氧化苦参碱提高乳腺癌MCF-7细胞对NK-92MI细胞杀伤作用的敏感性
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摘要
目的:探讨氧化苦参碱(OMT)处理前后MCF-7细胞对NK-92MI细胞敏感性的变化及其分子机制。方法:采用CCK-8法检测OMT对MCF-7细胞的毒性作用,采用LDH法和流式细胞术检测NK-92MI细胞对OMT处理后MCF-7细胞的杀伤活性,采用流式细胞术检测OMT处理后MCF-7细胞表面ULBP1、ULBP2和MICA/B蛋白的表达,采用Western blot法检测MCF-7细胞中p65蛋白的磷酸化水平,采用ELISA法检测培养液上清中TNF-α和IFN-γ的含量。结果:OMT对乳腺癌MCF-7细胞活力具有显著的抑制作用,且呈剂量和时间依赖性(P<0. 05);在不同效靶比(5∶1、10∶1和20∶1)下,低浓度OMT可显著提高MCF-7细胞对NK-92MI细胞杀伤作用的敏感性(P<0. 05);低浓度OMT处理后的MCF-7细胞,ULBP1、ULBP2和MICA/B蛋白的表达水平以及p65蛋白的磷酸化水平都显著上调(P<0. 05),可促进NK-92MI细胞分泌更多的TNF-α和IFN-γ(P<0. 05),但该作用可被NF-κB抑制剂PDTC抑制(P<0. 05)。结论:低浓度OMT在体外提高乳腺癌MCF-7细胞对NK-92MI细胞杀伤作用的敏感性,这可能与其激活NF-κB信号通路有关,进而上调MCF-7细胞表面ULBP1、ULBP2和MICA/B蛋白表达,以及促进NK-92MI细胞分泌TNF-α和IFN-γ有关。
Objective: To investigate the effect of oxymatrine( OMT) on the sensibility of MCF-7 cells to the cytotoxicity of NK-92 MI cells and its possible mechanism. Methods: MCF-7 cells were cultured in vitro and treated with OMT at different concentrations for 24,48 and 72 h,and then the cell viability were detected by CCK-8 assay. LDH release assay was used to evaluate the effect of MCF-7 cells on the cytotoxic activity of NK-92 MI cells. Apoptosis and the expression levels of ULBP1,ULBP2 and MICA/B in MCF-7/ADM cells was analyzed by flow cytometry. The levels of IFN-γ and TNF-α in the co-cultured supernatant were detected by ELISA. Results: OMT remarkably reduced the cell viability of MCF-7 in dose-dependent and time-dependent manner( P<0. 05). The cytotoxicity of NK-92 MI cells against MCF-7 cells was higher following OMT pretreatment than without OMT pretreatment( P<0. 05).The expression of p-p65,ULBP1,ULBP2 and MICA/B in MCF-7 cells was upregulated by pretreatment with OMT( P <0. 05),which also increased the secretion of IFN-γ and TNF-α in NK-92 MI cells( P < 0. 05),and these effects could be alleviated by PDTC( P <0. 05). Conclusion: OMT enhances the sensibility of MCF-7 cells to the cytotoxicity of NK-92 MI cells,most likely through activating the NF-κB signaling pathway,and then up-regulating the expression levels of ULBP1,ULBP2 and MICA/B in MCF-7 cells and promoting the secretion of TNF-α and IFN-γ by NK-92 MI cells.
Objective: To investigate the effect of oxymatrine( OMT) on the sensibility of MCF-7 cells to the cytotoxicity of NK-92 MI cells and its possible mechanism. Methods: MCF-7 cells were cultured in vitro and treated with OMT at different concentrations for 24,48 and 72 h,and then the cell viability were detected by CCK-8 assay. LDH release assay was used to evaluate the effect of MCF-7 cells on the cytotoxic activity of NK-92 MI cells. Apoptosis and the expression levels of ULBP1,ULBP2 and MICA/B in MCF-7/ADM cells was analyzed by flow cytometry. The levels of IFN-γ and TNF-α in the co-cultured supernatant were detected by ELISA. Results: OMT remarkably reduced the cell viability of MCF-7 in dose-dependent and time-dependent manner( P<0. 05). The cytotoxicity of NK-92 MI cells against MCF-7 cells was higher following OMT pretreatment than without OMT pretreatment( P<0. 05).The expression of p-p65,ULBP1,ULBP2 and MICA/B in MCF-7 cells was upregulated by pretreatment with OMT( P <0. 05),which also increased the secretion of IFN-γ and TNF-α in NK-92 MI cells( P < 0. 05),and these effects could be alleviated by PDTC( P <0. 05). Conclusion: OMT enhances the sensibility of MCF-7 cells to the cytotoxicity of NK-92 MI cells,most likely through activating the NF-κB signaling pathway,and then up-regulating the expression levels of ULBP1,ULBP2 and MICA/B in MCF-7 cells and promoting the secretion of TNF-α and IFN-γ by NK-92 MI cells.
引文
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[3]Austreid E,Lonning PE,Eikesdal HP.The emergence of targeted drugs in breast cancer to prevent resistance to endocrine treatment and chemotherapy[J].Expert Opin Pharmacother,2014,15(5):681-700.
[4]Wu J,Cai Y,Li M,et al.Oxymatrine promotes S-phase arrest and inhibits cell proliferation of human breast cancer cells in vitro through mitochondria-mediated apoptosis[J].Biol Pharm Bull,2017,40(8):1232-1239.
[5]Li S,Zhang Y,Liu Q,et al.Oxymatrine inhibits proliferation of human bladder cancer T24 cells by inducing apoptosis and cell cycle arrest[J].Oncol Lett,2017,13(6):4453-4458.
[6]Liu F,Wang B,Wang J,et al.Oxymatrine inhibits proliferation and migration while inducing apoptosis in human glioblastoma cells[J].Biomed Res Int,2016,2016:1784161.
[7]Pei Z,Zeng J,Gao Y,et al.Oxymatrine inhibits the proliferation of CaSki cells via downregulating HPV16E7 expression[J].Oncol Rep,2016,36(1):291-298.
[8]Wang B,Han Q,Zhu Y.Oxymatrine inhibited cell proliferation by inducing apoptosis in human lung cancer A549 cells[J].Biomed Mater Eng,2015,26 Suppl 1:S165-172.
[9]Li J,Jiang K,Zhao F.Oxymatrine suppresses proliferation and facilitates apoptosis of human ovarian cancer cells through upregulating microRNA-29b and downregulating matrix metalloproteinase-2 expression[J].Mol Med Rep,2015,12(4):5369-5374.
[10]Tong AA,Hashem H,Eid S,et al.Adoptive natural killer cell therapy is effective in reducing pulmonary metastasis of Ewing sarcoma[J].Oncoimmunology,2017,6(4):e1303586.
[11]刘春香,张怡,芦慧颖.NK细胞的高效扩增及其对体外肿瘤模型的杀伤效果研究[J].中国免疫学杂志,2017,33(8):1186-1190.Liu CX,Zhang Y,Lu HY.Efficient amplification of NK cells and their anti-tumor cytotoxic activity in vitro[J].Chin J Immunol,2017,33(8):1186-1190.
[12]Lanier LL.NKG2D receptor and its ligands in host defense[J].Cancer Immunol Res,2015,3(6):575-582.
[13]Dhar P,Wu JD.NKG2D and its ligands in cancer[J].Curr Opin Immunol,2018,51:55-61.
[14]Fernández L,Valentín J,Zalacain M,et al.Activated and expanded natural killer cells target osteosarcoma tumor initiating cells in an NKG2D-NKG2DL dependent manner[J].Cancer Lett,2015,368(1):54-63.
[15]Feng H,Dong Y,Wu J,et al.Epirubicin pretreatment enhances NK cell-mediated cytotoxicity against breast cancer cells in vitro[J].Am J Transl Res,2016,8(2):473-484.
[16]Chen Z,Li Z,Chang Y,et al.Relationship between NF-κB,MMP-9,and MICA expression in pituitary adenomas reveals a new mechanism of pituitary adenomas immune escape[J].Neurosci Lett,2015,597:77-83.
[17]Huang YX,Chen XT,Guo KY,et al.Sunitinib induces NK-κB-dependent NKG2D ligand expression in nasopharyngeal carcinoma and hepatoma cells[J].J Immunother,2017,40(5):164-174.
[18]朱燕,石永进,赵玉亮,等.拓扑异构酶抑制剂通过ATM/ATR和NF-κB途径上调乳腺癌细胞MICA/B的表达[J].北京大学学报(医学版),2018,50(2):318-325.Zhu Y,Shi YJ,Zhao YL,et al.Topoisomerase inhibitor upregulates MICA/B expression in breast cancer cells[J].JPekjing Univ(Health Sciences),2018,50(2):318-325.
[19]Yuan L,Zhou C,Lu Y,et al.IFN-γ-mediated IRF1/miR-29b feedback loop suppresses colorectal cancer cell growth and metastasis by repressing IGF1[J].Cancer Lett,2015,359(1):136-147.
[20]Li J,Zhang Y,Chen L,et al.Cervical cancer He La cell autocrine apoptosis induced by coimmobilized IFN-γplus TNF-αbiomaterials[J].ACS Appl Mater Interfaces,2018,10(10):8451-8464.
[21]Hong J,Shao T,Sun X,et al.Interferon-γup-regulates major-histocompatibility-complex class I-related chain A expression and enhances major-histocompatibility-complex class I-related chain A-mediated cytolysis of human corneal epithelium by natural killer cells in vitro[J].J Interferon Cytokine Res,2012,32(3):115-120.
[22]Danforth DN,Zhu Y.Conversion of Fas-resistant to Fas-sensitive MCF-7 breast cancer cells by the synergistic interaction of interferon-gamma and all-trans retinoic acid[J].Breast Cancer Res Treat,2005,94(1):81-91.
[23]Bansal R,Tomar T,Ostman A,et al.Selective targeting of interferonγto stromal fibroblasts and pericytes as a novel therapeutic approach to inhibit angiogenesis and tumor growth[J].Mol Cancer Ther,2012,11(11):2419-2428.
[24]Guan YQ,Li Z,Yang A,et al.Cell cycle arrest and apoptosis of OVCAR-3 and MCF-7 cells induced by co-immobilized TNF-αplus IFN-γon polystyrene and the role of p53 activation[J].Biomaterials,2012,33(26):6162-6171.