M2巨噬细胞通过调控NF-κB信号通路对非小细胞肺癌A549细胞上皮-间质转化和顺铂耐药的促进作用

doi:10.13481/j.1671-587X.20250309

Abstract

Abstract:

Objective To discuss the role of M2 macrophages in epithelial-mesenchymal transition （EMT） and cisplatin （DDP） resistance in the non-small cell lung cancer （NSCLC）， and to clarify the regulatory mechanism of nuclear factor κB （NF-κB） signaling pathway. Methods The human monocytic leukemia THP-1 cells were selected and differentiated into M0 macrophages by phorbol myristate acetate （PMA） induction， followed by M2 macrophage polarization through interleukin （IL）-4 and IL-13 stimulation. Western blotting and immunofluorescence methods were used to detect the protein expression levels of CD163， CD86， and arginase-1 （Arg-1） in M0 and M2 macrophages.The human NSCLC A549 cells were co-cultured non-contactly with M0 or M2 macrophages in Transwell chambers， and the cells were divided into A549+M0 group （A549 cells co-cultured with M0 macrophages）， A549+M2 group （A549 cells co-cultured with M2 macrophages）， and A549+M2+BAY11-7082 group （A549 cells co-cultured with M2 macrophages and treated with 10 mmol·L^-1 NF-κB inhibitor BAY11-7082）. Wound healing assay was used to detect the wound healing rate of the A549 cells in various groups； Transwell assay was used to detect the number of invasion A549 cells in various groups； cell counting kit-8 （CCK-8） assay was used to detect the inhibitory rate of proliferation and half maximal inhibitory concentration （IC₅₀） value of the A549 cells after treated with DDP in the co-culture system； Western blotting method was used to detect the expression levels of vimentin， E-cadherin， N-cadherin， transcription factor Snail， phosphorylated P65 （p-P65）， P-glycoprotein （P-gp）， and programmed death-ligand 1 （PD-L1） proteins in the A549 cells in various groups. Results The Western blotting results showed that compared with M0 group， the expression levels of CD163 and Arg-1 proteins in the macrophages in M2 group were significantly increased （P<0.05）， while the expression level of CD86 protein was significantly decreased （P<0.05）. The immunofluorescence results showed that compared with M0 group， the expression of CD163 protein in the macrophages in M2 group was enhanced and the expression of CD86 protein was weakened. The wound healing assay results showed that at 24 and 48 h of culture， compared with A549+M0 group， the wound healing rate of the A549 cells in A549+M2 group was significantly increased （P<0.05）； in the co-culture system， compared with A549+M0 group， the wound healing rate of the A549 cells in A549+M2 group was significantly increased （P<0.05）； compared with A549+M2 group， the wound healing rate of the A549 cells in A549+M2+BAY11-7082 group was significantly decreased （P<0.05）. The Transwell assay results showed that compared with A549+M0 group， the number of invasion A549 cells in A549+M2 group was significantly increased （P<0.05）； compared with A549+M2 group， the number of invasion A549 cells in A549+M2+BAY11-7082 group was significantly decreased （P<0.05）； in the co-culture system， compared with A549+M0 group， the number of invasion A549 cells in A549+M2 group was significantly increased （P<0.05）. The CCK-8 assay results showed that after treated with 2.50， 5.00， 10.00， 20.00， and 40.00 mg·L^-1 DDP， compared with A549+M0 group， the inhibitory rate of proliferation of the A549 cells in A549+M2 group was significantly decreased （P<0.05 or P<0.01）， and the IC₅₀ value was significantly increased （P<0.01）； in the co-culture system， compared with A549+M0 group， the inhibitory rate of proliferation of the A549 cells in A549+M2 group was significantly decreased （P<0.05 or P<0.01）， and the IC₅₀ value was significantly increased （P<0.01）； compared with A549+M2 group， the inhibitory rate of proliferation of the A549 cells in A549+M2+BAY11-7082 group was significantly increased （P<0.05）， and the IC₅₀ value was significantly decreased （P<0.05）. The Western blotting results showed that compared with A549+M0 group， the expression level of E-cadherin proteins in the A549 cells in A549+M2 group was significantly decreased （P<0.05）， while the expression levels of N-cadherin， vimentin， and Snail proteins were significantly increased （P<0.05）； in the co-culture system， compared with A549+M0 group， the expression levels of vimentin， Snail， N-cadherin， and p-P65 proteins in the A549 cells in A549+M2 group were significantly increased （P<0.05）， while the expression level of E-cadherin proteins was significantly decreased （P<0.05）； compared with A549+M2 group， the expression levels of vimentin， N-cadherin， and p-P65 proteins in the A549 cells in A549+M2+BAY11-7082 group were significantly decreased （P<0.05）， while the expression level of E-cadherin proteins was significantly increased （P<0.05）； compared with A549+M0 group， the expression levels of P-gp and PD-L1 proteins in the A549 cells in A549+M2 group were significantly increased （P<0.05）； in the co-culture system， compared with A549+M0 group， the expression levels of P-gp and PD-L1 proteins in the A549 cells in A549+M2 group were significantly increased （P<0.05）； compared with A549+M2 group， the expression levels of P-gp and PD-L1 proteins in the A549 cells in A549+M2+BAY11-7082 group were significantly decreased （P<0.05）. Conclusion The M2 macrophages can regulate EMT in the NSCLC cells to promote the invasion and metastasis of tumor， and modulate the expressions of P-gp and PD-L1 to enhance DDP resistance， which is associated with the NF-κB signaling pathway.

Key words: M2 macrophage, Non-small cell lung cancer, Nuclear factor-κB, Epithelial-mesenchymal transition, Cisplatin

CLC Number:

R734.2

Xingxiang WANG,Ying ZHAO,Qiaotong REN,Hefei WANG,Gang PU,Chun LI. Promotive effect of M2 macrophages on epithelial-mesenchymal transition and cisplatin resistance in non-small cell lung cancer A549 cells by regulating NF-κB signaling pathway[J].Journal of Jilin University(Medicine Edition), 2025, 51(3): 642-652.

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References 26

[1]	HAN B F， ZHENG R S， ZENG H M， et al. Cancer incidence and mortality in China， 2022［J］. J Natl Cancer Cent， 2024， 4（1）： 47-53.
[2]	盖晓东，赵颖，王鹤霏，等. FOXP3调控非小细胞肺癌A549细胞对阿霉素敏感性的作用及其机制［J］. 吉林大学学报（医学版）， 2023， 49（5）： 1161-1167.
[3]	石安辉，朱广迎. 局部晚期非小细胞肺癌放化疗的现状与进展［J］. 临床外科杂志， 2016， 24（7）： 505-508.
[4]	何程远，杨红宇，谭钰晶，等. IL-17A在非小细胞肺癌组织中的表达及其通过NF-κB信号通路对VEGF表达的调控作用［J］. 吉林大学学报（医学版）， 2022， 48（4）： 1003-1009.
[5]	MAHMOUD S A， LEE A S， PAISH E C， et al. Tumour-infiltrating macrophages and clinical outcome in breast cancer［J］. J Clin Pathol， 2012， 65（2）： 159-163.
[6]	TARIQ M， ZHANG J Q， LIANG G K， et al. Macrophage polarization： anti-cancer strategies to target tumor-associated macrophage in breast cancer［J］. J Cell Biochem， 2017， 118（9）： 2484-2501.
[7]	CAO L L， CHE X F， QIU X S， et al. M2 macrophage infiltration into tumor islets leads to poor prognosis in non-small-cell lung cancer［J］. Cancer Manag Res， 2019， 11： 6125-6138.
[8]	CHEN S H， MORINE Y， TOKUDA K， et al. Cancer-associated fibroblast-induced M2-polarized macrophages promote hepatocellular carcinoma progression via the plasminogen activator inhibitor-1 pathway［J］. Int J Oncol， 2021， 59（2）： 59.
[9]	LIN S C， LIAO Y C， CHEN P M， et al. Periostin promotes ovarian cancer metastasis by enhancing M2 macrophages and cancer-associated fibroblasts via integrin-mediated NF-κB and TGF-β2 signaling［J］. J Biomed Sci， 2022， 29（1）： 109.
[10]	姜一弘，张丹，张天择，等. 核因子κB（NF-κB）信号通路在炎症与肿瘤中作用的研究进展［J］. 细胞与分子免疫学杂志， 2018， 34（12）： 1130-1135.
[11]	朱帅旗，段海潇，汪洋，等. NF-κB在非小细胞肺癌中的作用及治疗研究［J］. 生命科学， 2022， 34（4）： 409-419.
[12]	YAO M， MAO X H， ZHANG Z R， et al. Tumor-derived CircRNA_102191 promotes gastric cancer and facilitates M2 macrophage polarization［J］. Cell Cycle， 2023， 22（18）： 2003-2017.
[13]	MANTOVANI A， ALLAVENA P， MARCHESI F， et al. Macrophages as tools and targets in cancer therapy［J］. Nat Rev Drug Discov， 2022， 21（11）： 799-820.
[14]	万小英，周崧雯. M2型肿瘤相关巨噬细胞在肺癌中的研究进展［J］. 肿瘤防治研究， 2022， 49（7）： 733-737.
[15]	SUMITOMO R， HIRAI T， FUJITA M， et al. M2 tumor-associated macrophages promote tumor progression in non-small-cell lung cancer［J］. Exp Ther Med， 2019， 18（6）： 4490-4498.
[16]	BAO Z， ZENG W， ZHANG D， et al. Snail induces emt and lung metastasis of tumours secreting cxcl2 to promote the invasion of m2-type immunosuppressed macrophages in colorectal cancer［J］. Int J Biol Sci， 2022， 18（7）： 2867-2881.
[17]	张帆，郭咸希. 肿瘤微环境中肿瘤相关巨噬细胞与上皮间质化“对话”的研究进展［J］. 现代肿瘤医学， 2020， 28（20）： 3640-3646.
[18]	LI X， CHEN L， PENG X， et al. Progress of tumor-associated macrophages in the epithelial-mesenchymal transition of tumor［J］. Front Oncol， 2022， 12： 911410.
[19]	施鹏冲，祝先进，曹颖平. 化疗药物诱导肿瘤细胞耐药研究进展［J］. 中国免疫学杂志， 2021， 37（11）： 1400-1403.
[20]	CHEN Y B， SONG Y C， DU W， et al. Tumor-associated macrophages： an accomplice in solid tumor progression［J］. J Biomed Sci， 2019， 26（1）： 78.
[21]	SUN C， MEZZADRA R， SCHUMACHER T N. Regulation and function of the PD-L1 checkpoint［J］. Immunity， 2018， 48（3）： 434-452.
[22]	张润兵，史婷婷，伍杨，等. 自噬介导肝细胞癌耐药的相关机制［J］. 临床肝胆病杂志， 2024， 40（11）： 2315-2319.
[23]	TANG D F， ZHAO D D， WU Y， et al. The miR-3127-5p/p-STAT3 axis up-regulates PD-L1 inducing chemoresistance in non-small-cell lung cancer［J］. J Cell Mol Med， 2018， 22（8）： 3847-3856.
[24]	MIRZAEI S， SAGHARI S， BASSIRI F， et al. NF-κB as a regulator of cancer metastasis and therapy response： a focus on epithelial-mesenchymal transition［J］. J Cell Physiol， 2022， 237（7）： 2770-2795.
[25]	王方园，孔宪斌，杨玉莹，等. M2型TAMs激活NF-κB通路促进结肠癌细胞侵袭转移的实验研究［J］. 现代肿瘤医学， 2020， 28（21）： 3651-3656.
[26]	WEI R， ZHU W W， YU G Y， et al. S100 calcium-binding protein A9 from tumor-associated macrophage enhances cancer stem cell-like properties of hepatocellular carcinoma［J］. Int J Cancer， 2021， 148（5）： 1233-1244.

Group	Scratch healing rate
Group	(t/h) 24	48
A549+M0	21.83±2.80	28.26±2.68
A549+M2	29.02±3.17^*	39.64±2.24^*

Group	Scratch healing rate
Group	(t/h) 24	48
A549+M0	22.71±3.25	30.41±2.95
A549+M2	35.29±2.63^*	45.02±2.41^*
A549+M2+BAY11-7082	29.41±3.55^△	36.63±2.84^△

Group	Inhibitory rate of proliferation （η/%）						IC₅₀ value[ρ_B/(mg·L^-1)]
Group	1.25 mg·L^-1 DDP	2.50 mg·L^-1 DDP	5.00 mg·L^-1 DDP	10.00 mg·L^-1 DDP	20.00 mg·L^-1 DDP	40.00 mg·L^-1 DDP	IC₅₀ value[ρ_B/(mg·L^-1)]
A549+M0	2.29±0.60	7.37±0.46	12.94±1.19	48.34±2.18	54.88±1.29	65.77±0.46	17.54±0.28
A549+M2	2.18±0.15	5.41±0.44^*	9.40±1.47^**	36.43±3.36^**	45.31±3.11^**	54.46±3.36^**	26.55±2.94^**

Group	Inhibitory rate of proliferation （η/%）						IC₅₀ value[ρ_B/(mg·L^-1)]
Group	1.25 mg·L^-1 DDP	2.50 mg·L^-1 DDP	5.00 mg·L^-1 DDP	10.00 mg·L^-1 DDP	20.00 mg·L^-1 DDP	40.00 mg·L^-1 DDP	IC₅₀ value[ρ_B/(mg·L^-1)]
A549+M0	2.25±0.23	7.35±0.41	15.20±0.75	44.68±1.85	56.50±1.33	62.95±2.98	18.21±0.79
A549+M2	2.07±0.16	4.17±1.89^*	9.21±1.14^**	33.76±2.09^**	46.93±2.34^**	53.07±1.55^**	27.03±1.39^**
A549+M2+BAY11-7082	2.19±0.19	6.43±0.78^△	13.82±2.20^△	40.50±1.64^△	53.07±1.52^△	56.85±1.39^△	21.80±0.71^△

Promotive effect of M2 macrophages on epithelial-mesenchymal transition and cisplatin resistance in non-small cell lung cancer A549 cells by regulating NF-κB signaling pathway

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