钙调素拮抗剂EBB抑制MDA-MB-231人乳腺癌细胞骨转移和增殖活动的作用

Inhibitory effects of regucalcin antagonist EBB on bone metastasis and proliferation of human breast cancer MDA-MB-231 cells in vitro

目的通过体外实验探讨钙调素(regucalcin)拮抗剂抑制MDA-MB-231人乳腺癌细胞骨转移和增殖活动的作用。方法体外培养人乳腺癌细胞株MDA-MB-231细胞,取对数生长期的细胞,分为对照组、实验1组、实验2组[分别以fenbieyi regucalcin拮抗剂O-(4-乙氧基)-丁基-小檗胺(EBB) 10μmol/L、1μmol/L进行处理]。采用MTT法检测肿瘤细胞增殖抑制作用; Transwell细胞侵袭实验、细胞划痕实验检测肿瘤细胞侵袭,转移情况;流式细胞仪测定细胞周期;免疫印迹法测定骨转移相关蛋白(P38、JNK、AKT)表达情况。结果 MTT检测显示EBB能够抑制MDA-MB-231细胞的生长,且当剂量增加时这种抑制作用更加显著,MDA-MB-231细胞的半数抑制浓度(IC50)为(13. 22±2. 10)μmol/L。实验1组与实验2组的细胞划痕愈合率为(67. 03±3. 63)%和(72. 01±5. 20)%,明显少于空白对照组的(92. 22±5. 39)%(P <0. 01)。与对照组比较,不同浓度的EBB均能有效抑制MDA-MB-231细胞的转移(P <0. 01),其中实验1组的抑制效果明显优于实验2组(P <0. 01)。实验1组与实验2组的的S期比例明显高于对照组(P <0. 01),G2/M与GO/G1期比例明显低于对照组(P <0. 05),实验1组与实验2组之间比较无统计学差异(P> 0. 05)。实验1组与实验2组的JNK、AKT蛋白相对表达量明显低于对照组(P <0. 05,P <0. 01),三组P38蛋白相对表达量比较无统计学差异(P> 0. 05)。结论钙调素拮抗剂EBB能明显抑制MDA-MB-231人乳腺癌细胞骨转移和增殖活动,调节细胞周期,也能抑制成骨相关蛋白的表达。

Objective To investigate the inhibitory effects of regucalcin antagonist on bone metastasis and proliferation of human breast cancer cell line MDA-MB-231 cells in vitro. Methods Human breast cancer cell line MDA-MB-231 cells were cultured in vitro,and the cells in logarithmic growth phase were divided into control group,experiment group 1 and experiment group 2(treated with 10 μmol/L and 1 μmol/L fenbieyi regucalcin antagonist O-( 4-ethoxy)-butyl-berbamine( EBB),respectively)MTT method was used to detect the inhibitory effect of tumor cell proliferation. Transwell cell invasion test and cell wound healing test were used to detect the invasion and metastasis of tumor cells. Flow cytometry was used to detect the cell cycle. Western blot method was used to detect the expressions of bone metastasis related proteins P38,JNK and AKT. Results MTT method showed that EBB can inhibit the growth of MDA-MB-231 cells,and this inhibitory effect was more significant when the dose of EBB increases. The 50% inhibiting concentration( IC50) of MDAMB-231 cells was( 13. 22 ± 2. 10) μmol/L. Cell scratch healing rate in experiment group 1 [( 67. 03 ± 3. 63) %]and experiment group 2[( 72. 01 ± 5. 20) %] were significantly lower than that in control group[( 92. 22 ± 5. 39) % ]( P <0. 01). Compared with control group,EBB at different concentrations could effectively inhibit the metastasis of MDA-MB-231 cells( P < 0. 01) in which the inhibition effect in experiment group 1 was significantly better than that in experiment group 2( P < 0. 01). Compared with control group,the ratios of S-phase increased,and the ratios of G2/M-phase and GO/G1-phase decreased in experiment group 1 and experiment group 2( P < 0. 05,P < 0. 01),while there were no significant differences in them between experiment group 1 and experimental group 2( all P > 0. 05). The relative expressions of JNK and AKT proteins in experiment group 1 and experiment group 2 were significantly lower than those in control group( P <0. 05,P < 0. 01),while there was no significant difference in the relative expression level of P38 protein among three groups( P > 0. 05). Conclusion Regucalcin antagonist EBB can significantly inhibit bone metastasis and proliferation of MDAMB-231 human breast cancer cells,regulate cell cycle,and inhibit the expressions of osteogenic associated proteins.