The Effect And Mechanism Of NK1R And Annexin A1 In Colon Cancer

Colorectal cancer（CRC）,including colon cancer and rectum cancer,is the one of the most common malignancies in the world.CRC is the third most commonly diagnosed and the second leading cause of cancer-related death all over the world.Neurokinin receptors includes NK1R,NK2R and NK3R,which are involved in various physiological and pathological processes.And SR140333 is an antagonist of NK1R with high specificity and selectivity.Our research group has proved before that NK1R antagonist SR140333 can inhibit the growth of colon cancer cells in a time-and dose-dependent manner and can also induce cell cycle arrest at G₀/G₁ phase and cell apoptosis.However,there are few research report about the in-depth mechanism of the proliferate inhibition effect of the SR140333 in colon cancer cells.In present study,we depleted NK1R by shRNA in HCT116 cells whose viability was significantly reduced.This finding was in accordance with the inhibition effect after blocking NK1R by SR140333.The NK1R protein expression level in epitheliums of mucosa from 50patients using IHC was significantly high in the carcinoma tissues.And the NK1R-positive patients had a significantly poor outcome than NK1R-negative ones（P=0.004）.The NK1R protein expression level was increased significantly in colon cell lines compared to hemorrhoids as normal control detected by Western blot.C-Myc expression level after treatment with SR140333 was decreased significantly and stable overexpression of c-Myc in HCT116 cells can effectively rescued the cell death,indicating that overexpression of c-Myc in colon cell lines could resist the cytotoxic effect of SR140333.The expression level of the pERK1/2 decreased significantly and the expression level of p AKT was increased after SR140333 treatment.Furthermore,MEK1/2inhibitor U0126 but not PI3K inhibitor LY294002 had the ability to synergistically inhibit the cell proliferation of HCT116 cells with SR140333.What’s more,the expression levels of pERK1/2and c-Myc after treatment with U0126 in HCT116 cells showed both decreased significantly in a time-dependent manner.And the expression level of pERK1/2 decreased significantly after 1 hour while the c-Myc significantly decreased after 3 hours,which suggested pERK1/2 regulated c-Myc directly in HCT116.Next,we found the degradation of c-Myc was blocked in the present of pretreatment with MG132 by Western blot.The examination of intracellular calcium mobilization after SR140333 treatment in HCT116 cells showed a rapid and high-intensity rise of cytosolic calcium followed by a moderate and transient mitochondrial calcium elevation in a dose-dependent manner.In contrast,application of SP caused a very weak cytosolic calcium and a hardly detectable mitochondrial calcium.Furthermore,calcium chelating reagent BAPTA and IP₃R inhibitor 2-APB increased cell viability in response to blocking NK1R by SR140333 whereas DIDS,the pharmacological inhibitor of voltage-dependent anion channel type 1 in the outer mitochondrial membrane didn’t,suggesting that cytosolic calcium instead of mitochondrial calcium contributes to cell apoptosis.However,we detected no obvious ROS increase in response to NK1R inhibition.Moreover,BAPTA could block the decreasing expression levels of pERK1/2 and c-Myc after SR140333 treatment,indicating that cytosolic calcium overload contributes to cell apoptosis through pERK1/2-c-Myc signaling axis in response to NK1R blocking.Besides,the results of xenograft in vivo showed that SR140333 group was significantly inhibited compared to the vehicle group and there were no obvious side effects after injection of SR140333.Moreover,H&E and TUNEL staining showed SR140333 inhibited HCT116 xenograft growth and induced apoptosis.Also,the results of Western blot showed SR140333 could regulate cell cycle-and apoptosis-related proteins as well as pERK1/2 and c-Myc,which indicated that blocking NK1R could inhibit the growth and induce apoptosis through pERK1/2-c-Myc signaling axis in vivo which is consistent with our in vitro results.Annexins（ANXs）are a super family of calcium-and phospholipid-binding proteins,playing an important role in a series of vital physiological and pathological processes.Annexin A1 might specifically function either as a tumor suppressor or a tumor promoter candidate for certain cancers depending on the particular type of tumor cells/tissues.However,there is few article related to the effect of Annexin A1 in colon cancer at present.The Annexin A1 protein expression level in epitheliums of mucosa from 50 patients using IHC was significantly high in the carcinoma tissues and the Annexin A1-positive patients had a significantly poor outcome（P=0.005）.To study the function of Annexin A1 in colon cancer cell,we chose HCT116 and SW620 to construct Annexin A1-knockdonw and-overexpressing cells.The results of growth curves indicated that Annexin A1has the ability to promote the proliferation of colon cancer cells.The results of flow cytometry showed that knockdown expression of Annexin A1 could block cell in the G₀/G₁ phase and overexpression of Annexin A1 could increase the cell number in S phase.However,neither over-nor knockdown-expression of Annexin A1 had significant relationship with cell apoptosis and metastasis.In addition,HCT116 cells with the knockdown expression of Annexin A1 have the lower growth and proliferation property compared with HCT116 itself after subcutaneous injection in BALB/c nude mice.And then we used SR140333 to treat the constructed colon cancer cells,and the results of MTT showed that the cell lines with the property of knockdown the expression level of Annexin A1 were more sensitive to the drug with the low concertration of IC₅₀.In sum,our results showed that both the expression level of NK1R and Annexin A1 in carcinoma tissues were higher than para-carcinoma tissue and had associations with clinicopathological factors and prognosis.For NK1R,blocking NK1R induced proliferation inhibition by regulating stability of c-Myc via MAPK signaling pathway in colonic cancer.As for Annexin A1,it could promote the proliferation of colon cells both in vivo and in vitro by regulating cell cycle.However,neither over-nor knockdown-expression of Annexin A1 had significant relationship with cell apoptosis and metastasis.Besides,cell lines with the property of knockdown the expression level of Annexin A1 were more sensitive to the NK1R antagonist SR140333.All of our present results will provide the basic theoretical data for the subsequent development of NK1R and Annexin A1 as new single or multi targets for the treatment of colon cancer.Moreover,further and systematic study about the function and mechanism of NK1R and Annexin A1 will bring new sight to diagnosis and treatment of colon cancer.