肥大细胞调控IDO诱导的肺癌细胞转移
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摘要
建立了小鼠肥大细胞体外诱导体系。探究肥大细胞对肿瘤细胞侵袭转移能力的影响。Transwell迁移和侵袭实验检测了与肥大细胞共培养后的细胞2LL侵袭转移能力变化。实验结果表明:肥大细胞能够抑制IDO诱导的细胞2LL的侵袭转移能力。生物信息学分析发现基质金属蛋白酶抑制剂-1(TIMP1)能够抑制促肿瘤转移的基质金属蛋白酶-1(MMP1)、基质金属蛋白酶-2(MMP2)以及基质金属蛋白酶-9(MMP9)的表达。Western blot结果表明肥大细胞通过抑制STAT3磷酸化而减少IDO的表达;并通过TIMP1信号通路抑制MMP2、MMP9和VEGF的表达。这些结果表明肥大细胞通过抑制STAT3信号通路磷酸化调控IDO表达和抑制下游TIMP1、MMP2、MMP9信号通路抑制细胞2LL转移能力。
The aim of this study is to investigate IDO expression and metastasis ability of lung carcinoma cells after co-culturing with mast cells. Mast cells were obtained from C57 BL/6 mice(aged 4—6 weeks), and a cellular phenotype analysis was performed by flow cytometry. IDO expression in lung carcinoma cells was detected by real-time PCR and western blotting. Transwell migration and invasion assays were used to assess the metastasis capacity of lung carcinoma cells. Moreover, the effects of culturing lung carcinoma cells with mast cells on the STAT3 and TIMP/MMP signaling pathways were determined by western blotting. Afterwards,metastatic-associated gene expression data were obtained from Gene Expression Omnibus database with 2 metastatic samples and 2 healthy samples. We subsequently analyzed differentially expressed genes(DEGs) in metastatic colonization using R software. IDO expression in lung carcinoma cells was attenuated after coculturing with mast cells. The migration and invasion abilities of lung carcinoma cells in the co-culture system were decreased. The identified DEGs including 1 368 mRNAs were dysregulated in metastatic nodes. TIMP1 was down-regulated in metastatic nodes. The mechanisms by which mast cells restrain the metastasis behavior of lung carcinoma cell as well as the STAT3 signaling pathway, and limit MMP2/MMP9 expression by promoting TIMP1 expression. In conclusion, Mast cells restrain the expression of IDO in lung carcinoma cells by inhibiting STAT3 phosphorylation. The migration and invasion ability of lung carcinoma cells were restricted by the facilitated TIMP1 expression. Our study demonstrates a novel mechanism by which mast cells limit the lung carcinoma angiogenesis.
The aim of this study is to investigate IDO expression and metastasis ability of lung carcinoma cells after co-culturing with mast cells. Mast cells were obtained from C57 BL/6 mice(aged 4—6 weeks), and a cellular phenotype analysis was performed by flow cytometry. IDO expression in lung carcinoma cells was detected by real-time PCR and western blotting. Transwell migration and invasion assays were used to assess the metastasis capacity of lung carcinoma cells. Moreover, the effects of culturing lung carcinoma cells with mast cells on the STAT3 and TIMP/MMP signaling pathways were determined by western blotting. Afterwards,metastatic-associated gene expression data were obtained from Gene Expression Omnibus database with 2 metastatic samples and 2 healthy samples. We subsequently analyzed differentially expressed genes(DEGs) in metastatic colonization using R software. IDO expression in lung carcinoma cells was attenuated after coculturing with mast cells. The migration and invasion abilities of lung carcinoma cells in the co-culture system were decreased. The identified DEGs including 1 368 mRNAs were dysregulated in metastatic nodes. TIMP1 was down-regulated in metastatic nodes. The mechanisms by which mast cells restrain the metastasis behavior of lung carcinoma cell as well as the STAT3 signaling pathway, and limit MMP2/MMP9 expression by promoting TIMP1 expression. In conclusion, Mast cells restrain the expression of IDO in lung carcinoma cells by inhibiting STAT3 phosphorylation. The migration and invasion ability of lung carcinoma cells were restricted by the facilitated TIMP1 expression. Our study demonstrates a novel mechanism by which mast cells limit the lung carcinoma angiogenesis.
引文
[1]SIEGEL R L,MILLER K D,JEMAL A.Cancer statistics,2017[J].CA:A Cancer Journal for Clinicians,2017,67(1):7-30.
[2]JING S W,WANG Y D,CHEN L Q,et al.Hypoxia suppresses E-cadherin and enhances matrix metalloproteinase-2expression favoring esophageal carcinoma migration and invasion via hypoxia inducible factor-1 alpha activation[J].Dis Esophagus,2013,26(1):75-83.
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[7]黎晴,杨梅,曹桂明.肥大细胞与乳腺浸润性导管癌淋巴结转移及淋巴管生成的关系探讨[J].海南医学,2012,23(9):7-9.
[8]LIANG X,YIN G,MA Y,et al.The critical role of mast cell-derived hypoxia-inducible factor-1αin regulating mast cell function[J].Journal of Pharmacy&Pharmacology,2016,68(11):1409-1416.
[9]柳雅玲,赵苗青,侯刚,等.肥大细胞的浸润对胃癌生长转移的影响[J].第一军医大学学报,2005,25(7):809-811.
[10]DA S E,JAMUR M C,OLIVER C.Mast cell function:Anew vision of an old cell[J].Journal of Histochemistry&Cytochemistry,2014,62(10):698-738.
[11]RODRIGUES C P,FERREIRA A C F,PINHO M P,et al.Tolerogenic IDO+Dendritic cells are induced by PD-1-expressing mast cells[J].Frontiers in Immunology,2016,7:1-17.
[12]MULLER A J,DUHADAWAY J B,DONOVER P S,et al.Inhibition of indoleamine 2,3-dioxygenase,an immunoregulatory target of the cancer suppression gene Bin1,potentiates cancer chemotherapy[J].Nature Medicine,2005,11(3):312-319.
[13]ZHANG Y,WANG Y,WU G,et al.Prolonged skin grafts survival time by IFN-gamma in allogeneic skin transplantation model during acute rejection through IFN-gamma/STAT3/IDO pathway in epidermal layer[J].Biochemical and Biophysical Research Communications,2017,496(2):436-442.
[14]GUO H,KUANG S,SONG Q L,et al.Cucurbitacin Iinhibits STAT3,but enhances STAT1 signaling in human cancer cells in vitro through disrupting actin filaments[J].Acta Pharmacologica Sinica,2018,39(3):425-437.
[15]OKAZAKI H,TOKUMARU S,HANAKAWA Y,et al.Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation[J].Biochemical and Biophysical Research Communications,2011,412(3):441-445.
[16]V?LIM?KI J,UUSITALO H.Matrix metalloproteinases(MMP-1,MMP-2,MMP-3 and MMP-9,and TIMP-1,TIMP-2 and TIMP-3)and markers for vascularization in functioning and non-functioning bleb capsules of glaucoma drainage implants[J].Acta Ophthalmologica,2015,93(5):450-456.
[17]赵芹,薛长湖,杨延存,等.海参皂苷Echinoside A通过MMP-9信号通路抑制肿瘤转移[J].华东理工大学学报(自然科学版),2011,37(4):444-452.
[18]AMMENDOLA M,SACCO R,DONATO G,et al.Mast cell positivity to tryptase correlates with metastatic lymph nodes in gastrointestinal cancer patients treated surgically[J].Oncology,2013,85(2):111-116.
[19]ROJIANI M V,ALIDINA J,ESPOSITO N,et al.Expression of MMP-2 correlates with increased angiogenesis in CNS metastasis of lung carcinoma[J].International Journal of Clinical and Experimental Pathology,2010,3(8):775.
[20]WEI L H,KUO M L,CHEN C A,et al.Interleukin-6 promotes cervical tumor growth by VEGF-dependent angiogenesis via a STAT3 pathway[J].Oncogene,2003,22(10):1517-1527.
[21]曾辉.VEGFR-2在肿瘤血管生成中的作用及其研究进展[J].世界肿瘤杂志,2006,2(4):286-292.
[22]SEYFARTH H,SACK U,GESSNER C,et al.Angiogenin,bFGF and VEGF:Angiogenic markers in breath condensate of patients with pulmonary hypertension[J].Pneumologie,2015,69(4):207-211.
[2]JING S W,WANG Y D,CHEN L Q,et al.Hypoxia suppresses E-cadherin and enhances matrix metalloproteinase-2expression favoring esophageal carcinoma migration and invasion via hypoxia inducible factor-1 alpha activation[J].Dis Esophagus,2013,26(1):75-83.
[3]SMITH C,CHANG M Y,PARKER K H,et al.IDO is a nodal pathogenic driver of lung cancer and metastasis development[J].Cancer Discovery,2012,2(8):722-735.
[4]MAUTINO M R,LINK C J,VAHANIAN N N,et al.Abstract 5023:Synergistic antitumor effects of combinatorial immune checkpoint inhibition with anti-PD-1/PD-L antibodies and the IDO pathway inhibitors NLG-919 and indoximod in the context of active immunotherapy[J].Cancer Research,2014,74(19 Supplement):5023-5023.
[5]吴俊军,徐跃洋.吲哚胺-2-3-双加氧酶IDO1抑制剂研究进展[J].中国新药杂志,2016,25(4):425-432.
[6]PRENDERGAST G C.Immune escape as a fundamental trait of cancer:Focus on IDO[J].Oncogene,2008,27(28):3889-3900.
[7]黎晴,杨梅,曹桂明.肥大细胞与乳腺浸润性导管癌淋巴结转移及淋巴管生成的关系探讨[J].海南医学,2012,23(9):7-9.
[8]LIANG X,YIN G,MA Y,et al.The critical role of mast cell-derived hypoxia-inducible factor-1αin regulating mast cell function[J].Journal of Pharmacy&Pharmacology,2016,68(11):1409-1416.
[9]柳雅玲,赵苗青,侯刚,等.肥大细胞的浸润对胃癌生长转移的影响[J].第一军医大学学报,2005,25(7):809-811.
[10]DA S E,JAMUR M C,OLIVER C.Mast cell function:Anew vision of an old cell[J].Journal of Histochemistry&Cytochemistry,2014,62(10):698-738.
[11]RODRIGUES C P,FERREIRA A C F,PINHO M P,et al.Tolerogenic IDO+Dendritic cells are induced by PD-1-expressing mast cells[J].Frontiers in Immunology,2016,7:1-17.
[12]MULLER A J,DUHADAWAY J B,DONOVER P S,et al.Inhibition of indoleamine 2,3-dioxygenase,an immunoregulatory target of the cancer suppression gene Bin1,potentiates cancer chemotherapy[J].Nature Medicine,2005,11(3):312-319.
[13]ZHANG Y,WANG Y,WU G,et al.Prolonged skin grafts survival time by IFN-gamma in allogeneic skin transplantation model during acute rejection through IFN-gamma/STAT3/IDO pathway in epidermal layer[J].Biochemical and Biophysical Research Communications,2017,496(2):436-442.
[14]GUO H,KUANG S,SONG Q L,et al.Cucurbitacin Iinhibits STAT3,but enhances STAT1 signaling in human cancer cells in vitro through disrupting actin filaments[J].Acta Pharmacologica Sinica,2018,39(3):425-437.
[15]OKAZAKI H,TOKUMARU S,HANAKAWA Y,et al.Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation[J].Biochemical and Biophysical Research Communications,2011,412(3):441-445.
[16]V?LIM?KI J,UUSITALO H.Matrix metalloproteinases(MMP-1,MMP-2,MMP-3 and MMP-9,and TIMP-1,TIMP-2 and TIMP-3)and markers for vascularization in functioning and non-functioning bleb capsules of glaucoma drainage implants[J].Acta Ophthalmologica,2015,93(5):450-456.
[17]赵芹,薛长湖,杨延存,等.海参皂苷Echinoside A通过MMP-9信号通路抑制肿瘤转移[J].华东理工大学学报(自然科学版),2011,37(4):444-452.
[18]AMMENDOLA M,SACCO R,DONATO G,et al.Mast cell positivity to tryptase correlates with metastatic lymph nodes in gastrointestinal cancer patients treated surgically[J].Oncology,2013,85(2):111-116.
[19]ROJIANI M V,ALIDINA J,ESPOSITO N,et al.Expression of MMP-2 correlates with increased angiogenesis in CNS metastasis of lung carcinoma[J].International Journal of Clinical and Experimental Pathology,2010,3(8):775.
[20]WEI L H,KUO M L,CHEN C A,et al.Interleukin-6 promotes cervical tumor growth by VEGF-dependent angiogenesis via a STAT3 pathway[J].Oncogene,2003,22(10):1517-1527.
[21]曾辉.VEGFR-2在肿瘤血管生成中的作用及其研究进展[J].世界肿瘤杂志,2006,2(4):286-292.
[22]SEYFARTH H,SACK U,GESSNER C,et al.Angiogenin,bFGF and VEGF:Angiogenic markers in breath condensate of patients with pulmonary hypertension[J].Pneumologie,2015,69(4):207-211.