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The Effects of BMP9 Expressions on Proliferation and Apoptosis of Human Breast Cancer Cell Line MDA-MB-231

Author: FengHongLei
Tutor: ZhangYan
School: Chongqing Medical University
Course: Clinical Laboratory Science
Keywords: BMP9 Breast cancer Proliferation Cell cycle Apoptosis
CLC: R737.9
Type: Master's thesis
Year: 2011
Downloads: 30
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Abstract

Objective To investigate the effects of BMP9 on the proliferation, cell cycle and apoptosis of breast cancer cell line MDA-MB-231 in vivo and in vitro . To investigate the potential signal pathway and downstream target molecule of BMP9 in MDA-MB-231 and provide experimental evidence for the new therapeutic agent or inhibitor of breast cancer.Methods The human breast cancer cell line MDA-MB-231 were infected with AdBMP9 (MDA-MB-231/BMP9 as experimental groups) and AdGFP (MDA-MB-231/GFP as control groups) respectively, non-infected MDA-MB-231 cell were used as control groups too.MTT assay was used to examine cell proliferation, flow cytometry was used to detect cell cycle distribution. To detect the percentage of apoptosis in three groups, AnnexinⅤ-PE/7-AAD double staining and Hoechst33258 staining were carried out. Spectrophotometer method was employeed to examine the activity of caspase-3 .Western Blot was used to examine the level of total protein and phosphorylated protein of Smad1、P38、ERK1/2 and JNK in MDA-MB-231/BMP9 cells; Real time PCR was used to examine the expression of c-jun、c-fos、c-myc、bcl-2、bax、Fas、P21、P53 which were related to cell proliferation and apoptosis .A nude mice xenograft model was constructed to confirm the inhibitory effects of BMP9 in vivo. The MDA-MB-231/BMP9 cells and MDA-MB-231/GFP cells were injected subcutaneously into nude mice respectively. After two weeks, when tumors can be observed, the diameter of the tumors was recorded every three days. The mice were sacrificed and tumors were obtained after one month. The tumors were dissected and processed for further TUNEL analysis.Results The MDA-MB-231/BMP9 cells showed an inhibited proliferation. On the fourth day, the growth rate of experimental groups(0.7142±0.1324)was significantly lower than that of control groups(0.9480±0.0164)(P<0.05). The results of flow cytometry showed that, The MDA-MB-231/BMP9 cells were arrested in G2/M phase. On the second day and third day , the cell number of G2/M phase increased significantly, which were 3.2 fold and 2.4 fold that of control groups respectively. It’s also found that after 72h , the apoptosis rate of experimental groups(31.55±8.26)% was significantly higher than that of control groups (3.80±0.46)%(P<0.05). Hoechst33258 staining showed that, after 72h ,the percentage of positive cell of experimental groups(15.5±0.8)% was significantly higher than that of control groups (4.75±0.77)%(P<0.05). After 72h, the caspase-3 activity of experimental groups(0.289±0.158) was significantly higher than that of control groups(0.180±0.094)(P<0.05).Western Blot showed that BMP9 can promote the expression of P-Smad1、P-P38、P-JNK and inhibit the expression of P-ERK1/2 in MDA-MB-231/BMP9 cells , but not their total protein levels;The expression of c-jun and c-fos gene were up-regulated in MDA-MB-231/BMP9 cells , which were ( 3.0017±0.4374 ) fold and (2.7147±1.0881)fold(P<0.05)of control groups respectively, and the expression of c-myc and bcl-2 were down-regulated, which were (0.2035±0.0242)fold and(0.5974±0.1702)fold(P<0.05) of control groups respectively.The results of nude mice xenograft model showed that the time of subcutaneous tumor formation in MDA-MB-231/BMP9 groups were longer than that of control groups, and the volume of subcutaneous tumor in MDA-MB-231/BMP9 groups(0.792±0.065)cm~3 were smaller than that of control groups (2.595±0.242)cm~3 (P<0.05). The TUNEL assay of tumor tissue section showed that the apoptotic index of MDA-MB-231/BMP9 groups (95.7±2.4)% were higher than that of control groups (61.6±7.3)% (P<0.05) significantly.Conclusion BMP9 can cause growth inhibition and apoptosis of MDA-MB-231 cell in vivo and in vitro , these effect of BMP9 may developed through activating the Smad and MAPKs signal pathways, up-regulating c-jun and c-fos and down-regulating c-myc and bcl-2.

The Effects of BMP9 Expressions on Proliferation and Apoptosis of Human Breast Cancer Cell Line MDA-MB-231

Abstract

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