P Substance And Its Receptor Nk-1 Expression In Breast Cancer And Role

Substance P is a member of neurokinin family.It has been demonstrated that both preprotachykinin A(PPT-A) and neurokinin-1(NK-1) mRNA were increased in several breast cancer cell lines and certain NK-1 antagonists could reduce proliferation of several cancer cell lines,however,little is known about the cellular localization of substance P and NK-1 on human breast carcinoma tissues and human breast cancer cell line T47D. Additionally,it is unknown whether SR140333 exerts proliferation inhibition action on this cell line.Objective 1.To study the expression of substance P(SP) and its receptor, neurokinin-1(NK-1) in breast carcinomas and human breast cancer cell line T47D.2.To evaluate the effect of potent selective NK-1 agonist[Sar9,Met(O2)11]substance P(SMSP) and selective antagonist SR140333 on human breast cancer cell line T47D in vitro.3.To investigate the potential function of substance P and NK-1 in human breast cancer cell line T47D.Methods The immunohistochemistry study was performed to detect the immunolocation of substance P and neurokinin-1 in breast carcinomas(n=103) and corresponding para-cancer tissues.Several benign tumors were also selected to study as follows:fibroadenoma(n=5),adenosis(n=6),intraductal papilloma(n=3),epithelial atypical hyperplasia(n=5).And hence we carried out an in vitro immunocytochemical study to investigate the localization of substance P and NK-1 in T47D cells.Thereafter, various concentrations of SMSP(10¹⁰M-10(-6)M) and SR140333(10^-9M-10^-5M) were applied alone and combined.Hemacytometer and MTT assay were used to detect the cytoactivation and to confirm growth curves.The Hoechst33258 method and HE staining was performed to determine apoptosis.Rhodamine 123 staining and flow cytometry analysis were designed to investigate the status of the mitochondrial membrane potential. Results We show here that breast carcinoma could co-express both substance P and NK-1.The immunopositive substance P cells were distributed diffusely and the majority of SP labeling was localized within the cytoplasma of breast carcinoma.However,NK-1 could be observed in both plasma membrane and/or cytoplasma.The immunoreactive NK-1 cells were widely distributed.Moreover,atypical hyperplasia tissues also bear positive substance P and NK-1 expression.2/3 intraductal papilloma tissues have positive immunoreactive NK-1.However,the immunolablling substance P and NK-1 were not detected on fibroadenoma and adenosis tissues.In addition,intratumoral and peritumoral blood vessels also beared NK-1 expression.In T47D cells,we found immunoreactive substance P was mainly presented on cytoplasm and NK-1 on both membrane and plasma.At a concentration of 10^-8M SMSP exerted maximum proliferation action and SR140333(10^-9-10^-5M) inhibited cell growth in a dose dependent manner.Furthermore, SR140333 could counteract SMSP induced proliferation.Hoechst33258 and HE staining revealed the presence of apoptosis after SR140333 treatment.The flow cytometry analysis indicated mitochondria may act as a player of SR140333 induced T47D apoptosis.Growth curves suggest that from the third day SMSP exert a significant growth promotion effect while SR140333 show growth inhibition effect from the fourth day.Conclusion 1.Given that both breast carcinoma tissues and T47D cell line bear over expression of substance P and NK-1,it is suggested that neuroendocrine mechanisms participate the development of breast carcinoma.The present study also provides novel diagnostic markers for breast carcinomas.2.SR140333 could blunt cell growth and inhibit SMSP induced proliferation via NK-1 in vitro.3.Substance P may stimulate cell proliferation through NK-1 in vivo;NK-1 may serve as a critical gate to regulate substance P related cell proliferation and a new target for therapy.