IL-4协同雌二醇对小鼠乳腺癌细胞生长的促进作用及其机制

doi:10.13481/j.1671-587x.20200318

摘要/Abstract

摘要： 目的：探讨白细胞介素4（IL-4）协同雌二醇对小鼠乳腺癌4T1细胞对其生物学行为的影响，并阐明其作用机制。方法：体外培养4T1细胞，加入不同浓度（0、12.5、25.0、50.0和100.0 μg·L^-1） IL-4或雌二醇（0、6.25、12.50、25.00、50.00 nmol·L^-1），作用72 h后MTT法检测乳腺癌4T1细胞增殖率。将乳腺癌4T1细胞分为对照组（不进行任何处理）、IL-4组（加入50.0 μg·L^-1 IL-4）、雌二醇组（加入12.50 nmol·L^-1雌二醇）和联合组（加入50.0 μg·L^-1 IL-4+12.50 nmol·L^-1雌二醇），MTT法检测各组乳腺癌4T1细胞增殖率，流式细胞术检测各组不同细胞周期乳腺癌4T1细胞百分比，Western blotting法检测各组乳腺癌4T1细胞中STAT6、p-STAT6、ERα、Erk、p-Erk、P70S6K、p-P70S6K、S6和p-S6蛋白表达水平。结果：与0 μg·L^-1 IL-4组比较，25.0、50.0和100.0 μg·L^-1IL-4组乳腺癌4T1细胞增殖率升高（P<0.05）。与0 nmol·L^-1雌二醇组比较，12.50、25.00、50.00 nmol·L^-1雌二醇组乳腺癌4T1细胞增殖率升高（P<0.05）。与对照组比较，IL-4组乳腺癌4T1细胞增殖率升高（P<0.05）；与对照组比较，雌二醇组乳腺癌4T1细胞增殖率升高（P<0.05）；与IL-4组或雌二醇组比较，联合组乳腺癌4T1细胞增殖率升高（P<0.05）。与对照组比较，IL-4组乳腺癌4T1细胞中S期和G₂/M期细胞百分比升高（P<0.05），G₀及G₁期细胞百分比降低（P<0.05）；雌二醇组乳腺癌4T1细胞中S期细胞百分比明显升高（P<0.05），G₀及G₁期细胞百分比降低（P<0.05）。与IL-4组或雌二醇组比较，联合组乳腺癌4T1细胞中S期和G₂/M期细胞百分比升高（P<0.05），G₀及G₁期细胞百分比降低（P<0.05）。与对照组比较，IL-4组乳腺癌4T1细胞中ERα、p-Erk、p-P70S6K和p-S6蛋白表达水平升高，雌二醇组乳腺癌4T1细胞中p-STAT6、ERα、p-Erk、p-P70S6K、S6和p-S6蛋白表达水平升高（P<0.05）；联合组乳腺癌4T1细胞中STAT6、p-STAT6、ERα、p-Erk、p-P70S6K和p-S6蛋白表达水平升高（P<0.05）。结论：IL-4协同雌二醇可促进小鼠4T1乳腺癌细胞膜IL-4受体（IL-4R）和雌激素受体（ER）表达，增强乳腺癌4T1细胞中Erk1和p70S6K激酶的激活及下游分子S6蛋白的磷酸化。

关键词: 白细胞介素4, 雌激素受体, 乳腺癌4T1细胞, 巨噬细胞, 肿瘤微环境, 协同作用

Abstract: Objective: To investigate the effect of interleukin-4(IL-4) and estradiol on the biological behavior of breast cancer 4T1 cells of the mice, and to elucidate its mechanism. Methods: The 4T1 cells were cultured in vitro and added with different concentrations (0, 12.5, 25.0, 50.0 and 100.0μg·L^-1) of IL-4 or estradiol (0, 6.25, 12.50, 25.00 and 50.00 nmol·L^-1).The proliferation rate of the breast cancer 4T1 cells was measured by MTT method after treated for 72 h.The breast cancer 4T1 cells were divided into control group (without any treatment), IL-4 group (treated with 50.0μg·L^-1IL-4), estradiol group (treated with 12.50 nmol·L^-1 estradiol) and combination group (treated with 50.0μg·L ^-1 IL-4+ 12.50 nmol·L^-1 estradiol). MTT method was used to detect the proliferation rates of the breast cancer 4T1 cells in various groups, and flow cytometry was used to detect the percentages of the breast cancer 4T1 cells in different cell cycles in various groups,and Western blotting method was used to detect the expression levels of STAT6, p-STAT6, ERα, Erk, p-Erk, P70S6K, p-P70S6K, S6,and p-S6 in the breast cancer 4T1 cells in various groups. Results: Compared with 0μg·L^-1 IL-4 group, the proliferation rates of the breast cancer 4T1 cells in 25.0, 50.0 and 100.0μg·L^-1 IL-4 groups were increased (P<0.05);compared with 0 nmol·L^-1 estradiol groups, the proliferation rates of the breast cancer 4T1 cells in 12.50, 25.00 and 50.00 nmol·L^-1 estradiol groups were increased (P<0.05).Compared with control group, the proliferation rate of the breast cancer 4T1 cells in IL-4 group was increased (P<0.05); compared with control group, the proliferation rate of the breast cancer 4T1 cells in estradiol group was increased (P<0.05); compared with IL-4 group or estradiol group, the proliferation rate of the breast cancer 4T1 cells in combination group was increased (P<0.05).Compared with control group, the percentages of the breast cancer 4T1 cells at S phase and G₂/M phase in IL-4 group were increased (P<0.05),and the percentage of the breast cancer 4T1 cells at G₀ and G₁ phases were decreased(P<0.05); compared with control group, the percentage of the breast cancer 4T1 cells at S phase in estradiol group was increased(P<0.05),and the percentages of the breast cancer 4T1 cells at G₀ and G₁ phases were decreased (P<0.05).Compared with control group, the expression levels of ERα, p-Erk, p-P70S6K, and p-S6 in the breast cancer 4T1 cells in IL-4 group were increased(P<0.05), while the expression levels of p-STAT6, ERα, p-Erk, p-P70S6K, S6,and p-S6 in the breast cancer 4T1 cells in estradiol group were increased (P<0.05); the expression levels of STAT6, p-STAT6, ERα, p-ERK, p-P70S6K, and p-S6, in the breast cancer 4T1 cells in combination group were increased (P<0.05). Conclusion: The combination of IL-4 and estradiol can increase the expressions of IL-4 receptor(IL-4R) and estrogen receptor (ER),and enhance the activation of Erk1, p70S6K kinase and phosphorylation of downstream S6 protein in the breast cancer 4T1 cells.

Key words: interleukin-4, estrogen receptor, breast cancer 4T1 cells, macrophages, tumor microenvironment, synergistic effect

中图分类号:

R737.9

冯磊, 张韩特, 李响, 孟繁平, 李妍. IL-4协同雌二醇对小鼠乳腺癌细胞生长的促进作用及其机制[J]. 吉林大学学报(医学版), 2020, 46(03): 536-542.

FENG Lei, ZHANG Hante, LI Xiang, MENG Fanping, LI Yan. Promotion effect of IL-4 and estradiol on growth of breast cancer cells in mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2020, 46(03): 536-542.

参考文献

[1] JEONG H, HWANG I, KANG S H,et al.Tumor-associated macrophages as potential prognostic biomarkers of invasive breast cancer[J].Breast Cancer,2019, 22(1):38-51.
[2] 马守宝,林丹丹,刘海燕.炎症细胞因子在肿瘤微环境中的作用及其作为治疗靶点的研究进展[J].生命科学,2016,28(2):182-191.
[3] MOONEY S M,RAJAGOPALAN K,RANGARAJAN G, et al. Cancer/testis antigens and obligate participation in multiple hallmarks of cancer:an update[J]. Asian J Androl, 2016, 18(5):711-712.
[4] 魏智民,孙玉发,李刚,等.癌症相关性炎症与肿瘤微环境相关研究进展[J].中国肿瘤临床,2018,45(21):1117-1121.
[5] KITAMURA T,QIAN B Z,POLLARD J W.Immune cellpromotion of metastasis[J]. Nat Rev Immunol,2015,15(2):73-86.
[6] SHEN M,WANG J,YU W W,et al. A novel MDSC-induced PD-1-PD-L1+B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties[J].Oncoimmunology,2018,7(4):e1413520.
[7] LUONG B,SCHWENK R,BRAUTIGAM J,et al.The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen[J].PLoS One,2018,13(9):e0203053.
[8] LEBLEU V S,KALLURI R.A peek into cancer-associated fibroblasts:origins, functions and translational impact[J]. Dis Model Mech,2018,11(4):dmm029447.
[9] YANG J E,LU Y,LIN Y Y,et al.Vascular mimicry formation is promoted by paracrine TGF-β and SDF₁ of cancer-associated fibroblasts and inhibited by miR-101 in hepatocellular carcinoma[J].Cancer Lett,2016,383(1):18-27.
[10] DELGADO-BELLIDO D,SERRANO-SAENZ S,FERNÁNDEZ-CORTÉ S,et al.Vasculogenic mimicry signaling revisited:focus on non-vascular VE-cadherin[J].Mol Cancer,2017,16(1):65.
[11] MILLS C D. Anatomy of a discovery:m1 and m2 macrophages[J].Front Immunol,2015,6:212.
[12] SORMENDI S,WIELOCKX B.Hypoxia pathway proteins as central mediators of metabolism in the tumor cells and their microenvironment[J].Front Immunol,2018,9:40-47.
[13] 孙林冲,高平.代谢重编程在调控肿瘤免疫微环境中的作用[J].生物化学与生物物理进展,2017,44(8):688-696.
[14] ZHANG LJ, LU R Q, SONG YN, et al. Knockdown of anion exchanger 2 suppressed the growth of ovarian cancer cells via mTOR/p70S6K1 signaling[J]. Sci Rep, 2017, 7(1):6362.
[15] WANG X,LI M.Correlate tumor mutation burden with immunes ignatures in human cancers[J].BMC Immunol,2019,20(1):4.
[16] 李杏,王丹丹,唐琪,等.巨噬细胞极化的转录调控及其对相关疾病影响的研究进展[J].吉林大学学报(医学版),2016,42(3):622-626.
[17] TSAI H F,TRUBELJA A,SHEN A Q,et al.Tumour-on-a-chip:microfluidic models of tumour morphology,growth and microenvironment[J]. J R Soc Interface,2017,14(131):20170137.
[18] YATES LR,KNAPPSKOG S,W EDGE D,et al.Genomic evolution of breast cancer metastasis and relapse[J].Cancer Cell,2017,32(2):169-184.
[19] SOMASUNDARAM R,ZHANG G,FUKUN M,et al.Tumor-associated B-cells induce tumor heterogeneity and therapy resistance[J]. Nat Commun,2017,8(1): 607-615.
[20] HIDA K,MAISHI N,ANNAN D,et al.Contribution of tumor endothelial cells in cancer progression[J]. Int J Mol Med,2018,19(5):2306-2317.