汉黄芩素对皮肤鳞状细胞癌A431细胞株增殖与侵袭的影响及其作用机制
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摘要
目的探讨汉黄芩素对皮肤鳞癌细胞的增殖和侵袭能力的影响及其潜在作用机制。方法检测正常皮肤组织和皮肤鳞癌组织中Notch1 mRNA和蛋白表达水平。使用0、5、10、和20μmol/L浓度的汉黄芩素处理皮肤鳞癌A431细胞系24、48、72 h,分别用MTT法检测各组细胞增殖能力;使用0、5、10、和20μmol/L浓度的汉黄芩素处理细胞48 h,随后采用Transwell侵袭试验检测各组细胞侵袭能力,Real time PCR (RT-PCR)和Western blot法检测Notch1基因的mRNA及蛋白表达。使用Notch1激活剂多西环素处理后,进一步采用MTT法检测各组细胞增殖能力、Transwell法检测各组细胞侵袭能力。结果与正常皮肤组织相比,皮肤鳞癌组织中Notch1基因的mRNA及蛋白表达水平均显著升高,差异具有统计学意义(P <0. 05)。汉黄芩素能够抑制皮肤鳞癌细胞的增殖和侵袭,且呈剂量依赖性,差异具有统计学意义(P <0. 05)。汉黄岑素能够抑制Notch1基因的表达,而应用Notch1激活剂多西环素处理后,明显减弱了汉黄芩素对细胞增殖与侵袭能力的抑制作用,差异具有统计学意义(P <0. 05)。结论汉黄芩素能够抑制皮肤鳞癌细胞的增殖及侵袭进程,可能主要通过抑制Notch1基因来发挥作用。
Objective To investigate the role of wogonin on proliferation and invasion of skin squamous-cell carcinoma and to explore the potential mechanism. Methods The mRNA and protein expression levels of Notch1 were detected in normal skin tissues and skin squamous-cell carcinoma tissues. Skin squamous-cell carcinoma cell line A431 were treated with 0,5,10 and 20 μmol/L wogonin for 24,48,72 hours. Cell proliferation capacities were detected by MTT assay. Cell invasion abilities were monitored by Transwell invasion assay. The mRNA and protein expression of Notch1 gene were detected by Real time PCR( RT-PCR) and Western blot. After treatment with Notch1 activator doxycycline,cell proliferation ability and invasion ability of each group were further detected by MTT assay and Transwell assay.Results Compared with normal skin tissue,mRNA and protein expression levels of Notch1 gene in skin squamous cell carcinoma were significantly increased,with statistically significant differences( P < 0. 05). Wogonin inhibited the proliferation and invasion ability of skin squamous-cell carcinoma cells in a dose dependent manner,with statistically significant differences( P < 0. 05). Wogonin inhibited the expression of Notch1 gene,and the treatment with Notch1 activator doxycycline significantly reduced the inhibitory effect of Wogonin on cell proliferation and invasion,with statistically significant differences( P < 0. 05). Conclusion Wogonin can inhibite the proliferation and invasion of skin squamous-cell carcinoma primarily by inhibiting the Notch1 gene.
Objective To investigate the role of wogonin on proliferation and invasion of skin squamous-cell carcinoma and to explore the potential mechanism. Methods The mRNA and protein expression levels of Notch1 were detected in normal skin tissues and skin squamous-cell carcinoma tissues. Skin squamous-cell carcinoma cell line A431 were treated with 0,5,10 and 20 μmol/L wogonin for 24,48,72 hours. Cell proliferation capacities were detected by MTT assay. Cell invasion abilities were monitored by Transwell invasion assay. The mRNA and protein expression of Notch1 gene were detected by Real time PCR( RT-PCR) and Western blot. After treatment with Notch1 activator doxycycline,cell proliferation ability and invasion ability of each group were further detected by MTT assay and Transwell assay.Results Compared with normal skin tissue,mRNA and protein expression levels of Notch1 gene in skin squamous cell carcinoma were significantly increased,with statistically significant differences( P < 0. 05). Wogonin inhibited the proliferation and invasion ability of skin squamous-cell carcinoma cells in a dose dependent manner,with statistically significant differences( P < 0. 05). Wogonin inhibited the expression of Notch1 gene,and the treatment with Notch1 activator doxycycline significantly reduced the inhibitory effect of Wogonin on cell proliferation and invasion,with statistically significant differences( P < 0. 05). Conclusion Wogonin can inhibite the proliferation and invasion of skin squamous-cell carcinoma primarily by inhibiting the Notch1 gene.
引文
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[21]Chen SS,Michael A,Butler-Manuel SA.Advances in the treatment of ovarian cancer:a potential role of antiinflammatory phytochemicals[J].Discov Med,2012,13(68):7-17.
[22]Hong M,Cheng H,Song L,et al.Wogonin suppresses the activity of matrix metalloproteinase-9 and inhibits migration and invasion in human hepatocellular carcinoma[J].Molecules,2018,23(2):384.doi:10.3390/molecules23020384.
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[27]Afshar Y,Stanculescu A,Miele L,et al.The role of chorionic gonadotropin and Notch1 in implantation[J].J Assist Reprod Genet,2007,24(7):296-302.doi:10.1007/s10815-007-9149-2.
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[29]Sakamoto K.Notch signaling in oral squamous neoplasia[J].Pathol Int,2016,66(11):609-617.doi:10.1111/pin.12461.
[30]Demehri S,Turkoz A,Kopan R.Epidermal Notch1 loss promotes skin tumorigenesis by impacting the stromal microenvironment[J].Cancer Cell,2009,16(1):55-66.doi:10.1016/j.ccr.2009.05.016.
[31]Wang Z,Liu L,Wang M,et al.NOTCH1 regulates migration and invasion of skin cancer cells by E-cadherin repression[J].Mol Cell Biochem,2012,362(1-2):35-41.doi:10.1007/s11010-011-1125-6.
[32]Shu Q,Zhuang H,Fan J,et al.Wogonin induces retinal neuron-like differentiation of bone marrow stem cells by inhibiting Notch-1 signaling[J].Oncotarget,2017,8(17):28431-28441.doi:10.18632/oncotarget.16085.
[2]Goyal N,Thatai P,Sapra B.Skin cancer:symptoms,mechanistic pathways and treatment rationale for therapeutic delivery[J].Ther Deliv,2017,8(5):265-287.doi:10.4155/tde-2016-0093.
[3]Fogel AL,Sarin KY,Teng JMC.Genetic diseases associated with an increased risk of skin cancer development in childhood[J].Curr Opin Pediatr,2017,29(4):426-433.doi:10.1097/MOP.0000000000000514.
[4]Watson M,Holman DM,Maguire-Eisen M.Ultraviolet radiation exposure and its impact on skin cancer risk[J].Semin Oncol Nurs,2016,32(3):241-254.doi:10.1016/j.soncn.2016.05.005.
[5]Wernli KJ,Henrikson NB,Morrison CC,et al.Screening for skin cancer in adults:updated evidence report and systematic review for the US preventive services task force[J].JAMA,2016,316(4):436-447.doi:10.1001/jama.2016.5415.
[6]Behan JW,Sutton A,Wysong A.Management of skin cancer in the high-risk patient[J].Curr Treat Options Oncol,2016,17(12):60.doi:10.1007/s11864-016-0435-z.
[7]Soleymani T,Abrouk M,Kelly KM.An analysis of laser therapy for the treatment of nonmelanoma skin cancer[J].Dermatol Surg,2017,43(5):615-624.doi:10.1097/DSS.0000000000001048.
[8]Tai MC,Tsang SY,Chang LY,et al.Therapeutic potential of wogonin:a naturally occurring flavonoid[J].CNS Drug Rev,2005,11(2):141-150.doi:10.1111/j.1527-3458.2005.tb00266.x.
[9]Chen B,Wu X,Zhang H,et al.Advances of wogonin,an extract from Scutellaria baicalensis,for the treatment of multiple tumors[J].Onco Targets Ther,2016,9:2935-2943.doi:10.2147/ott.s105586.
[10]Chirumbolo S.Anticancer properties of the flavone wogonin[J].Toxicology,2013,314(1):60-64.doi:10.1016/j.tox.2013.08.016.
[11]Winkelmann RR,Farberg AS,Glazer AM,et al.Integrating skin cancer-related technologies into clinical practice[J].Dermatol Clin,2017,35(4):565-576.doi:10.1016/j.det.2017.06.018.
[12]Soura E,Chasapi V,Stratigos AJ.Pharmacologic treatment options for advanced epithelial skin cancer[J].Expert Opin Pharmacother,2015,16(10):1479-1493.doi:10.1517/14656566.2015.1052743.
[13]Perez HC,Benavides X,Perez JS,et al.Basic aspects of the pathogenesis and prevention of non-melanoma skin cancer in solid organ transplant recipients:a review[J].Int J Dermatol,2017,56(4):370-378.doi:10.1111/ijd.13409.
[14]Wollina U.Update of cetuximab for non-melanoma skin cancer[J].Expert Opin Biol Ther,2014,14(2):271-276.doi:10.1517/14712598.2013.876406.
[15]Asgari MM.Utility and limitations of large population-based data for skin cancer outcomes[J].J Invest Dermatol,2016,136(11):2128-2130.doi:10.1016/j.jid.2016.07.031.
[16]Gracia-Caza1a T,Salazar N,Zamarrón A,et al.Resistance of nonmelanoma skin cancer to nonsurgical treatments.PartⅡ:photodynamic therapy,vismodegib,cetuximab,intralesional methotrexate,and radiotherapy[J].Actas dermo-sifiliograficas,2016,107(9):740-750.doi:10.1016/j.ad.2016.04.020.
[17]Rudnick EW,Thareja S,Cherpelis B.Oral therapy for nonmelanoma skin cancer in patients with advanced disease and large tumor burden:a review of the literature with focus on a new generation of targeted therapies[J].Int J Dermatol,2016,55(3):249-258.doi:10.1111/ijd.12961.
[18]Kirby JS,Miller CJ.Intralesional chemotherapy for nonmelanoma skin cancer:a practical review[J].J Am Acad Dermatol,2010,63(4):689-702.doi:10.1016/j.jaad.2009.09.048.
[19]Hayasaka S,Kodama T,Ohira A.Traditional Japanese herbal(kampo)medicines and treatment of ocular diseases:a review[J].Am JChin Med,2012,40(5):887-904.doi:10.1142/S0192415X12500668.
[20]Huynh DL,Sharma N,Kumar Singh A,et al.Anti-tumor activity of wogonin,an extract from Scutellaria baicalensis,through regulating different signaling pathways[J].Chin J Nat Med,2017,15(1):15-40.doi:10.1016/S1875-5364(17)30005-5.
[21]Chen SS,Michael A,Butler-Manuel SA.Advances in the treatment of ovarian cancer:a potential role of antiinflammatory phytochemicals[J].Discov Med,2012,13(68):7-17.
[22]Hong M,Cheng H,Song L,et al.Wogonin suppresses the activity of matrix metalloproteinase-9 and inhibits migration and invasion in human hepatocellular carcinoma[J].Molecules,2018,23(2):384.doi:10.3390/molecules23020384.
[23]Liu X,Tian S,Liu M,et al.Wogonin inhibits the proliferation and invasion,and induces the apoptosis of HepG2 and Bel7402 HCC cells through NF-κB/Bcl-2,EGFR and EGFR downstream ERK/AKT signaling[J].Int J Mol Med,2016,38(4):1250-1256.doi:10.3892/ijmm.2016.2700.
[24]Zhao K,Wei L,Hui H,et al.Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways[J].PLo S One,2014,9(9):e106458.doi:10.1371/journal.pone.0106458.
[25]Kim MS,Bak Y,Park YS,et al.Wogonin induces apoptosis by suppressing E6 and E7 expressions and activating intrinsic signaling pathways in HPV-16 cervical cancer cells[J].Cell Biol Toxicol,2013,29(4):259-272.doi:10.1007/s10565-013-9251-4.
[26]Palomero T,Ferrando A.Therapeutic targeting of NOTCH1 signaling in T-cell acute lymphoblastic leukemia[J].Clin Lymphoma Myeloma,2009,9(Suppl 3):S205-S210.doi:10.3816/CLM.2009.s.013.
[27]Afshar Y,Stanculescu A,Miele L,et al.The role of chorionic gonadotropin and Notch1 in implantation[J].J Assist Reprod Genet,2007,24(7):296-302.doi:10.1007/s10815-007-9149-2.
[28]Psyrri A,Seiwert TY,Jimeno A.Molecular pathways in head and neck cancer:EGFR,PI3K,and more[J].Am Soc Clin Oncol Educ Book,2013:246-255.doi:10.1200/EdBook_AM.2013.33.246.
[29]Sakamoto K.Notch signaling in oral squamous neoplasia[J].Pathol Int,2016,66(11):609-617.doi:10.1111/pin.12461.
[30]Demehri S,Turkoz A,Kopan R.Epidermal Notch1 loss promotes skin tumorigenesis by impacting the stromal microenvironment[J].Cancer Cell,2009,16(1):55-66.doi:10.1016/j.ccr.2009.05.016.
[31]Wang Z,Liu L,Wang M,et al.NOTCH1 regulates migration and invasion of skin cancer cells by E-cadherin repression[J].Mol Cell Biochem,2012,362(1-2):35-41.doi:10.1007/s11010-011-1125-6.
[32]Shu Q,Zhuang H,Fan J,et al.Wogonin induces retinal neuron-like differentiation of bone marrow stem cells by inhibiting Notch-1 signaling[J].Oncotarget,2017,8(17):28431-28441.doi:10.18632/oncotarget.16085.