Antitumor activity of Verteporfin in renal cell carcinoma 769-P cells and its mechanism
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摘要:
目的 研究维替泊芬影响肾癌769-P细胞的作用及潜在机制。 方法 采用不同浓度（0、1、5、10 μmol/L）维替泊芬处理肾癌细胞株769-P，分别采用CCK8检测维替泊芬对769-P细胞活性的抑制作用；流式细胞术分析维替泊芬促进769-P细胞凋亡及抑制细胞周期；划痕实验观察维替泊芬影响769-P细胞迁移能力；克隆形成实验观察维替泊芬抑制769-P细胞集落形成能力；荧光定量PCR法及免疫印迹法分别分析维替泊芬作用于769-P细胞后，CDK4、Bax、CyclinD1、Bcl-2、YAP、TEAD及caspase-3的表达量变化情况。 结果 维替泊芬对769-P细胞的半数抑制浓度为4.917 μmol/L（P<0.05）。维替泊芬可诱导769-P细胞凋亡并阻滞细胞生长周期，抑制细胞增殖作用呈浓度及时间依赖性（P<0.05）。维替泊芬处理后769-P细胞迁移能力及克隆形成能力亦呈浓度依赖性降低（P<0.05）。维替泊芬处理后769-P细胞的转录和翻译水平均受影响，caspase-3被激活，Bax及CDK4表达量增加，YAP、Bcl-2、cyclinD1和TEAD表达量减少（P<0.05）。 结论 维替泊芬具有杀伤肾癌细胞的能力，为探寻肾癌新药开拓了新方向。 Abstract:Objective To explore the ability of Verteporfin killing renal cell carcinoma 769-P cells and its potential mechanism. Methods 769-P was treated with different concentrations (0, 1, 5 and 10 μmol/L) of Verteporfin. The effect of Verteporfin on 769-P cell proliferation was assessed by CCK8. Flow cytometry was applied to examine the effect of Verteporfin on 769-P cell cycle and apoptosis. Wound healing assay and colony forming assay were applied to evaluate the capacity of cell migration and colony formation, respectively. Quantitative Real-time PCR and Western blotting were applied to determine the mRNA and protein levels of CDK4, Bax, CyclinD1, Bcl-2, YAP, TEAD and caspase-3. Results Verteporfin significantly inhibited the growth of 769-P cells with an IC50 of 4.917 μM. Verteporfin induced both apoptosis and cell cycle arrest of 769-P cells in a dose-dependent manner. The migration and colony forming ability were also inhibited by Verteporfin in a dose-dependent manner. Verteporfin regulated both transcriptional and translational level in 769-P cells. CDK4 and Bax were up-regulated while CyclinD1, Bcl-2, YAP and TEAD were down-regulated. Moreover, caspase-3 was activated. Conclusion Verteporfin has significant antitumor effect against renal tumor cells, suggesting it’s a novel therapeutic agent for renal cell carcinoma. -
Key words:
- renal cell carcinoma /
- verteporfin /
- cell cycle /
- cell apoptosis
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图 1 维替泊芬对769-P细胞增殖、凋亡及周期的影响
A, B: 不同浓度、不同时间维替泊芬作用于769-P细胞后细胞活性变化; C, D: 不同浓度维替泊芬作用于769-P细胞后细胞凋亡及周期变化, *P<0.05.
图 2 细胞划痕实验
A: 刚划痕, 不加维替泊芬处理对照组; B: 刚划痕, 加1 μmol/L维替泊芬处理组; C: 刚划痕, 加5 μmol/L维替泊芬处理组; D: 刚划痕, 加10 μmol/L维替泊芬处理组; E: 划痕24 h后, 不加维替泊芬处理对照组; F: 划痕24 h后, 加1 μmol/L维替泊芬处理对照组; G: 划痕24 h后, 加5 μmol/L维替泊芬处理对照组; H: 划痕24 h后, 加10 μmol/L维替泊芬处理对照组.
图 3 不同浓度维替泊芬对对769-P细胞克隆形成能力的影响
不同浓度维替泊芬（0,1,5,10 μmol/L）作用于769-P细胞, 随着维替泊芬浓度的升高, 细胞克隆形成能力减弱, *P<0.050 vs 0 μmol/L.
图 4 维替泊芬对相关基因及蛋白表达的影响
A: 维替泊芬对769-P细胞YAP、TEAD、Bcl-2、CyclinD1、Bax、Caspase-3及CDK4 mRNA表达的影响; B: 维替泊芬对769-P细胞YAP、TEAD、Bcl-2、CyclinD1、Bax、Caspase-3及CDK4蛋白表达的影响.
表 1 实时荧光定量PCR引物序列
Gene Primer sequence（5′-3′） YAP Forward primer GCCCAGTTATACCTCAGTGTTGTAG Reverse primer GGTCTCCTTCAGGTCAGTACAG TEAD Forward primer GCCCATGTGCGAGTACCT Reverse primer TCCAGGACGCTGTTCATCA Bcl-2 Forward primer GAGCACAGAAGATGGGAACACT Reverse primer AGGTGCATGGATTTACTCAGTATC CyclinD1 Forward primer CTGGGTCTGTGCATTTCTGGTT Reverse primer CTGCTGGAAACATGCCGGTTA Bax Forward primer GATGCGTCCACCAAGAAGC Reverse primer CACGGCGGCAATCATCCT Caspase-3 Forward primer GGCTGAGCTGCCTGTAACTTG Reverse primer TGGCTCTGCCTTCATGGAAC CDK4 Forward primer GCTGCCATGGAAGGAAGAAA Reverse primer GCCTCAGAGATAAAGGCAAAGATTG GAPDH Forward primer CCTGCACCACCAACTGCTTAG Reverse primer TGAGTCCTTCCACGATACCAA 
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