S100A6通过巨噬细胞促结直肠癌细胞增殖的作用及机制
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摘要
目的:探讨微环境中钙周期素S100A6是否通过影响巨噬细胞(macrophages,M_φ)进而促进结直肠癌(colorectal cancer,CRC)细胞的增殖及其机制。方法:制备(原核表达)并鉴定带GST(glutathione S-transferase,谷胱甘肽S-转移酶)标签的人重组S100A6蛋白(recombinant GSTh S100A6,r S100A6)和对照蛋白GST;采用台盼兰计数、CCK8和结晶紫染色检测CRC细胞系HCT116的增殖能力;用定量实时聚合酶链反应检测M_φ中IL-6 mRNA水平;用Western blot检测M_φ中IL-6的蛋白水平、HCT116细胞中JAK2和STAT3及其磷酸化水平。结果:(1)成功制备r S100A6和GST蛋白。(2)与经r S100A6处理的M_φ(即A6-M_φ)共培养后,HCT116细胞的增殖能力增强(P <0. 05);同时,HCT116细胞中的JAK2和STAT3水平无明显变化,但其磷酸化水平提高(P <0. 05)。(3) A6-M_φ中,IL-6的mRNA和蛋白水平均升高(P <0. 05)。(4)在HCT116与A6-M_φ的共培养体系中加入IL-6R封闭肽后,A6-M_φ促HCT116细胞的活力和增殖能力的作用被部分逆转(P <0. 05)。结论:微环境中的S100A6可通过上调巨噬细胞中IL-6的表达、进而激活HCT116细胞中IL-6/JAK2/STAT3信号通路来促进CRC细胞的增殖。
Objective: To explore whether Calcyclin S100A6 in tumor microenvironment promotes cell proliferation of colorectal cancer (CRC) through affecting macrophages (M_φ) and its mechanism. Methods:Prokaryotic expression was used to prepare recombinant human protein GST-S100A6 (r S100A6) and GST (as control). THP-1 were induced to M_φ by PMA (Phorbol-12-myristate-13-acetate).A6-M_φ were the macrophages which were treated with r S100A6 for 24 h. The proliferation of CRC HCT116 cells was detected by Trypan blue staining,CCK8 and crystal violet staining. IL-6 mRNA and protein level inA6-M_φ were tested with quantitative polymerase chain reaction (qPCR) and Western blot,respectively. The protein levels of total JAK2 and STAT3 (t-JAK2 and t-STAT3) and the phosphorylated JAK2 and STAT3 (p-JAK2 and p-STAT3) in HCT116 cells were detected by Western blot. Results: (1) rS100A6 and GST were prepared successfully. (2)A6-M_φ promoted proliferation of HCT116 cells (P < 0. 05). (3) r S100A6 upregulated IL-6 expression in macrophages (P < 0. 05). (4) IL-6 R blocking antibody partly reversed the facilitation ofA6-M_φ to proliferation of HCT116 cells (P < 0. 05). (5)A6-M_φ increased protein levels of p-JAK2 and p-STAT3 in HCT116 cells (P < 0. 05),but not t-JAK2 and t-STAT3. Conclusion: S100A6 in tumor microenvironment facilitates proliferation of HCT116 cells through upregulating IL-6 expression in macrophages and activating IL-6/JAK2/STAT3 pathway in HCT116 cells.
Objective: To explore whether Calcyclin S100A6 in tumor microenvironment promotes cell proliferation of colorectal cancer (CRC) through affecting macrophages (M_φ) and its mechanism. Methods:Prokaryotic expression was used to prepare recombinant human protein GST-S100A6 (r S100A6) and GST (as control). THP-1 were induced to M_φ by PMA (Phorbol-12-myristate-13-acetate).A6-M_φ were the macrophages which were treated with r S100A6 for 24 h. The proliferation of CRC HCT116 cells was detected by Trypan blue staining,CCK8 and crystal violet staining. IL-6 mRNA and protein level inA6-M_φ were tested with quantitative polymerase chain reaction (qPCR) and Western blot,respectively. The protein levels of total JAK2 and STAT3 (t-JAK2 and t-STAT3) and the phosphorylated JAK2 and STAT3 (p-JAK2 and p-STAT3) in HCT116 cells were detected by Western blot. Results: (1) rS100A6 and GST were prepared successfully. (2)A6-M_φ promoted proliferation of HCT116 cells (P < 0. 05). (3) r S100A6 upregulated IL-6 expression in macrophages (P < 0. 05). (4) IL-6 R blocking antibody partly reversed the facilitation ofA6-M_φ to proliferation of HCT116 cells (P < 0. 05). (5)A6-M_φ increased protein levels of p-JAK2 and p-STAT3 in HCT116 cells (P < 0. 05),but not t-JAK2 and t-STAT3. Conclusion: S100A6 in tumor microenvironment facilitates proliferation of HCT116 cells through upregulating IL-6 expression in macrophages and activating IL-6/JAK2/STAT3 pathway in HCT116 cells.
引文
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[16]Wang H,Zhang L,Zhang I Y,et al.S100B promotes glioma growth through chemoattraction of myeloid-derived macrophages.Clinical Cancer Research,2013,19(14):3764-3775.
[17]Wunderlich C M,Ackermann P J,Ostermann A L,et al.Obesity exacerbates colitis-associated cancer via IL-6-regulated macrophage polarisation and CCL-20/CCR-6-mediated lymphocyte recruitment.Nature Communication,2018,9(1):1646.
[18]Waldner M J,Sebastian F,Neurath M F.Interleukin-6-A key regulator of colorectal cancer development.International Journal of Biological Sciences,2012,8(9):1248-1253.
[2]Komohara Y,Fujiwara Y,Ohnishi K,et al.Tumor-associated macrophages:Potential therapeutic targets for anti-cancer therapy.Advanced Drug Delivery Reviews,2016,99(Pt B):180-185.
[3]Erreni M,Mantovani A,Allavena P.Tumor-associated macrophages(TAM)and inflammation in colorectal cancer.Cancer Microenvironment,2011,4(2):141-154.
[4]Nakayama Y,Nagashima N,Minagawa N,et al.Relationships between tumor-associated macrophages and clinicopathological factors in patients with colorectal cancer.Anticancer Research,2002,22(6C):4291-4296.
[5]Donato R,Sorci G,Giambanco I.S100A6 protein:Functional roles.Cellular&Molecular Life Sciences,2017,74(4):2749-2760.
[6]Duan L,Wu R,Zou Z,et al.S100A6 stimulates proliferation and migration of colorectal carcinoma cells through activation of the MAPK pathways.International Journal of Oncology,2014,44(3):781-90.
[7]Donato R,Cannon B R,Sorci G,et al.Functions of S100proteins.Current Molecular Medicine,2013,13(1):24-57.
[8]Byun K,Yoo Y,Son M,et al.Advanced glycation end-products produced systemically and by macrophages:A common contributor to inflammation and degenerative diseases.Pharmacology&Therapeutics,2017,177:44-55.
[9]Nasser M W,Qamri Z,Deol Y S,et al.S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways.Cancer Research,2012,72(3):604-615.
[10]Nasser M W,Wani N,Ahirwar D K,et al.RAGE mediates S100A7-induced breast cancer growth and metastasis by modulating the tumor microenvironment.Cancer Research,2015,75(6):974-985.
[11]Chavakis T,Bierhaus A,Nawroth P P.RAGE(receptor for advanced glycation end products):A central player in the inflammatory response.Microbes Infection,2004,6(13):1219-1225.
[12]Duan L,Wu R,Zou Z,et al.S100A6 stimulates proliferation and migration of colorectal carcinoma cells through activation of the MAPK pathways.International Journal of Oncology,2014,44(3):781-790.
[13]Zha H,Sun H,Li X,et al.S100A8 facilitates the migration of colorectal cancer cells through regulating macrophages in the inflammatory microenvironment.Oncology Reports,2016,36(1):279-290.
[14]Forman D,Ferlay J,Jemal A,et al.Global cancer statistics.Ca A Cancer Journal for Clinicians,2015,65(2):87-108.
[15]Kampan N C,Xiang S D,Mcnally O M,et al.Immunotherapeutic interleukin-6 or interleukin-6 receptor blockade in cancer:Challenges and opportunities.Current Medicinal Chemistry,2018,25(36):1-22.
[16]Wang H,Zhang L,Zhang I Y,et al.S100B promotes glioma growth through chemoattraction of myeloid-derived macrophages.Clinical Cancer Research,2013,19(14):3764-3775.
[17]Wunderlich C M,Ackermann P J,Ostermann A L,et al.Obesity exacerbates colitis-associated cancer via IL-6-regulated macrophage polarisation and CCL-20/CCR-6-mediated lymphocyte recruitment.Nature Communication,2018,9(1):1646.
[18]Waldner M J,Sebastian F,Neurath M F.Interleukin-6-A key regulator of colorectal cancer development.International Journal of Biological Sciences,2012,8(9):1248-1253.