| Leukemia,a kind of malignant cancer of hematopoietic system,is a serious threat to human health.The number of cases is more than 300 thousand per year worldwide.As for the mortality,leukemia ranks first in malignant cancer among children and adults under 35 years old.Meanwhile,bone cancer pain induced by leukemia has been affecting the quality of life of patients severely.However,at the present,clinical medication of leukemia is inefficient insufficient and poor selective.Therefore,it has always been a hot pot to find and develop more effective drugs and reduce the side and toxic effects as well as improve the cure rate.The classical members of tachykinin include substance P(SP)and Neurokinin Receptor 1(NK1R),playing a role in various physiological or pathological events.For example,SP is a important kind of excitatory neurotransmitter;the expression of NK1 R is increased in a number of tumor types so that NK1 R could be a new cancer therapeutic target and a potential anti-cancer agent.On our study,we used SR140333,the third generation of NK1 R antagonist with the properties of non-peptide and high affinity and selectivity,to block the NK1 R selectively in order to investigate the mechanism of cell proliferation inhibition and the effects in bone cancer pain.Our study shows that the expression of NK1 R is increased significantly in leukocyte of peripheral blood among the patients of leukemia.And the results of MTT show SR140333 inhibits the proliferation in K562 and HL-60 cell.In addition,the results of cell cycle distribution tested by flow cytometry shows that SR140333 blocks the cell cycle in G0/G1 phase significantly in a dose-dependent manner.Furthermore,the results of Western Blot show variation in cell cycle related protein including Cyclin D1?Cyclin D2?Cyclin D3?Cyclin E1?Cyclin B1?CDK4?CDK5?CDK7?Cdc25A?P15?P16?P18?P21 after treatment with SR140333.What's more,blocking NK1 R selectively with SR140333 induce apoptosis in K562.The effect of SR140333 on k562 cell apoptosis was tested by Hoechst 33342 uptake assay,which shows that evident apoptosis bodies.And flow cytometry shows a distinct increased ration of early and late stage of apoptosis in carcinoma cells,with the variation in cell apoptosis related protein including PARP/Cleaved PARP?Bcl-2?Bcl-xL?Cleaved caspase 3?Bax?Bim.Also,model of transplanted tumor using K562 and DLuciferin-K562 on nude mouse was adopted to study the anticancer effect in vivo,showing that SR140333 inhibit the growth of K562 significantly.In our study,model of bone cancer pain using K562 on mice was adopted to investigate the effects on bone cancer pain induced by leukemia after SR140333 blocking NK1 R,the results of which shows notable remission.In addition,the results of HE uptake assay show that there are damage of bone tissue injected K562 cells including breaking marginal bone and dropping cortical bone,which are in accordance with behavior index of cancer pain.And the results of Western Blotting show that cell cycle arrest and cell apoptosis play an important role in the remission of bone cancer pain induced by leukemia after treatment with SR140333.What's more,the results of WB and IHC also show the mechanism of remission is closely related to up-regulated expression levels of endogenous opioid system proteins like POMC and MOR.Moreover,proteomics and transcriptomics were adopted to investigate the effects on regulation of K562 growth at the omics level after treatment with SR140333.The results show that there are 5310 protein groups discrepant before and after treatment,among which,the expression of 713 protein groups up-regulate and 401 down-regulate significantly.And at the lysine acetylation level,we identified 1571 protein groups and then found there are 3474 sites of lysine acetylation being modified discrepantly,among which,the modification of 120 sites up-regulate in 92 protein groups and 336 sites down-regulated in 229 protein groups significantly.Besides,the results of transcriptomics show that there are 810 genes discrepant before and after treatment,among which,the expression of 117 genes up-regulate and 693 genes down-regulate significantly.In summary,SR140333,the third generation of NK1 R antagonist with the properties of non-peptide and high affinity and selectivity,could inhibit the proliferation of K562 at cell and animal level and remit the bone cancer pain induced by leukemia,the mechanism of which is related with arresting cell cycle and inducing cell apoptosis.Meanwhile,quantitative proteomics and lysine acetylation analysis,as well as transcriptomics,provide a large number of differentially expressed proteins related to cell cycle and cell apoptosis,basing on which,more studies can be carried out to investigate the mechanism of inhibiting leukemia cell proliferation at a much deeper level.Further studies are warranted for SR140333 as a new cancer therapeutic target and a potential agent. |
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