The Effect And Mechannism Of Two Kinds Of Neurokinin Receptor Antagonists On Colonic Cancer Cells

Tachykinins are a group of small and common conservative structurally related peptides characterized by the specific C-terminal sequence, Phe-X-Gly-Lue-Met-NH2, including SP, neurokinin A, neurokinin B, neuropeptide K and neuropeptide-γ. The peptides of the tachykinin family are widely distributed within the mammalian peripheral and central nervous systems and play a well-recognized role as excitatory neurotransmitters. The biological actions of tachykinins are mediated through three types of NK1 receptor, NK2 receptor and NK3 receptor that belong to the family of G protein-coupled receptors.Many evidences indicated that tachykinin and neurokinin receptors have been implicated in numerous facets of cancer initiation and progression. An increase in SP and the NK1 receptor expression has now been reported in a number of tumor types. After binding to the neurokinin-1(NK1) receptor, substance P(SP) induces tumor cell proliferation, angiogenesis, and the migration of tumor cells for invasion and metastasis. After binding to NK1 receptor, NK1 receptor antagonists inhibit tumor cell proliferation, angiogenesis and the migration of tumor cells. Experimentally, a number of NK1 antagonists could inhibit specific tumor cell proliferation(L-733,060, Aprepitant, L-732,138 and MEN 11467). However, there is only one NK1 antagonist, Aprepitant which is a broad-spectrum drug that is currently approved for human use as an antiemetic and specifically approved for use in cancer patients. In addition, recent researchs have shown that raised NKA is a good predictor of prognosis in metastatic neuroendocrime tumors of the midgut and lowering NKA by treatment improves prognosis significantly, persistently elevated(>50 pg/m L)plasma NKA levels are associated with poor short-term survival in patients with midgut neuroendocrine neoplasms.Although earlier in vitro studies have demonstrated that NK-1receptor antagonists could inhibit the growth of certain tumor cells in presence or absence of apoptosis, no study has been carried out on the antitumor action of specific NK1 antagonist SR140333 and NK2 antagonist SR48968 in human colonic cancer cells. Therefore, this study for the first time identified the novel cytotoxic effect and mechanism of SR140333 and SR48968 in colonic cancer cells. In this study, six kinds of colorectal cancer cell were used to investigate the effect of proliferative inhibition induced by SR48968 and SR140333 and HCT-116 cell to the mechanism. We demonstrated that SR140333 and SR48968 inhibited cellular growth of HCT-116 cells significantly in a dose- and time-dependent manner, also inhibited other colonic cancer cells growth selectively. We further systematically analyzed the mechanisms of cell death induced by SR140333 and SR48968. The classical features of apoptosis, including chromatin condensation, loss of membrane asymmetry in the early phases of apoptosis, were observed in HCT-116 cells in response to SR140333 and SR48968, indicating that apoptosis is a major mechanism of SR140333 and SR48968-induced HCT-116 cell death, also it was obtained from Hoechst 33342 staining assay and flow cytometry. In addition, the results of cell cycle distribution tested by flow cytometry showed that SR140333 and SR48968 could block the cell cycle in G0/G1 phase significantly in a dose-dependent manners. Furthermore, after the treatment with SR140333, the results from Western Blot assays showed that SR140333 inhibited phosphorylation of ERK1/2 significantly. Then the results of calcium flux assay showed that SR140333 induced rapid and robust Ca2+ response in HCT-116 cells, however, SR48968 can not induce Ca2+ response in HCT-116 cells. Most importantly, treatment of a CT-26 xenograft mouse mode with 10mg/kg/two day SR140333 and SR48968 for 14 days results in a striking reduction of tumor growth.In short, the results of this study show that NK1 antagonist SR140333 and NK2 antagonist SR48968 can inhibit the growth of colonic cancer cells in vitro and vivo, HCT-116 cells are most sensitive to SR140333 and SW480 cells to SR48968. Additionally, SR140333 could induce the cell cycle arrest and cell apoptosis of HCT-116 cells, activate Ca2+ signal pathway, and inhibit MEK-MAPK pathway, SR48968 inhibited the growth of HCT-116 cells by an apoptosis,cell cycle arrest and Ca2+-independent pathway. Further studies are warranted for tachykinins receptor as a new cancer therapeutic target and tachykinins receptor antagonist as a potential anti-cancer agent.