| Breast cancer is one of the common tumors in women. Bone is one of the favored metastatic location in breast cancer. The incidence of bone metastasis in breast cancer could be up to 70-80%. Many patients with bone metastasis could suffer pathologicalfracture and have serious bone pain . The bad symptoms had lowered the life quality of patients with bone metastasis. The regular treatment including chemotherapy, radiotherapy and surgery cannot prolong the survival of patients with bone metastasis in advanced breast cancer.Thus, the study of molecular and cellular mechanism of bone metastasis in breast cancer , especially the molecular mechanism of early bone metastasis, are very important for early diagnosis of bone metastasis , making the effective comprehensive therapy to control the occurrence of bone pain and pathologicalfracture , and improving the life quality and prolonging the survival in patients with advanced breast cancer.Presently, the exact molecular mechanism of bone metastasis is not still clear. Recent studies showed that the following factors could be important for the occurrence and development of bone metastasis. These factors included the adhesion of cancer cells to microvasculature of metaphyseal bone;cancer cells migration out of the vasculature and adhesion to bone stromal structure;interaction with cancer cells, bone cells and bone marrow microenvironment. But owing to the absence of suitable animal model and complexity of bone structure, the research of mechanism for cancer bone metastasis was limited largely.Following by the progress in neurology, more and more studies hadrevealed that neurokinin and neurotransraitter not only regulated the physiological action of located tissue , but also participate in signal transduction by the combination with special receptors on surface of cell membrance. Moreover, neurokinin and neurotransmitter could effect on the differentiation, growth, metabolizability, cellular framework and gene expression of target cell. Recently, the regulation effect and mechanism for neurokinin and neurotransmitter to the profiltration and differentiation of cancer cells, the relationship between the expression of neurokinin recptors on the surface of cancer cells and tumor infiltration or migration, had caught the reseachers' s attention. Thus, our study firstly focused on the expression of PPT-I, NK-1, NK-2 in breast cancer tissue and the effect on proliferation of breast cancer cells. Furthermore, we primarily studied possible mechanism for PPT-I, NK-1, NK-2 on early events of bone metastasis in breast cancer.. Our research could provide the important theoretical arguments on the revealment for molecular mechanism of bone metastasis in breast cancer, the prevention and early diagnosis of bone metastasis, and making the effective comprehensive therapy.Part I The expression of preprotachykinin-I gene (PPT-I), the tachykinin receptors (NK-1 and NK-2) in breast cancer tissue and cell linesObjective: To study the expression of PPT-I, NK-1 and NK-2 in breast cancer tissue and paracarcinoma tissue, breast cancer cell lines T-47D ,MCF-7 and MDA-MB-435, nontumourigenic mammary epithelial cell line HBL-100. To research the relationship between the expression of PPT-I, NK-1, NK-2 and the neoplastic biological behavior in breast cancer.Methods: We studied the expression of PPT-I, NK-1, NK-2 in breastcancer tissue and paracarcinoma tissue of 37 patients, breast cancer cell lines T-47D , MCF-7 , normal mammary epithelial cell line HBL-100 by immunhistochemistry . In addition, by realtime PCR and western bolt , we researched the mRNA and protein expression of PPT-I, NK-1, NK-2 in breast cancer cell lines T-47D , MCF-7 , MDA-MB-435 and mammary epithelial cell line HBL-100.Results: The positive expression rate of PPT-I, NK-1, NK-2 in breastcancer tissue and paracarcinoma tissue are 83. 3%, 81. 1%, 86. 5, and 18. 8%, 75%, 21.9%. The expression rate of NK-1 and PPT-I are higher in cancer tissue than paracarcinoma tissue (p<0. 01). The expression of NK-2 is no difference in cancer and paracarcinoma tissue.The expression of PPT-I , NK-1 is higher in breast cancer cell lines than mammary epithelial cell line HBL-100. The expression of NK-2 is no difference in them.Conclusion: The expression of PPT-I and NK-1 could be relevant to the capability of malignant proliferation in breast cancer.Part II The influence of NK-1 and NK-2 receptor antagonists on proliferation of breast cancer cell line T-47DObjective: By the research of action of NK-1 and NK-2 receptorantagonists on the proliferation of breast cancer cell line T-47D, to study the important effect of tachykinins biological network on the malignant proliferation of breast cancer.Methods: We divided breast cancer cell line T-47D into four groups,including control group, NK-1 receptor antagonist group, NK-2 receptor antagonist group, NK-1 and NK-2 receptor antagonists combination group. By the test of clone formation and the flow cytometeric analysis of cell cycle distribution, we studied the changes of proliferation capability and cell cycle of T-47D with and without being given NK-1 or/and NK-2 receptor antagonists.Results: The test of clone formation showed that the proliferatingcapability was greatly inhibited after being dealt with NK-1 or/and NK-2 receptor antagonists.But we observed that the two antagonists couldnot fully cut down the growth of T-47D. So we thought that the proliferation of breast cancer cell line T-47D could be partly regulated by the other pathway yet. The flow cytometeric analysis was given to investigate the changes of cell cycle of T-47D after beingdealt with NK-1 or/and NK-2 receptor antagonists. The results showed that the cell cycle distribution had significant changes with and without being given the two agents. Thenumber of T-47D with G2/M stage was sharply decreased. On the other hand, the proportion of T-47D cell with S stage and GO/G1 stage was greatly increased.Conclusion: The tachykinins receptors NK-1 and NK-2 could play animportant role in the proliferation of breast cancer cell line T-47D. These results suggested that breast cancer cells could autosecret and parasecret tachykinins to improve itself growth.Part III The discuss of the relationship between the expression of PPT-I, NK-1, NK-2 and bone metastasis in breast cancerObjective: To study the potential relationship between the expression of PPT-I, NK-1, NK2 and bone metastasis in breast cancer.Methods: The cocultrues of breast cancer cell line T-47D andmarrow-derived mesencheymal stem cell (MSC) were established with the equal number. At 48 hours and 96 hours after cocultrue, we divided positively T-47D cell from cocultrue system by MACS magnetic cell sorting(MicroBeads). Furthermore, we studied the expressing changes of PPT-I, NK-1, NK-2 in T-47D between before and after coculture by realtime PCR and western bolt. In addition , we observed the changes of cellular ultrastructure of T-47D between before and after cocultrue by electron microscopic analysis. Finally, we also studied the changes of cell cycle distributionthe by flow cytometeric analysis and growth curve after andbefore cocultrue.Results: After cocultrue of T-47D and MSC, the expression of mRNA andprotein of PPT-I were significantly upregulated, and the expression of mRNA and protein of NK-1 and NK-2 were greatly downregulated. The analysis of cell cycle distribution by flow cytometery showed that the proportion of T-47D with S stage was increased and the number of G2/M stage was sharply decreased. By electron microscopic analysis, we observed that the synthesization of hereditary material was increased, but hepatin granule was largely stacked in T-47D cell. These results suggested that although the synthesization of DNA was increased, the proliferation of cell was inhibited after coculture. The cell growth curve also confirmed the outcome of electron microscopic analysis and flow cytometeric analysis.Conclusion: Breast cancer cell could sustain themselves survival by theupregulation of the expression of preprotachykinin-I gene during early bone metastasis in breast cancer. The phenomenon of breast cell ' s growth was suppressed after coculture could be induced by the downregulation of the expression of NK-1 and NK-2 receptors.
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