麦胚凝集素对人乳腺癌MDA-MB-231细胞生长及凋亡基因表达的影响

Influence of wheat germ agglutinin on cell growth and expressions of apoptosis genes in human breast cancer MDA-MB-231 cells

目的探讨麦胚凝集素(WGA)对人乳腺癌MDA-MB-231细胞的生长及凋亡基因表达的影响。方法实验分为对照组、0.035μmol/LWGA组、0.07μmol/LWGA组、0.14μmol/LWGA组和0.28μmol/LWGA组,应用酸性磷酸酶法(APA)、流式细胞术(FCM)和Western blotting法,探讨WGA对MDA-MB-231细胞生长、细胞凋亡率、线粒体膜电位、细胞凋亡相关基因蛋白聚腺苷酸二磷酸核糖转移酶(PARP)、细胞色素C表达的影响。结果不同浓度WGA作用24、48和72h时可抑制MDA-MB-231细胞生长;与人乳腺上皮细胞HBL-100相比,WGA对MDA-MB-231细胞的抑制作用尤为显著。光镜下观察0.14μmol/LWGA组与0.28μmol/LWGA组作用24h时,细胞密度稀疏,体积缩小;早期凋亡率分别为(28.7±3.48)%和(30.15±3.96)%;晚期凋亡率分别为(53.66±4.21)%和(63.18±5.53)%;线粒体膜电位平均荧光强度显著降低;凋亡相关蛋白PARP呈现剪切带,细胞色素C表达上调。结论 WGA抑制人乳腺癌MDA-MB-231细胞增殖,通过干预线粒体途径诱导细胞发生凋亡。

Objective To study the influence of wheat germ agglutinin （WGA） on cell growth and expressions of apoptosis genes in MDA-MB-231 cells. Methods MDA-MB-231 cells were cultured in DMEM containing 10% fetal bovine serum at 37℃ in a humidified atmosphere with 5% CO2. The experiment was performed on five groups： WGA groups were given 0. 035, 0. 07,0.14,0. 28μmol/L WGA respectively, while in the control group the cells were merely grown in DMEM medium solution. Acid phosphatase assay （APA） , flow cytometry（FCM） and Western blotting were used to detect the changes of cell viability, apoptosis ratio, mitochondrial transmembrane potential, the expressions of apoptosisrelated protein poly ADP-ribose polymerase （PARP） and cytochrome C. Each experiment was done four times independently. Results Cell growth was inhibited after MDA-MB-231 cells were co-cultured with different concentrations of WGA for 24, 48, and 72 hours. Compared with human breast epithelia cells line HBL-100, MDA-MB-231 cells were more sensitive to WGA. After cells were treated with WGA for 24 hours, cell numbers reduced, volumes diminished. Early apoptosis ratio caused by WGA （0. 14μmo//L, 0. 28μmol/L, 24 hours） were （ 28.7 ± 3.48 ）% and （ 30. 15 ± 3.96）% , while the late apoptosis rates were （ 53.66 ± 4.21 ） % and （ 63.18 ± 5.53 ） %. Mean fluorescence intensity （MFI） of mitochondrial transmembrane potentials in MDA-MB-231 cells decreased. Apoptosis-related protein PARP showed an cleavage and cytochrome C expression increased in a dose-depandance manner. Conclusion WGA could significally restrain cell growth of breast cancer cell line MDA-MB-231. WGA can induce cleavage of PARP and activate mitochondrial signaling pathway which would induce apoptosis,but the mechanism of WGA is yet to be studied in detail.