周期进程的影响。蛋白质印迹分析用于检查对Bax、Bcl-2以及与Wnt/β-连环蛋白信号通路相关的蛋白质的影响。
结果表明，7-甲氧基七叶茶碱浓度依赖性地抑制Y-79细胞的活力，IC50值为15.5μM。DAPI阳性细胞的百分比显著升高，这让人联想到Y-79视网膜母细胞瘤细胞中的凋亡。7-甲氧基七叶茶碱还导致Y-79视网膜母细胞瘤细胞出现相当多的核碎片化，这是凋亡的特征。随着7-甲氧基七叶茶碱的剂量从0增加到12、24和48μM，凋亡百分比显著增加。该分子导致Y-79视网膜母细胞瘤细胞中Bax上调和Bcl-2下调。7-甲氧基七叶茶碱还导致S期细胞周期停滞，同时细胞周期蛋白A、E和D1的表达水平呈浓度依赖性下降。还发现，7-甲氧基七叶茶碱剂量增加导致wnt-13a和β-连环蛋白的表达水平呈剂量依赖性下降。
总之，人们认为该分子可能被证明是一种有前途的用于治疗视网膜母细胞瘤的抗癌药物。
