姜黄素对乳腺癌MDA-MB-231细胞侵袭的抑制作用

Inhibitory effect of curcumin on invasion of human breast cancer MDA-MB-231 cells

目的研究姜黄素对侵袭相关因子中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin,NGAL)表达的调控,初步探讨姜黄素在防治乳腺癌侵袭转移中可能存在的新的作用机制。方法以高侵袭性乳腺癌细胞株MDA-MB-231为研究对象,采用不同剂量姜黄素处理,并使用核转录因子-κB(nuclear transcription factor-κB,NF-κB)特异性抑制剂Bay-117082(20μmol/L)对比观察。MTT法检测5、10、15、20、25、30、35、40、50μmol/L姜黄素作用24、48、72 h对细胞的毒性作用;Transwell小室侵袭试验法检测5、10、15μmol/L姜黄素对细胞的侵袭能力,黏附试验法检测对细胞的黏附能力;Western blot法检测5、10、15μmol/L姜黄素、20μmol/L Bay-117082及15μmol/L姜黄素+20μmol/L Bay-117082对细胞中核转录因子κB抑制剂α(inhibitorαofκB,IκBα)磷酸化的IκBα(phosphorylated-IκBα,p-IκBα)及NGAL蛋白表达水平的影响,RT-PCR法检测对细胞中NGAL基因mRNA转录水平的影响。结果姜黄素浓度>15μmol/L,作用时间>24 h时,对细胞的生长抑制呈时间和剂量依赖性(P<0.001),当姜黄素浓度≤15μmol/L,作用时间为24 h时,对细胞无明显毒性作用,细胞存活率>90%;随着姜黄素浓度的增加,细胞侵袭能力和黏附能力逐渐降低,以15μmol/L姜黄素作用效果最明显(P<0.05);姜黄素对p-IκBα、NGAL蛋白表达的抑制作用与NF-κB特异性抑制剂Bay-117082相似,且呈剂量依赖性(P<0.05),而对IκBα蛋白的表达无明显影响;姜黄素可明显抑制NGAL基因mRNA转录水平,其抑制作用呈剂量依赖性(P<0.05),且Bay-117082抑制NGAL基因mRNA转录水平的作用与15μmol/L姜黄素相似。结论姜黄素可通过抑制NF-κB信号通路的活化及此信号通路靶基因NGAL的表达降低乳腺癌MDA-MB-231细胞的侵袭性。

Objective To investigate the regulatory effect of curcumin on expression of neutrophil gelatinase-associated lipoealin （NGAL） as well as the possible mechanism of eurcumin in prevention and treatment of breast cancer. Methods Highly invasive MDA-MB-231 cells were treated with curcumin at various dosages, using the specific inhibitor Bay-117082（20 μmol/L） of nuclear transcription factor-κB（NF-κB） as control. The eytotoxie effects of treatment with 5, 10, 15, 20, 25, 30, 35, 40 and 50 μmol/L curcumin for 24, 48 and 72 h on MDA-MB-231 cells were measured by MTF assay. The invasion abilities of cells treated with 5, 10 and 15 μmol/L curcumin were measured by Transwell test, while the adhesive ability by cell adhesion assay. The effects of 5, 10 and 15 μmol/L curcumin, 20 μmol/L Bay-117082 and 15 μmol/L cureumin ＋ 20 μmol/L Bay-117082 on expression levels of inhibitor αofκB （IκBα）, phosphorylated IκB and NGAL were determined by Western blot, while those on transcription level of NGAL mRNA by RT-PCR. Results The treatment with curcumin at a dosage of more than 15 μmol/L for more than 24 h inhibited the growth of MDA-MB-231 cells in time-and dose-dependent patterns （P 〈 0. 001 ）. The treatment with curcumin at a dosage of not less than 15 μmol / L for 24 h showed no significant cytotoxic effect on the cells, while the survival rate of cells was more than 90%. The invasion and adhesive abilities of cells decreased gradually with the increasing curcumin concentration. The curcumin at a concentration of 15 μmol/L showed the strongest inhibitory effect on the two abilities （P 〈 0. 05）. The in-hibitory effects of curcumin on expressions of p-IκBα and NGAL were similar to those of Bay-117082, which was dose-de-pendent （P 〈 0. 05）, while showed no significant effect on expression of IκBα. Curcumin showed significantly dose-depen-dent inhibitory effect on the transcription level of NGAL mRNA （P 〈 0. 05）. However, the inhibitory effect of Bay-117082 on transcription level of NGAL mRNA was similar to that of 15 μmol/L curcumin. Conclusion Curcumin inhibited the invasive ability of MDA-MB-231 ceils by inhibiting the activation of NF-κB signal pathway and expression of NGAL as a target gene of the pathway.