Study Of The Proliferative Inhibition Induced By Two Kinds Of Neurokinin Receptor Antagonists On Chronic Myeloid Leukemia Cells

Tachykinin is a peptide family shares common conservative structure. By combining its receptor, tachykinin regulates various physiological or pathological events including immune regulation, inflammatory response, respiratory and gastrointestinal muscle contraction and tumor progression. The classical members of tachykinin family are substance P, neurokinin A and neurokinin B. The biological actions of tachykinin are mediated through three types of receptors denoted NK1R, NK2R and NK3R that belong to the family of G protein-coupled receptors. Given neurokinin receptors are widely involved in various physiological activities, potent and selective neurokinin receptor antagonists have been developed in the past years and many efforts have been made to authenticate their efficacy in the treatment of various diseases. Currently, neurokinin receptor antagonists have been developed to the third generation, in which the antagonists in the first and second generation are peptides with the properties of easy degradation and side effects, while the antagonists in the third generation are small molecule compounds with the properties of non-peptides and high affinity and selectivity for NKRs. The representative antagonists of the third generation are SR140333(neurokinin receptor1antagonist) and SR48968(neurokinin receptor2antagonist) which are in clinical phase â…¡ research for treatment of asthma.Many evidences indicate that tachykinin and its receptors are closely related with tumor progression. In some kinds of cancers, the expression of neurokinin receptor1significantly raises, and the expression of NK1R is correlated with the degree of malignancy. Substance P and NK1R are involved in many tumor progressions including induction of mitogenesis, vasculogenesis, tumor cell migration and transfer. Raised neurokinin A (NKA) is a good predictor of prognosis in neuroendocrine tumours of the midgut, colon and ileum. Several experiments demonstrate that some antagonists for neurokinin receptor1(for example, Aprepitant, L-733,060, L-732,138and MEN11467) have antitumor effect, in which, especially Aprepitant, it may be a broad-spectrum anti-tumor drug. Likewise, there are several reports about the anti-tumor effects of SR140333on breast and colon cancer, and no report about the anti-tumor effect of SR48968. Moreover, the mechanisms of anti-tumor effects induced by neurokinin receptor antagonists are needed to be clarified.In this thesis, we investigated the mechanism of the proliferative inhibition induced by SR140333and SR48968on human chronic myelogenous leukemia cells (K.562cell line). The results of MTT test and optical microscopy observation showed that SR140333and SR48968inhibited the growth of K562cells in a dose-and time-dependent manner. Moreover, the inhibited effects of the growth of leukaemia cells induced by SR140333and SR48968may be mediated through an apoptosis pathway, which was obtained from the Hoechst33342staining test and flow cytometry. Furthermore, after the treatment with SR140333and SR48968, the results from Western blot assays showed that the expression of c-Myc was inhibited significantly, the expression of Bax and Bcl-2which are associated with cell apoptosis was raised significantly, the expression of cyclin-dependent kinase4was reduced significantly, however the expression of NK1R and NK2R were steady. All of these results indicate that SR140333and SR48968could induce the cell cycle arrest and cell apoptosis of K562cells. Additionally, the data from two dimensional electrophoresis (2-DE) showed that the expression levels of50and71proteins were changed after treated by SR140333and SR48968, respectively. The results of this study will be helpful for the further investigating of the mechanism of proliferative inhibition induced by SR140333and SR48968on human chronic myelogenous leukemia cell line (K562cells). Plus, we have successfully constructed leukemia nude mouse model, and the anti-tumor effect of antagonists on animal level is in progress.In conclusion, SR140333and SR48968could inhibit the growth of K562cells in a dose-and time-dependent manner, and the mechanism involved in the antitumor effects induced by the antagonists may be mediated through the cell cycle arrest and cell apoptosis.