Antitumor activity of CPI203 in renal cell carcinoma ACHN cells and its mechanism
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摘要:
目的研究CPI203杀伤人肾癌ACHN细胞的能力及潜在机制。 方法不同浓度（0、0.1、0.5、1、5 μmol/L）CPI203药物作用于ACHN细胞24、48 h后，采用CCK8试剂盒检测CPI203抑制ACHN细胞增殖效果；采用流式细胞术分析CPI203对ACHN细胞凋亡及细胞周期的影响；划痕实验观察CPI203影响ACHN细胞迁移及侵袭能力；克隆形成实验检测CPI203抑制ACHN细胞克隆集落形成能力；荧光定量PCR法及免疫印迹法分别分析CPI203作用于ACHN细胞后，MYC、NOXA、AKT、ERK、CyclinD1和GSK3β的表达量变化情况。 结果CPI203明显抑制肾癌细胞株ACHN细胞增殖，并呈时间及浓度依赖性（P<0.05）；CPI203药物可促进ACHN细胞凋亡并抑制细胞生长周期（P<0.05）；CPI203呈浓度依赖性地降低ACHN细胞的克隆形成能力及迁移能力（P<0.05）；CPI203降低肾癌ACHN细胞中MYC、NOXA、AKT、ERK、CyclinD1和GSK3β表达量减少（P<0.05）。 结论CPI203可抑制ACHN细胞增殖，促进肾癌细胞凋亡并抑制其生长周期进展，明显抑制癌细胞迁移及克隆形成能力，其作用机制与CPI203影响肾癌ACHN细胞中MYC、NOXA、AKT、ERK、CyclinD1和GSK3β表达量有关。 Abstract:ObjectiveTo explore the ability of CPI203 killing renal cell carcinoma ACHN cells and its potential mechanism. MethodsACHN was treated with various concentrations (0, 0.1, 0.5, 1 and 5 μmol/L) of CPI203 for 24 and 48 h. The effect of CPI203 on ACHN cell proliferation was assessed by CCK8. Flow cytometry was applied to examine the effect of CPI203 on ACHN 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 evaluate the mRNA and protein expression of MYC, NOXA, AKT, ERK, CyclinD1 and GSK3β. ResultsCPI203 inhibited the growth of ACHN cells. CPI203 induced both apoptosis and cell cycle arrest of ACHN cells in a dose-dependent manner. The migration and colony forming ability were also inhibited by CPI203 in a dose-dependent manner. CPI203 decreased relating genes expression, such as MYC, NOXA, AKT, ERK, CyclinD1 and GSK3β. ConclusionCPI203 has significant antitumor effect against renal tumor cells via inhibition of MYC, NOXA, AKT, ERK, CyclinD1 and GSK3β. -
Key words:
- renal cell carcinoma /
- CPI203 /
- ACHN cell /
- cell apoptosis /
- cell cycle /
- cell proliferation
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图 1 ACHN细胞划痕实验
A: 刚划痕, 不加CPI203处理对照组; B: 刚划痕, 加0.1 μmol/L CPI203处理组; C: 刚划痕，加0.5 μmol/L CPI203处理组; D: 刚划痕, 加1 μmol/L CPI203处理组; E: 刚划痕，加5 μmol/L CPI203处理组; F: 划痕48 h后, 不加CPI203处理对照组; G: 划痕48 h后, 加0.1 μmol/L CPI203处理对照组; H: 划痕48 h后, 加0.5 μmol/L CPI203处理对照组; I: 划痕48 h后, 加1 μmol/L CPI203处理对照组; J: 划痕48 h后, 加5 μmol/L CPI203处理对照组.
图 3 小分子抑制剂CPI203作用于肾癌ACHN细胞后相关基因表达的影响
CPI203作用于肾癌ACHN细胞后降低MYC、NOXA、AKT、ERK、GSK3β和CyclinD1基因表达量；*P<0.05 vs Control.
图 4 不同浓度CPI203药物对肾癌ACHN细胞克隆形成能力的影响
越高浓度的CPI203药物作用于肾癌ACHN细胞后，细胞克隆形成数目越少，*P<0.05 vs 0 μmol/L.
图 5 小分子抑制剂CPI203作用于肾癌ACHN细胞后相关蛋白表达的影响
CPI203作用于肾癌ACHN细胞后降低MYC、NOXA、AKT、ERK、GSK3β和CyclinD1蛋白表达量.
表 1 qRT-PCR引物序列
Gene Primer sequence（5’-3’） MYC Forward primer CACATGCCCAAGATTCACTGATAG Reverse primer GAGGTGGCTTGGACAGGTTAG GSK3β Forward primer GGCAGCATGAAAGTTAGCAGA Reverse primer TTTCTTGATGGCGACCAGTTCT ERK Forward primer TGCTCTGCATGTGGTAACTTG Reverse primer GAACCCTAGGAGCACTGACATC CyclinD1 Forward primer CTGGGTCTGTGCATTTCTGGTT Reverse primer CTGCTGGAAACATGCCGGTTA AKT Forward primer GGCCTCAGCCCTCAGAAC Reverse primer TGCCACATTGCGCATAGCT NOXA Forward primer CCAGCAGAGCTGGAAGTCG Reverse primer CTTCCGTTTCCAAGGGCACC GAPDH Forward primer CCTGCACCACCAACTGCTTAG Reverse primer TGAGTCCTTCCACGATACCAA 
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表 2 CCK8法检测CPI203抑制ACHN细胞增殖效果（%，Mean±SD）
CPI203浓度（μmol/L） 24 h 48 h 0 100±2.36 100±2.41 0.1 87.29±0.98* 79.33±1.27* 0.5 76.52±1.35* 60.45±0.87* 1 63.24±0.82* 46.28±1.25* 5 52.61±1.51* 31.42±1.36* *P<0.05 vs 0 μmol/L
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