TGF-β1通过激活NOX4/ROS通路促进宫颈癌细胞的侵袭和迁移
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摘要
目的探讨转化生长因子β1/尼克酰胺腺嘌呤二核苷酸磷酸氧化酶/活性氧(TGF-β1/NOX4/ROS)信号通路在宫颈癌细胞侵袭和迁移中的作用。方法采用5 ng/mL TGF-β1处理培养的SiHa、 CaSki、 HeLa、 C4-I和C33A宫颈癌细胞4 h或8 h, 2′, 7′-二氯荧光素二乙酸酯(DCFH-DA)染色结合流式细胞术检测处理前后SiHa、 CaSki、 HeLa、 C4-I和C33A宫颈癌细胞ROS水平;转染NOX4小干扰RNA(NOX4-siRNA)或加入NOX4信号通路抑制剂二亚苯基碘氯化物(DPI), Western blot法检测宫颈癌HeLa细胞NOX4蛋白水平, Transwell~(TM)侵袭和迁移实验检测TGF-β1对宫颈癌细胞侵袭和迁移能力的影响。结果 TGF-β1显著上调HPV阳性SiHa、 CaSki、 HeLa和C4-I宫颈癌细胞NOX4蛋白表达,促进ROS产生,并增强宫颈癌细胞的侵袭和迁移性;敲低NOX4基因或使用NOX4通路抑制剂DPI可逆转这种作用。结论 TGF-β1激活NOX4/ROS信号通路增强宫颈癌细胞的侵袭和迁移能力。
Objective To investigate the role of transforming growth factor β1/nicinamide adenine dinucleotide phosphate oxidase/reactive oxygen species(TGF-β1/NOX4/ROS) signaling pathway in the invasion and migration of cervical cancer cells. Methods The cultured SiHa, CaSki, HeLa, C4-I and C33A cervical cancer cells were treated with 5 ng/mL TGF-β1 for 4 hours or 8 hours. ROS levels in these cervical cancer cells were detected using 2′, 7′-dichlorofluorescein diacetate(DCFH-DA) staining combined with flow cytometry. After the transfection of NOX4 small interfering RNA(NOX4-siRNA) into HeLa cells or using of NOX4 signaling pathway inhibitor diphenylidene iodine chloride(DPI), NOX4 protein expression was measured by Western blotting, and the effect of TGF-β1 on migration and invasion ability of cervical cancer cells were analyzed by Transwell~(TM) assay. Results TGF-β1 significantly up-regulated NOX4 protein expression, increased ROS production in HPV-positive cervical cancer cells(SiHa, CaSki, HeLa and C4-I) and promoted the migration and invasion abilities of cervical cancer cells. Silencing the NADPH oxidase gene or using the NOX4 pathway inhibitor DPI could reverse this effect. Conclusion TGF-β1 can activate NOX4/ROS signaling pathway to enhance the invasion and migration abilities of cervical cancer cells.
Objective To investigate the role of transforming growth factor β1/nicinamide adenine dinucleotide phosphate oxidase/reactive oxygen species(TGF-β1/NOX4/ROS) signaling pathway in the invasion and migration of cervical cancer cells. Methods The cultured SiHa, CaSki, HeLa, C4-I and C33A cervical cancer cells were treated with 5 ng/mL TGF-β1 for 4 hours or 8 hours. ROS levels in these cervical cancer cells were detected using 2′, 7′-dichlorofluorescein diacetate(DCFH-DA) staining combined with flow cytometry. After the transfection of NOX4 small interfering RNA(NOX4-siRNA) into HeLa cells or using of NOX4 signaling pathway inhibitor diphenylidene iodine chloride(DPI), NOX4 protein expression was measured by Western blotting, and the effect of TGF-β1 on migration and invasion ability of cervical cancer cells were analyzed by Transwell~(TM) assay. Results TGF-β1 significantly up-regulated NOX4 protein expression, increased ROS production in HPV-positive cervical cancer cells(SiHa, CaSki, HeLa and C4-I) and promoted the migration and invasion abilities of cervical cancer cells. Silencing the NADPH oxidase gene or using the NOX4 pathway inhibitor DPI could reverse this effect. Conclusion TGF-β1 can activate NOX4/ROS signaling pathway to enhance the invasion and migration abilities of cervical cancer cells.
引文
[1] Bray F,Ferlay J,Soerjomataram I,et al.Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin,2018,68(6):394-424.
[2] Hemmat N,Baghi H B.Human papillomavirus E5 protein,the undercover culprit of tumorigenesis[J/OL].Infect Agent Cancer,2018,13:31.DOI:10.1186/s13027-018-0208-3.eCollection 2018.
[3] Wu Z,Yu L,Lei X,et al.The association between human papillomavirus 16,18 DNA load and E6 protein expression in cervical intraepithelial neoplasia and cancer[J].J Clin Virol,2018,108:6-11.
[4] Kruk J,Aboul-Enein H Y.Reactive oxygen and nitrogen species in carcinogenesis:implications of oxidative stress on the progression and development of several cancer types[J].Mini Rev Med Chem,2017,17(11):904-919.
[5] Hu Z,Ma D.The precision prevention and therapy of HPV-related cervical cancer:new concepts and clinical implications[J].Cancer Med,2018,7(10):5217-5236.
[6] Brandes R P,Weissmann N,Schr?der K.Nox family NADPH oxidases:Molecular mechanisms of activation[J].Free Radic Biol Med,2014,76:208-226.
[7] Hajjar C,Cherrier M V,Dias Mirandela G,et al.The NOX family of proteins is also present in bacteria[J/OL].MBio,2017,8(6):e01487-17.DOI:10.1128/mBio.01487-17.
[8] Ju H Q,Ying H,Tian T,et al.Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma[J/OL].Nat Commun,2017,8:14437.DOI:10.1038/ncomms14437.
[9] Cho S Y,Kim J S,Eun H S,et al.Expression of NOX family genes and their clinical significance in colorectal cancer[J].Dig Dis Sci,2018,63(9):2332-2340.
[10] Du S,Miao J,Zhu Z,et al.NADPH oxidase 4 regulates anoikis resistance of gastric cancer cells through the generation of reactive oxygen species and the induction of EGFR[J/OL].Cell Death Dis,2018,9(10):948.DOI:10.1038/s41419-018-0953-7.
[11] Tang C T,Lin X L,Wu S,et al.NOX4-driven ROS formation regulates proliferation and apoptosis of gastric cancer cells through the GLI1 pathway[J].Cell Signal,2018,46:52-63.
[12] Shanmugasundaram K,Nayak B K,Friedrichs W E,et al.NOX4 functions as a mitochondrial energetic sensor coupling cancer metabolic reprogramming to drug resistance[J/OL].Nat Commun,2017,8(1):997.DOI:10.1038/s41467-017-01106-1.
[13] Xie X,Percipalle P.Elevated transforming growth factor β signaling activation in β-actin-knockout mouse embryonic fibroblasts enhances myofibroblast features[J].J Cell Physiol,2018,233(11):8884-8895.
[14] Jeong B Y,Park S R,Cho S,et al.TGF-β-mediated NADPH oxidase 4-dependent oxidative stress promotes colistin-induced acute kidney injury[J].J Antimicrob Chemother,2018,73(4):962-972.
[15] Zhao Y,Ma J,Fan Y,et al.TGF-β transactivates EGFR and facilitates breast cancer migration and invasion through canonical Smad3 and ERK/Sp1 signaling pathways[J].Mol Oncol,2018,12(3):305-321.
[16] Peng X,Liu G,Peng H,et al.SOX4 contributes to TGF-β-induced epithelial-mesenchymal transition and stem cell characteristics of gastric cancer cells[J].Genes Dis,2017,5(1):49-61.
[17] Staudacher J J,Bauer J,Jana A,et al.Activin signaling is an essential component of the TGF-β induced pro-metastatic phenotype in colorectal cancer[J/OL].Sci Rep,2017,7(1):5569.DOI:10.1038/s41598-017-05907-8.
[18] Cao W,Peng T,Zhou Y.Long noncoding RNA activated by transforming growth factor-β promotes cancer development and is a prognostic marker in cervical cancer[J].J Cancer Res Ther,2017,13(5):801-806.
[19] Moreno-Càceres J,Caballero-Díaz D,Nwosu Z C,et al.The level of caveolin-1 expression determines response to TGF-β as a tumour suppressor in hepatocellular carcinoma cells[J/OL].Cell Death Dis,2017,8(10):e3098.DOI:10.1038/cddis.2017.469.
[20] Lu J,Risbood P,Kane C T Jr,et al.Characterization of potent and selective iodonium-class inhibitors of NADPH oxidases[J].Biochem Pharmacol,2017,143:25-38.
[21] 秦源,卫静,张怡,等.铁过载抑制人肝癌Huh7.5 细胞增殖和促进细胞凋亡的机制[J].细胞与分子免疫学杂志,2017,33(8):1056-1061.Qin Y,Wei J,Zhang Y,et al.Mechanism for the inhibition of proliferation and promotion of apoptosis in Huh7.5 cells by iron overload[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2017,33(8):1056-1061.
[22] Ebrahimi S,Soltani A,Hashemy S I.Oxidative stress in cervical cancer pathogenesis and resistance to therapy[J].J Cell Biochem,2019,120(5):6868-6877.
[23] Sirokmány G,Donkó á,Geiszt M.Nox/Duox family of NADPH oxidases:Lessons from knockout mouse models[J].Trends Pharmacol Sci,2016,37(4):318-327.
[24] Moreno-Càceres J,Caballero-Díaz D,Nwosu Z C,et al.The level of caveolin-1 expression determines response to TGF-β as a tumour suppressor in hepatocellular carcinoma cells[J/OL].Cell Death Dis,2017,8(10):e3098.DOI:10.1038/cddis.2017.469.
[25] Cruz-Gregorio A,Manzo-Merino J,Gonzaléz-García M C,et al.Human papillomavirus types 16 and 18 early-expressed proteins differentially modulate the cellular redox state and DNA damage[J].Int J Biol Sci,2018,14(1):21-35.
[26] Prevc A,Niksic Zakelj M,Kranjc S,et al.Electrochemotherapy with cisplatin or bleomycin in head and neck squamous cell carcinoma:Improved effectiveness of cisplatin in HPV-positive tumors[J].Bioelectrochemistry,2018,123:248-254.
[27] Du S,Liu K,Gao P,et al.Differential anticancer activities of arsenic trioxide on head and neck cancer cells with different human papillomavirus status[J].Life Sci,2018,212:182-193.
[28] Morikawa M,Derynck R,Miyazono K.TGF-β and the TGF-β Family:Context-dependent roles in cell and tissue physiology[J/OL].Cold Spring Harb Perspect Biol,2016,8(5):a021873.DOI:10.1101/cshperspect.a021873.
[29] Zheng H,Jarvis I W H,Bottai M,et al.TGF beta promotes repair of bulky DNA damage through increased ERCC1/XPF and ERCC1/XPA interaction[J].Carcinogenesis,2018 Nov 12.DOI:10.1093/carcin/bgy156.[Epub ahead of print].
[30] Chen W,Ten Dijke P.Immunoregulation by members of the TGFβ superfamily[J].Nat Rev Immunol,2016,16(12):723-740.
[31] Colak S,Ten Dijke P.Targeting TGF-β signaling in cancer[J].Trends Cancer,2017,3(1):56-71.
[32] Wang X,Wang G,Zhang L,et al.LncRNA PVT1 promotes the growth of HPV positive and negative cervical squamous cell carcinoma by inhibiting TGF-β1[J/OL].Cancer Cell Int,2018,18:70.DOI:10.1186/s12935-018-0567-2.eCollection 2018.
[33] Han S,Wang Y,Shi X,et al.Negative roles of B7-H3 and B7-H4 in the microenvironment of cervical cancer[J].Exp Cell Res,2018,371(1):222-230.
[34] Tang C T,Gao Y J,Ge Z Z.NOX4,a new genetic target for anti-cancer therapy in digestive system cancer[J].J Dig Dis,2018,19(10):578-585.
[35] Witte D,Bartscht T,Kaufmann R,et al.TGF-β1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38 MAPK[J].Oncol Rep,2017,38(6):3693-3701.
[36] Zong L,Li J,Chen X,et al.Lipoxin A4 attenuates cell invasion by inhibiting ROS/ERK/MMP pathway in pancreatic cancer[J/OL].Oxid Med Cell Longev,2016,2016:6815727.DOI:10.1155/2016/6815727.Epub 2015 Nov 16.
[37] Tamborindeguy M T,Matte B F,Ramos G O,et al.NADPH-oxidase-derived ROS alters cell migration by modulating adhesions dynamics[J].Biol Cell,2018,110(10):225-236.
[2] Hemmat N,Baghi H B.Human papillomavirus E5 protein,the undercover culprit of tumorigenesis[J/OL].Infect Agent Cancer,2018,13:31.DOI:10.1186/s13027-018-0208-3.eCollection 2018.
[3] Wu Z,Yu L,Lei X,et al.The association between human papillomavirus 16,18 DNA load and E6 protein expression in cervical intraepithelial neoplasia and cancer[J].J Clin Virol,2018,108:6-11.
[4] Kruk J,Aboul-Enein H Y.Reactive oxygen and nitrogen species in carcinogenesis:implications of oxidative stress on the progression and development of several cancer types[J].Mini Rev Med Chem,2017,17(11):904-919.
[5] Hu Z,Ma D.The precision prevention and therapy of HPV-related cervical cancer:new concepts and clinical implications[J].Cancer Med,2018,7(10):5217-5236.
[6] Brandes R P,Weissmann N,Schr?der K.Nox family NADPH oxidases:Molecular mechanisms of activation[J].Free Radic Biol Med,2014,76:208-226.
[7] Hajjar C,Cherrier M V,Dias Mirandela G,et al.The NOX family of proteins is also present in bacteria[J/OL].MBio,2017,8(6):e01487-17.DOI:10.1128/mBio.01487-17.
[8] Ju H Q,Ying H,Tian T,et al.Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma[J/OL].Nat Commun,2017,8:14437.DOI:10.1038/ncomms14437.
[9] Cho S Y,Kim J S,Eun H S,et al.Expression of NOX family genes and their clinical significance in colorectal cancer[J].Dig Dis Sci,2018,63(9):2332-2340.
[10] Du S,Miao J,Zhu Z,et al.NADPH oxidase 4 regulates anoikis resistance of gastric cancer cells through the generation of reactive oxygen species and the induction of EGFR[J/OL].Cell Death Dis,2018,9(10):948.DOI:10.1038/s41419-018-0953-7.
[11] Tang C T,Lin X L,Wu S,et al.NOX4-driven ROS formation regulates proliferation and apoptosis of gastric cancer cells through the GLI1 pathway[J].Cell Signal,2018,46:52-63.
[12] Shanmugasundaram K,Nayak B K,Friedrichs W E,et al.NOX4 functions as a mitochondrial energetic sensor coupling cancer metabolic reprogramming to drug resistance[J/OL].Nat Commun,2017,8(1):997.DOI:10.1038/s41467-017-01106-1.
[13] Xie X,Percipalle P.Elevated transforming growth factor β signaling activation in β-actin-knockout mouse embryonic fibroblasts enhances myofibroblast features[J].J Cell Physiol,2018,233(11):8884-8895.
[14] Jeong B Y,Park S R,Cho S,et al.TGF-β-mediated NADPH oxidase 4-dependent oxidative stress promotes colistin-induced acute kidney injury[J].J Antimicrob Chemother,2018,73(4):962-972.
[15] Zhao Y,Ma J,Fan Y,et al.TGF-β transactivates EGFR and facilitates breast cancer migration and invasion through canonical Smad3 and ERK/Sp1 signaling pathways[J].Mol Oncol,2018,12(3):305-321.
[16] Peng X,Liu G,Peng H,et al.SOX4 contributes to TGF-β-induced epithelial-mesenchymal transition and stem cell characteristics of gastric cancer cells[J].Genes Dis,2017,5(1):49-61.
[17] Staudacher J J,Bauer J,Jana A,et al.Activin signaling is an essential component of the TGF-β induced pro-metastatic phenotype in colorectal cancer[J/OL].Sci Rep,2017,7(1):5569.DOI:10.1038/s41598-017-05907-8.
[18] Cao W,Peng T,Zhou Y.Long noncoding RNA activated by transforming growth factor-β promotes cancer development and is a prognostic marker in cervical cancer[J].J Cancer Res Ther,2017,13(5):801-806.
[19] Moreno-Càceres J,Caballero-Díaz D,Nwosu Z C,et al.The level of caveolin-1 expression determines response to TGF-β as a tumour suppressor in hepatocellular carcinoma cells[J/OL].Cell Death Dis,2017,8(10):e3098.DOI:10.1038/cddis.2017.469.
[20] Lu J,Risbood P,Kane C T Jr,et al.Characterization of potent and selective iodonium-class inhibitors of NADPH oxidases[J].Biochem Pharmacol,2017,143:25-38.
[21] 秦源,卫静,张怡,等.铁过载抑制人肝癌Huh7.5 细胞增殖和促进细胞凋亡的机制[J].细胞与分子免疫学杂志,2017,33(8):1056-1061.Qin Y,Wei J,Zhang Y,et al.Mechanism for the inhibition of proliferation and promotion of apoptosis in Huh7.5 cells by iron overload[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2017,33(8):1056-1061.
[22] Ebrahimi S,Soltani A,Hashemy S I.Oxidative stress in cervical cancer pathogenesis and resistance to therapy[J].J Cell Biochem,2019,120(5):6868-6877.
[23] Sirokmány G,Donkó á,Geiszt M.Nox/Duox family of NADPH oxidases:Lessons from knockout mouse models[J].Trends Pharmacol Sci,2016,37(4):318-327.
[24] Moreno-Càceres J,Caballero-Díaz D,Nwosu Z C,et al.The level of caveolin-1 expression determines response to TGF-β as a tumour suppressor in hepatocellular carcinoma cells[J/OL].Cell Death Dis,2017,8(10):e3098.DOI:10.1038/cddis.2017.469.
[25] Cruz-Gregorio A,Manzo-Merino J,Gonzaléz-García M C,et al.Human papillomavirus types 16 and 18 early-expressed proteins differentially modulate the cellular redox state and DNA damage[J].Int J Biol Sci,2018,14(1):21-35.
[26] Prevc A,Niksic Zakelj M,Kranjc S,et al.Electrochemotherapy with cisplatin or bleomycin in head and neck squamous cell carcinoma:Improved effectiveness of cisplatin in HPV-positive tumors[J].Bioelectrochemistry,2018,123:248-254.
[27] Du S,Liu K,Gao P,et al.Differential anticancer activities of arsenic trioxide on head and neck cancer cells with different human papillomavirus status[J].Life Sci,2018,212:182-193.
[28] Morikawa M,Derynck R,Miyazono K.TGF-β and the TGF-β Family:Context-dependent roles in cell and tissue physiology[J/OL].Cold Spring Harb Perspect Biol,2016,8(5):a021873.DOI:10.1101/cshperspect.a021873.
[29] Zheng H,Jarvis I W H,Bottai M,et al.TGF beta promotes repair of bulky DNA damage through increased ERCC1/XPF and ERCC1/XPA interaction[J].Carcinogenesis,2018 Nov 12.DOI:10.1093/carcin/bgy156.[Epub ahead of print].
[30] Chen W,Ten Dijke P.Immunoregulation by members of the TGFβ superfamily[J].Nat Rev Immunol,2016,16(12):723-740.
[31] Colak S,Ten Dijke P.Targeting TGF-β signaling in cancer[J].Trends Cancer,2017,3(1):56-71.
[32] Wang X,Wang G,Zhang L,et al.LncRNA PVT1 promotes the growth of HPV positive and negative cervical squamous cell carcinoma by inhibiting TGF-β1[J/OL].Cancer Cell Int,2018,18:70.DOI:10.1186/s12935-018-0567-2.eCollection 2018.
[33] Han S,Wang Y,Shi X,et al.Negative roles of B7-H3 and B7-H4 in the microenvironment of cervical cancer[J].Exp Cell Res,2018,371(1):222-230.
[34] Tang C T,Gao Y J,Ge Z Z.NOX4,a new genetic target for anti-cancer therapy in digestive system cancer[J].J Dig Dis,2018,19(10):578-585.
[35] Witte D,Bartscht T,Kaufmann R,et al.TGF-β1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38 MAPK[J].Oncol Rep,2017,38(6):3693-3701.
[36] Zong L,Li J,Chen X,et al.Lipoxin A4 attenuates cell invasion by inhibiting ROS/ERK/MMP pathway in pancreatic cancer[J/OL].Oxid Med Cell Longev,2016,2016:6815727.DOI:10.1155/2016/6815727.Epub 2015 Nov 16.
[37] Tamborindeguy M T,Matte B F,Ramos G O,et al.NADPH-oxidase-derived ROS alters cell migration by modulating adhesions dynamics[J].Biol Cell,2018,110(10):225-236.
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