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目的 :探讨根皮素对前列腺癌PC-3细胞凋亡和周期的影响,及对其裸鼠移植瘤生长的影响。方法 :不同浓度的根皮素(0、20、50和100μmol/L)分别作用于人正常前列腺基质细胞WPMY-1和前列腺癌PC-3和DU145细胞24 h后,倒置光学显微镜下观察细胞形态的变化,并采用CCK-8法检测各组细胞活性的变化。用不同浓度的根皮素(0、20、50和100μmol/L)处理PC-3细胞24 h后,采用DAPI染色法观察细胞核破裂的情况,FCM法检测根皮素处理后前列腺癌PC-3细胞凋亡率和细胞周期分布的变化;同时,采用蛋白质印迹法检测细胞凋亡相关蛋白剪切型聚腺苷二磷酸核糖聚合酶1(cleaved-poly ADP-ribose polymerase-1,cleaved-PARP-1)、cleaved-caspase 3、cleaved-caspase 8、cleaved-caspase 9、X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)、survivin、Bcl-2、Bax以及细胞周期蛋白cyclin B1表达水平的变化。建立人前列腺癌PC-3细胞裸鼠皮下移植瘤模型,观察根皮素腹腔注射治疗后小鼠移植瘤生长体积以及体质量的变化,并计算抑瘤率。采用蛋白质印迹法检测小鼠肿瘤组织中凋亡相关蛋白的表达水平。结果:根皮素浓度为50和100μmol/L时,对人正常前列腺基质细胞WPMY-1的形态没有影响,仅对前列腺癌PC-3和DU145细胞的形态有影响。与空白对照组相比,根皮素浓度为20、50和100μmol/L时对前列腺癌PC-3和DU145细胞活力均有明显的抑制作用(P值均<0.01)。采用浓度为20、50和100μmol/L的根皮素处理PC-3细胞24 h后,PC-3细胞的核破裂率分别为(27.0±2.0)%、(47.0±1.5)%和(63.0±3.0)%,明显高于空白对照组的(3.0±1.0)%(P值均<0.001);凋亡率分别为(20.0±1.0)%、(32.0±2.0)%和(42.6±1.5)%,明显高于空白对照组的(4.0±1.0)%(P值均<0.001)。根皮素(100μmol/L)处理PC-3细胞24 h后,细胞中cleaved-PARP-1、cleaved-caspase 3、cleaved-caspase 8和cleaved-caspase 9蛋白的表达水平均较空白对照组明显升高(P值均<0.01),而XIAP、survivin和Bcl-2蛋白的表达水平均明显降低(P值均<0.001)。PC-3细胞被阻滞在G2/M期,并且cyclin B1蛋白的表达水平明显降低(P<0.001)。根皮素可以抑制裸鼠移植瘤的生长(P<0.05),并上调肿瘤组织中cleaved-PARP-1和cleaved-caspase 3蛋白的表达水平,下调survivin蛋白的表达水平(P值均<0.001),但是对小鼠体质量无明显影响(P>0.05)。结论 :根皮素通过下调cyclin B1、XIAP、survivin和Bcl-2蛋白水平,以及上调cleaved-PARP-1、cleaved-caspase 3、cleaved-caspase 8和cleaved-caspase 9蛋白的表达水平,诱导PC-3细胞周期阻滞和细胞凋亡,最终抑制肿瘤细胞生长。
Objective: To investigate the effects of phloretin on the apoptosis and cell cycle of prostate cancer PC-3 cells and its effect on the growth of xenografts in nude mice. METHODS: Phosphatidylinositol (0, 20, 50 and 100 μmol / L) at different concentrations were applied to human prostatic stromal cells WPMY-1 and PC-3 and DU145 respectively for 24 h. The changes of cell viability were detected by CCK-8 assay. PC-3 cells were treated with different concentrations of phloretin (0, 20, 50 and 100 μmol / L) for 24 h, and the nucleus rupture was observed by DAPI staining. PC-3 Apoptosis rate and cell cycle distribution were detected by Western blotting.At the same time, Western blotting was used to detect the expression of cleaved-poly ADP-ribose polymerase-1 (cleaved-PARP- 1, cleaved-caspase 3, cleaved-caspase 8, cleaved-caspase 9, X-linked inhibitor of apoptosis protein (XIAP), survivin, Bcl- Horizontal changes. A subcutaneous xenograft tumor model of human prostate cancer PC-3 cells was established in nude mice. The growth volume and body weight of transplanted tumor were observed after percutaneous injection of phloretin and the tumor inhibition rate was calculated. Western blotting was used to detect the expression of apoptosis-related proteins in mouse tumor tissues. Results: When phloretin concentration was 50 and 100 μmol / L, the morphology of WPMY-1 in human normal prostate stromal cells was not affected, and the morphological changes of PC-3 and DU145 cells were only affected. Compared with the blank control group, the cell viability of PC-3 and DU145 cells were significantly inhibited at the concentrations of phloretin 20, 50 and 100 μmol / L (all P <0.01). PC-3 cells were treated with 20, 50 and 100 μmol / L phloretin for 24 h, the rates of nuclear rupture were (27.0 ± 2.0)%, (47.0 ± 1.5)% and (63.0 ± (P <0.01). The apoptotic rates were (20.0 ± 1.0)%, (32.0 ± 2.0)% and (42.6 ± 1.5)% respectively in the control group (3.0% , Which was significantly higher than that of the control group (4.0 ± 1.0)% (P <0.001). The expression levels of cleaved-PARP-1, cleaved-caspase 3, cleaved-caspase 8 and cleaved-caspase 9 in PC-3 cells after treatment with 100 μmol / L phloretin for 24 h were significantly higher than those in blank control group (P <0.01), while the expression of XIAP, survivin and Bcl-2 protein were significantly lower (P <0.001). PC-3 cells were arrested in G2 / M phase, and cyclin B1 protein expression was significantly reduced (P <0.001). Phloretin could inhibit the growth of xenografts in nude mice (P <0.05), and upregulate the expressions of cleaved-PARP-1 and cleaved-caspase 3 and down-regulate the expression of survivin protein in tumor tissues (all P <0.001) , But had no significant effect on the body weight of mice (P> 0.05). CONCLUSION: Ephrenin can induce the expression of cyclin B1, XIAP, survivin and Bcl-2 and upregulate the expressions of cleaved-PARP-1, cleaved-caspase 3, cleaved-caspase 8 and cleaved-caspase 9, 3 cell cycle arrest and apoptosis, eventually inhibiting tumor cell growth.