| Objective To explore the effects of bone-stored growth factors, BMP9, ononcology behavior of human breast cancer cells MDA-MB-231in the bonemicroenvironment. To study its possible molecular mechanisms anddesign a novel therapeutic approaches for bone metastases of breast cancer.Methods We chose the high metastatic breast cancer cell lineMDA-MB-231as the experimental model. And constructed a co-culturesystem composed of breast cancer cells MDA-MB-231and bone marrowstromal cells HS-5. We tried to elucidate the effects of BMP9onMDA-MB-231cells from gene silencing and gene overexpressing aspects.We set up five groups in our experiment, including groupCo-Blank (MDA-MB-231+HS-5),Co-GFP (MDA-MB-231/AdGFP+HS-5),Co-RFP (MDA-MB-231/AdRFP+HS-5),Co-BMP9(MDA-MB-231/AdBMP9+HS-5)Co-siBMP9(MDA-MB-231/AdsiBMP9+HS-5).MTT assay was used to investigate the growth ability of breast cancer cells in different groups; Cell wounding assay and transwell invasion assaywere carried out to detect the changes in migration and invasion ofMDA-MB-231in the coculture system. Flow Cytometry (FCM) wascarried out to detect the percentage of apoptosis in each group. To identifythe important factors that may mediated by BMP9in breast cancer cells, weused QRT-PCR to screen metastasis-related genes: interleukin-6(IL-6),stromal cell-derived factor-1(SDF-1) and monocyte chemotactic protein1(MCP-1). The activities of MMPs in the conditioned media weredetermined by gelatin zymography. Immunofluorescence was used toinvestigate the EMT-associated molecules (Vimentin, Fibronectin) andCXCR4receptor of breast cancer MDA-MB-231cells. Western blot wascarried out to detect EMT-related molecules and the activated state ofSDF/CXCR4-PI3K signaling pathway.The human femur fragments obtained from freshly discarded tissue at thetime of surgery in female patients undergoing total hip replacement werewashed with PBS and dissected into pieces. Then the femur pieces weresubcutaneously implanted in female nude mice. After1week, theembedded bone appears successfully implanted, as evidenced by noinflammation and necrosis. Next, the mixed cell suspension ofMDA-MB-231cells (1×107) and HS-5cells (1×107) from each group wasinjected directly nestle up human adult femur fragments implanted in nudemice. After1week, when tumors were observable, the tumor diameters were recorded every5days. After41days, tumors and implanted femurfragments were harvested and fixed in buffered formaldehyde, embedded inparaffin, sectioned for further histological and immunohistochemicalanalysis.Result Firstly, we investigated the effect of BMP9on proliferation,migration and invasion of MDA-MB-231cells in bone microenvironment.We used BMP9interference (AdsiBMP9) and overexpression (AdBMP9)technology to successfully regulated the BMP9expression of breast cancerMDA-MB-231cells, which co-cultured with bone marrow stromal cellsHS-5.①The result of MTT showed that on the day5, group Co-BMP9cells proliferation rate was slightly reduced than those of Co-GFP cells;②The result of wound healing showed that the healing rate of Co-BMP9MDA-MB-231cells (46.33±2.87)%were markedly lower than those of theCo-GFP cells (84.67±4.64)%and the healing rate of Co-siBMP9cells wasElevated;③The result of transwell without matrigel showed that thenumber on the low chamber of Co-BMP9MDA-MB-231cells（161±3.2）were significantly lower than those of Co-GFP cells（215±15.1）and themigrated MDA-MB-231cells in Co-siBMP9group was increased slightly;④The number of invading MDA-MB-231cells which moved across thematrix barrier was decreased from control group （189±7.4） toexperimental Co-BMP9group（112±9.6）(P＜0.05), and the invadedMDA-MB-231cells in Co-siBMP9group was increasing.⑤The apoptosis rate of MDA-MB-231cells in Co-BMP9group（31.3±5.8）%was significantly higher than that of control group（s14.7±7.5）%(P＜0.05),and the apoptosis cell in Co-siBMP9group was decreasing.Secondly, we investigated the underlying mechanism of BMP9antitumor effect in bone microenvironment.①The BMP9overexpressiongroup MDA-MB-231cells showed a typical epithelial cuboidal shapecompare to the control group which exhibit a fibroblast-like shape;②Theresult of immunofluorescence showed that Vimentin and Fibronectindecreased in MDA-MB-231cells overexpressed BMP9;③We foundMMP9and MMP2activity were both cut down on BMP9overexpressiongroup by gelatin zymographic analysis;④By QRT-PCR We found thatelevated BMP9in the co-culture system can decreased IL-6and MCP-1ineither HS-5cells or MDA-MB-231cell. While, SDF-1only decreased inHS-5cells.⑤Immunofluorescence staining showed that CXCR4waspositive expression on MDA-MB-231cells.⑥By Western blot we foundthat the protein expressions of EMT marker (Vimentin, Fibronectin) andMMPs were decreased in Co-BMP9group, while increased slightly inCo-siBMP9group. Western blot analysis showed that BMP9suppressedSDF-1and the downstream molecule of SDF-1/CXCR4-PI3K signalingpathway, whereas they increased in Co-siBMP9group.Finally, we further explore the effect of BMP9in vivo.①Micexenograft model showed that there was no significant difference in tumor volume of each group;②Histological and histomorphometric analysisshowed that BMP9can reduce the damage of embedded bone;③Immunohistochemical study revealed that Vimentin, MMP2, SDF-1decreased in Co-BMP9group. There was no difference of these factorsbetween Co-siBMP9group and the control group.④The TUNEL assayshowed that apoptotic index of Co-BMP9group (79.5±1.6)%were higherthan Co-GFP group (37.8±1.2)%(P＜0.05). The apoptotic index ofCo-siBMP9group was decreased，while had no difference with controlgroup.Conclusion BMP9has an inhibitory effect on breast cancer cellsMDA-MB-231in bone microenvironment through downregulating theexpression of metastasis related factors. And SDF-1/CXCR4-PI3Ksignaling pathway and EMT may involve in this inhibitroy process. |
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