熊果酸对人结直肠癌SW620细胞恶性生物学行为的影响及机制

Effects and mechanism of ursolic acid on malignant biological behavior of human colorectal cancer SW620 cells

目的探讨熊果酸(UA)对人结直肠癌SW620细胞生长、侵袭、凋亡和转移的影响,并研究其可能的分子机制。方法以SW620细胞为对象,以CCK-8法检测不同浓度(0、5、10、15、20、25、30μmol/L)UA作用不同时间(24、48、72 h)对细胞增殖的影响;以克隆形成实验检测不同浓度(0、10、15、20μmol/L)UA作用于SW620细胞10 d对细胞克隆形成的影响;以不同浓度(0、10、15、20μmol/L)UA作用于SW620细胞24 h后,分别以流式细胞术、Transwell侵袭实验、Western blot实验检测细胞的凋亡、侵袭情况和细胞中B细胞淋巴瘤2(Bcl-2)、裂解型多腺苷二磷酸核糖聚合酶(cleaved-PARP)、磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)、p38 MAPK、上皮钙黏素(E-cadherin)、神经钙黏素(N-cadherin)、锌指转录因子Snail蛋白的表达情况。考察p38 MAPK抑制剂SB203580与UA联用对细胞中p-p38 MAPK、Bcl-2、N-cadherin蛋白表达的影响。结果与0μmol/L UA比较,5~30μmol/L UA作用24、48、72 h均可显著降低细胞的存活率(P<0.05);15、20μmol/L UA作用10 d均可显著降低细胞的克隆形成率(P<0.05);经15、20μmol/L UA作用后,细胞的凋亡率和cleaved-PARP、E-cadherin蛋白的表达量以及p38 MAPK蛋白的磷酸化水平均升高,穿膜细胞数和Bcl-2、Snail、N-cadherin蛋白的表达量均减少或降低,大部分指标差异有统计学意义(P<0.05),部分指标变化有浓度依赖性(P<0.05)。SB203580可显著抑制UA对p38 MAPK蛋白表达的上调作用,逆转UA对Bcl-2、N-cadherin蛋白表达的抑制作用(P<0.05)。结论UA可抑制SW620细胞的生长、侵袭和转移并诱导其凋亡,上述作用可能与激活p38 MAPK信号通路相关。

OBJECTIVE To explore the effects of ursolic acid(UA)on the growth,invasion,apoptosis and metastasis of human colorectal cancer cells SW620 and find out the underlying molecular mechanisms.METHODS The effects of different concentrations(0,5,10,15,20,25,30μmol/L)of UA on the proliferation of SW620 cells for different durations(24,48,72 h)were detected by CCK-8 assay;the clone formation was detected by clone formation assay after SW620 cells were treated with different concentrations of UA(0,10,15,20μmol/L)for 10 days.After SW620 cells were treated with different concentrations of UA(0,10,15,20μmol/L)for 24 hours,flow cytometry,Transwell invasion assay and Western blot assay were adopted to detect apoptosis and invasion of SW620 cells,and the expressions of B cell lymphoma 2(Bcl-2),cleaved-poly(ADP-ribose)polymerase(cleaved-PARP),phosphorylated p38 mitogen-activated protein kinase(p-p38 MAPK),p38 MAPK,E-cadherin,N-cadherin and zinc finger transcription factor Snail.The effects of p38 MAPK inhibitor(SB203580)combined with UA on the protein expressions of p-p38 MAPK,Bcl-2 and N-cadherin were investigated.RESULTS Compared with 0μmol/L UA,the survival rates of SW620 cells treated with 5-30μmol/L UA for 24,48 and 72 h were significantly decreased(P<0.05).The clone formation rate of cells treated with 15μmol/L and 20μmol/L UA was significantly decreased(P<0.05).After being treated with 15μmol/L and 20μmol/L UA,the cell apoptosis rate,the protein expressions of cleaved-PARP and E-cadherin,and the phosphorylation of p38 MAPK protein were increased;but the number of transmembrane cells,and the protein expressions of Bcl-2,Snail and N-cadherin were decreased;there was statistical significance in difference of most indexes(P<0.05).Some indexes changed in a concentration-dependent manner(P<0.05).SB203580 could significantly inhibit the upregulation of p38 MAPK by UA and reverse the inhibitory effect of UA on the protein expressions of Bcl-2 and N-cadherin(P<0.05).CONCLUSIONS UA can inhibit the growth,invasion and metastasis of SW620 cells,and induce cell apoptosis,the mechanism of which may be attributed to the activation of p38 MAPK signaling pathway.