The Effects Of RhBMP-2 On The Proliferation, Metastasis And Apoptosis Of Breast Cancer

BMP-2(bone morphogenetic protein-2, BMP-2) is a kind of cytokine which can induce the new bone generation independently, it belongs to the super family of TGFβ.Bone fractures and bone defects are very common in clinical caused by kinds of reasons, BMPs are very important in the treatment of these bone repairing. There are kinds of recombinant human BMPs (such as BMP-2, BMP-7) on the market overseas. In China, the manufacture of rhBMPs is just on the beginning.According to our original producing process of rhBMP-2 with intellectual property, we chose bacteria as the prokaryotic expression system to express recombinant human BMP-2 highly as inclusion body, then got it with high purity after refolding and purification. The advantages of this producing process are high-yield, high-activity and low-cost, so it is good for large-scale production (See the patent in the appendix).Oxidative and reductive electrophoresis, mass spectrum and HPLC assays showed that the molecular weight of rhBMP-2 was identified with the theoretical value, the purity of rhBMP-2 was more than 95%. In vitro, rhBMP-2 could induce C2C12 cells transferring to osteoblastic cells, expressing large amount of ALP (early differentiation marker), bone matrix deposition, forming mineralized nodules. In vivo, rhBMP-2 could induce the ectopic bone formation in thigh muscle pouches of mice, exhibiting an excellent bone-inductive activity. In the experiment of repairing of the rabbit radial defects, composite materials mixed rhBMP-2 with the bone-filling material (made by Guanhao bio-technology limited company) could integrate the material into the autogenous bone by the generation of large amount of new bone, meanwhile the composite material could be bio-degraded largely. All these bone-repairing property index of composite material with rhBMP-2 are better than the bone-filling material without rhBMP-2.The experimental results above showed the mature producing process of rhBMP-2 reached the requirements of sFDA basically. The pharmaco-dynamic studies on rhBMP-2 showed its good bone-repairing ability and its board clinical application prospect. Meanwhile, since BMP-2 is an important growth factor in embryonic development and maintenance of normal physiological functions of cells which participates in basic life activities broadly, such as proliferation, differentiation, apoptosis and aging, it is necessary to study the role of rhBMP-2 in these life activities, especially the relationship between rhBMP-2 and the cancer which is of common concern in clinics.Breast cancer is a kind of common tumor in clinics. We choose MCF-7 cell line (ERa-66 positive, low-metastatic) and MDA-MB-231 cell line(ERa-66 negative, high-metastatic) as our study objects to discover the relationship between rhBMP-2 and the occurring and developing of breast cancer.The genes of BMPR1a, BMPR1b, BMPR2, Smad4 expressed in the two cell lines in the mRNA level. BMP-2 induced phosphorylation of Smad1/5/8 on WB assay showed that there are complete BMP-signal pathways in the two cell lines.BMP-2 inhibited markedly the growth of the breast cancer cells. MTT assay showed that when the dose of BMP-2 was 20ng/ml, the inhibitory rates were highest, which were 60% and 30% respectively in MCF-7 cells and MDA-MB-231 cells. In MCF-7 cells, E2βcould weaken the inhibitory effect of BMP-2 on the proliferation of breast cancer cells, made the inhibitory rate drop from 45% to 25%. The antagonist of E2β, tamoxifen, was not able to reverse this effect of E2p. MDA-MB-231 cells were not sensitive to E2p and tamoxifen.Analysis of cell cycle with Flow Cytometry showed that the proportion of cells in G1 phase increased, from 63.7% to70.8% in MCF-7 cells and from 46.7% to 62% in MDA-MB-231 cells. The results of real-time PCR showed that BMP-2 could up-regulate markedly the expression of P21, which is a inhibitor of cell cycle by arresting the cells in G1 phase to S phase. We suggest that the inhibitory effect of BMP-2 on the proliferation of breast cancer cells may mainly result from its inducing expression of P21, and causing the G1-phase arrest.BMP-2 also promote the apoptosis of breast cancer cells. Analysis of apoptosis with Flow Cytometry showed that the rate of early apoptotic cells was from 1.0% to 7.3% after induction of BMP-2 in MCF-7 cells, the rate of early apoptotic cells was from 10.32% to 34.26% after induction of BMP-2 in MDA-MB-231 cells. The results of real-time PCR showed that BMP-2 could up-regulate markedly the expression of Caspase-3, which is one of the executors of apoptosis in cells. We suggest that the BMP-2 can promote the apoptosis by up-regulating the expression of Caspase-3.Through all above experimental results, we found that BMP-2 could inhibit markedly the growth of breast cancer cells. By up-regulating the expressions of P21 and Caspase-3, BMP-2 could inhibit the proliferation and promote the apoptosis of breast cancer cells.Our cytological observation was confirmed further by animal testing. MDA-MB-231 cells was implanted on the breast of each mouse. After intraperitoneal injection of BMP-2 at the dosage of 20μg each mouse once a day for sixteen days, there were 7 mice in control group, tumor formed in 6 mice, not formed in 1 mouse; also there were 7 mice in BMP-2 treated group, tumor formed in 1 mouse, not formed in 6mice. These results showed that BMP-2 could inhibit markedly the growth of breast cancer in the early stage.This study also explored the role of BMP-2 in tumor metastasis. Since MDA-MB-231 cells are high-metastatic, we focus our study on MCF-7 cell line (low-metastatic).1-50ng/ml BMP-2 could change the morphology of MCF-7 cells, transferred from epithelial cells' morphology such as multangular cell configuration like paved-bricks to interstitial cells'morphology such as spindle-shaped and loose-connected between cells. Atomic force microscopic observation showed that MCF-7 cells became more narrow, more flat, lamellipodia arising, characterized by enhanced motility of MCF-7 cells.Scratch healing and transwell experiment showed that the motility of MCF-7 cells was highest after 30ng/ml BMP-2 treated 1day. Real-time PCR results showed that BMP-2 up-regulated the expressions of series of genes related to the migration of cells such as Pail and ID1. The result of WB showed that BMP-2 up-regulated the expression of CD44. All above suggested that BMP-2 promoted the motility of MCF-7 cells resulted from its induction of the expressions of kinds of genes related to cells'migration.Since nodes of metastatic tumor did not form in lungs in nude mice when MCF-7 cells and MDA-MB-231 cells inoculated via tail vein injection, we explored the role of BMP-2 in the metastasis of breast cancer by mouse 4T1 breast cancer cells on lung metastasis model. Nodes of BMP-2 treated group were more than control group. Immunohistochemistry detections of mice lung sections showed that the expressions of pi-Smad1/5/8,α-SMA and Vimentin in BMP-2 treated group were higher that control group, which suggested that BMP-2 could induce the expressions of marker proteins of interstitial cells, transformation from epithelial cells to interstitial cells by activation of BMP-signal pathway. The expression of E-Cadherin was a little higher than control group, which suggested that the tumors in lungs of mice originated from epithelial cells.We also studied on the crosstalk between estrogen-signal pathway and BMP-signal pathway since estrogen played a vital role in breast cancer. We found that BMP-2 could induce the expression of ERa-36 on the mRNA level. After analyzing the unique sequence on the C end of ERa-36, we designed and synthesized the short peptide ofⅣ10. Anti-Ⅳ10 antiseria were produced with theⅣ10-KLH conjugate injecting rabbits, and idiotype antibody(Ab) were achieved after purification. WB results showed that BMP-2 could induce the expression of ERa-36 on the protein level with dose-dependent manner, which was inhibited by noggin, the competitive inhibitor of BMP-2. Since ERa-36 participates in the resistance against endocrine therapy, BMP-2 may be related to it by induction of the expression of ERa-36.From the overall experimental results, the effects of BMP-2 on breast cancer could be summed up into two kinds, one is negative regulations which include promoting G1-phase arrest and apoptosis of cancer cells, the other is positive regulations which include promoting metastasis and maybe include also promoting the resistance against endocrine therapy by induction of the expression of ERa-36. However, the two things are opposed that maybe cancel each other out, which caused us to fail to see the actual effects of rhBMP-2 on the model of xenografts tumors in nude mice.Hence the conclusions are:rhBMP-2 produced by our lab has the good osteoinductive capacity to repair bone defects. BMP-2 is closely related to cancer. Since the negative and positive effects on cancer exist simultaneously and counteract, the actual fuction of BMP-2 for cancers may be very limited.