吴茱萸碱通过NF-κB-p65信号通路抑制人结肠癌细胞SW480的迁移和侵袭
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摘要
目的探讨吴茱萸碱(evodiamine,EVO)对人结肠癌细胞SW480迁移与侵袭的影响,并初步探究其潜在的分子机制。方法以CCK-8法测定不同浓度(0、0.375、0.75、1.5、3、6、12μmol/L)的EVO对SW480细胞处理不同时间(24、48、72 h)的增殖抑制作用;以划痕实验检测EVO对SW480细胞迁移的影响;Transwell实验检测EVO对SW480细胞侵袭的影响;Western blot检测IκBα、p-IκBα、NF-κB-p65、p-NF-κB-p65、MMP-9、MMP-2、MMP-13等蛋白的表达水平;免疫荧光实验检测EVO作用于SW480细胞后p-NF-κB-p65和MMP-9的蛋白表达情况;NF-κB-p65的激活剂LPS预处理SW480细胞后,Western blot检测EVO对IκBα、p-IκBα、NF-κB-p65、p-NF-κB-p65、MMP-9、MMP-2等蛋白表达的影响。结果 EVO呈时间及浓度依赖性抑制SW480细胞的增殖;经EVO作用后,SW480细胞的迁移能力下降(P<0.01),侵袭能力下降,呈浓度依赖性(P<0.01);EVO可以抑制NF-κB-p65信号通路(P<0.05);EVO对NF-κB-p65的抑制作用能被LPS削弱(P<0.05)。结论 EVO能抑制SW480细胞的迁移和侵袭,其机制可能是通过抑制IκBα的磷酸化,进而抑制NF-κB-p65的磷酸化,最终抑制下游基因MMP-9的表达。
Objective To investigate the effect of evodiamine(EVO) on the migration and invasion of colorectal cancer SW480 cells and explore the underlying mechanism. Methods SW480 cells were treated with different concentrations(0, 0.375, 0.75, 1.5, 3, 6, 12 μmol/L) of EVO for 24, 48, or 72 h, and the cell inhibition was detected using CCK-8 assay. SW480 cells treated with different concentrations of EVO for 24 h were examined for changes in migration and invasion using wound healing assay and Transwell assay and also for expression of IκBα, p-IκBα, NF-κB-p65, p-NF-κB-p65, MMP-9, MMP-2 and MMP-13 using Western blotting. Immunofluorescence staining was used to detect the expression of p-NF-κB-p65 and MMP-9 in SW480 cells treated with EVO for 24 h. The effects of lipopolysaccharide(LPS) challenge of SW480 cells prior to treatment with 1.5 μmol/L EVO on the expressions of IκBα, p-IκBα, NF-κB-p65, p-NF-κB-p65, MMP-9 and MMP-2 were investigated using Western blotting. Results EVO inhibited the proliferation of SW480 cells in a time-and dose-dependent manner. EVO treatment also dose-dependently inhibited the migration ability(P<0.01) and invasion(P<0.01) of SW480 cells, and significantly inhibited NF-κB-p65 signaling(P<0.05). The inhibitory effect of EVO on NF-κB-p65 signaling was obviously attenuated by LPS challenge of the cells(P<0.05). Conclusion EVO can inhibit the expression of MMP-9 via suppressing NF-κB-p65 signaling, and then cause ultimately inhibition of the migration and invasion of SW480 cells.
Objective To investigate the effect of evodiamine(EVO) on the migration and invasion of colorectal cancer SW480 cells and explore the underlying mechanism. Methods SW480 cells were treated with different concentrations(0, 0.375, 0.75, 1.5, 3, 6, 12 μmol/L) of EVO for 24, 48, or 72 h, and the cell inhibition was detected using CCK-8 assay. SW480 cells treated with different concentrations of EVO for 24 h were examined for changes in migration and invasion using wound healing assay and Transwell assay and also for expression of IκBα, p-IκBα, NF-κB-p65, p-NF-κB-p65, MMP-9, MMP-2 and MMP-13 using Western blotting. Immunofluorescence staining was used to detect the expression of p-NF-κB-p65 and MMP-9 in SW480 cells treated with EVO for 24 h. The effects of lipopolysaccharide(LPS) challenge of SW480 cells prior to treatment with 1.5 μmol/L EVO on the expressions of IκBα, p-IκBα, NF-κB-p65, p-NF-κB-p65, MMP-9 and MMP-2 were investigated using Western blotting. Results EVO inhibited the proliferation of SW480 cells in a time-and dose-dependent manner. EVO treatment also dose-dependently inhibited the migration ability(P<0.01) and invasion(P<0.01) of SW480 cells, and significantly inhibited NF-κB-p65 signaling(P<0.05). The inhibitory effect of EVO on NF-κB-p65 signaling was obviously attenuated by LPS challenge of the cells(P<0.05). Conclusion EVO can inhibit the expression of MMP-9 via suppressing NF-κB-p65 signaling, and then cause ultimately inhibition of the migration and invasion of SW480 cells.
引文
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[22] CHOI B H, LEE D H, KIM J, et al. Controls of nuclear factor-Kappa B signaling activity by 5’-AMP-activated protein kinase activation with examples in human bladder cancer cells[J]. Int Neurourol J, 2016, 20(3): 182-187. DOI: 10.5213/inj.1632718.359.
[23] XU Y H, LI Z L, QIU S F. IFN-γ induces gastric cancer cell proliferation and metastasis through upregulation of integrin β3-mediated NF-κB signaling[J]. Transl Oncol, 2018, 11(1): 182-192. DOI: 10.1016/j.tranon.2017.11.008.
[24] FANG R, WANG C, JIANG Q, et al. NEMO-IKKβ are essential for IRF3 and NF-κB activation in the cGAS-STING pathway[J].J Immunol, 2017, 199(9): 3222-3233. DOI: 10.4049/jimmunol.1700699.
[25] LU Y X, JU H Q, WANG F, et al. Inhibition of the NF-κB pathway by nafamostat mesilate suppresses colorectal cancer growth and metastasis[J]. Cancer Lett, 2016, 380(1): 87-97. DOI: 10.1016/j.canlet.2016.06.014.
[2] HEO S K, YUN H J, YI H S, et al. Evodiamine and rutaecarpine inhibit migration by LIGHT via suppression of NADPH oxidase activation[J]. J Cell Biochem, 2009, 107(1): 123-133. DOI: 10.1002/jcb.22109.
[3] CHIOU W F, KO H C, WEI B L. Evodia rutaecarpa and three major alkaloids abrogate influenza avirus (H1N1)-induced chemokines production and cell migration[J]. Evid Based Complement Alternat Med, 2011, 2011: 750513. DOI: 10.1093/ecam/nep238.
[4] SIEGEL R L, MILLER K D, JEMAL A. Cancer statistics, 2016[J]. CA Cancer J Clin, 2016, 66(1): 7-30. DOI: 10.3322/caac.21332.
[5] SIEGEL R, DESANTIS C, JEMAL A. Colorectal cancer statistics, 2014[J].CA Cancer J Clin, 2014, 64(2): 104-117. DOI: 10.3322/caac.21220.
[6] HU X, JIAO R, LI H, et al. Antiproliferative hydrogen sulfide releasing evodiamine derivatives and their apoptosis inducing properties[J]. Eur J Med Chem, 2018, 151: 376-388. DOI: 10.1016/j.ejmech.2018.04.009.
[7] YAO X, WANG Y, DUAN Y, et al. IGFBP2 promotes salivary adenoid cystic carcinoma metastasis by activating the NF-κB/ZEB1 signaling pathway[J]. Cancer Lett, 2018, 432: 38-46. DOI: 10.1016/j.canlet.2018.06.008.
[8] LIU J Y, ZENG Q H, CAO P G, et al. RIPK4 promotes bladder urothelial carcinoma cell aggressiveness by upregulating VEGF-A through the NF-κB pathway[J]. Br J Cancer, 2018, 118(12): 1617-1627. DOI: 10.1038/s41416-018-0116-8.
[9] SHIH R H, WANG C Y, YANG C M. NF-kappaB signaling pathways in neurological inflammation: amini review[J]. Front Mol Neurosci, 2015, 8: 77. DOI: 10.3389/fnmol.2015.00077.
[10] ZHU C, JIANG W, CHENG Q, et al. Hemeoxygenase-1 suppresses IL-1β-induced apoptosis through the NF-κB pathway in human degenerative nucleus pulposus cells[J]. Cell Physiol Biochem, 2018, 46(2): 644-653. DOI: 10.1159/000488632.
[11] LIN Q, GENG Y, ZHAO M, et al. MiR-21 regulates TNF-α-induced CD40 expression via the SIRT1-NF-κB pathway in renal inner medullary collecting duct cells[J].Cell Physiol Biochem, 2017, 41(1): 124-136. DOI: 10.1159/000455981.
[12] ZHAO N, TIAN K T, CHENG K G, et al. Antiproliferative activity and apoptosis inducing effects of nitric oxide donating derivatives of evodiamine[J]. Bioorg Med Chem, 2016, 24(13): 2971-2978. DOI: 10.1016/j.bmc.2016.05.001.
[13] LIN L, REN L, WEN L, et al. Effect of evodiamine on the proliferation and apoptosis of A549 human lung cancer cells[J].Mol Med Rep, 2016, 14(3): 2832-2838. DOI: 10.3892/mmr.2016.5575.
[14] WEN Z, FENG S, WEI L, et al. Evodiamine, a novel inhibitor of the Wnt pathway, inhibits the self-renewal of gastric cancer stem cells[J]. Int J Mol Med, 2015, 36(6): 1657-1663. DOI: 10.3892/ijmm.2015.2383.
[15] CHEN T C, CHIEN C C, WU M S, et al.Evodiamine from Evodia rutaecarpa induces apoptosis via activation of JNK and PERK in human ovarian cancer cells[J]. Phytomedicine, 2016, 23(1): 68-78. DOI: 10.1016/j.phymed.2015.12.003.
[16] ZHU S, ZHANG X, WEICHERT-LEAHEY N, et al. LMO1 synergizes with MYCN to promote neuroblastoma initiation and metastasis[J].Cancer Cell, 2017, 32(3): 310-323.e5. DOI: 10.1016/j.ccell.2017.08.002.
[17] BAEK S, LEE Y W, YOON S, et al. CDH3/P-Cadherin regulates migration of HuCCT1 cholangiocarcinoma cells[J]. Anat Cell Biol, 2010, 43(2): 110-117. DOI: 10.5115/acb.2010.43.2.110.
[18] ZENG Z S, COHEN A M, GUILLEM J G. Loss of basement membrane type Ⅳ collagen is associated with increased expression of metalloproteinases 2 and 9 (MMP-2 and MMP-9) during human colorectal tumorigenesis[J].Carcinogenesis, 1999, 20(5): 749-755. DOI: 10.1093/carcin/20.5.749.
[19] MROCZKO B, GROBLEWSKA M, BARCIKOWSKA M. The role of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the pathophysiology of neurodegeneration: a literature study[J].J Alzheimers Dis, 2013, 37(2): 273-283. DOI: 10.3233/JAD-130647.
[20] HERSZéNYI L, SIPOS F, GALAMB O, et al. Matrix metalloproteinase-9 expression in the normal mucosa-adenoma-dysplasia-adenocarcinoma sequence of the colon[J]. Pathol Oncol Res, 2008, 14(1): 31-37. DOI: 10.1007/s12253-008-9004-5.
[21] TAKAHRA T, SMART D E, OAKLEY F, et al. Induction of myofibroblast MMP-9 transcription in three-dimensional collagen I gel cultures: regulation by NF-kappaB, AP-1 and Sp1[J]. Int J Biochem Cell Biol, 2004, 36(2): 353-363. DOI: 10.1016/s1357-2725(03)00260-7.
[22] CHOI B H, LEE D H, KIM J, et al. Controls of nuclear factor-Kappa B signaling activity by 5’-AMP-activated protein kinase activation with examples in human bladder cancer cells[J]. Int Neurourol J, 2016, 20(3): 182-187. DOI: 10.5213/inj.1632718.359.
[23] XU Y H, LI Z L, QIU S F. IFN-γ induces gastric cancer cell proliferation and metastasis through upregulation of integrin β3-mediated NF-κB signaling[J]. Transl Oncol, 2018, 11(1): 182-192. DOI: 10.1016/j.tranon.2017.11.008.
[24] FANG R, WANG C, JIANG Q, et al. NEMO-IKKβ are essential for IRF3 and NF-κB activation in the cGAS-STING pathway[J].J Immunol, 2017, 199(9): 3222-3233. DOI: 10.4049/jimmunol.1700699.
[25] LU Y X, JU H Q, WANG F, et al. Inhibition of the NF-κB pathway by nafamostat mesilate suppresses colorectal cancer growth and metastasis[J]. Cancer Lett, 2016, 380(1): 87-97. DOI: 10.1016/j.canlet.2016.06.014.
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