肿瘤相关巨噬细胞通过激活IGF-1R信号通路诱导三阴性乳腺癌细胞对白蛋白紫杉醇耐药的研究

doi:10.11958/20230438

天津医药 ›› 2023, Vol. 51 ›› Issue (11): 1158-1163.doi: 10.11958/20230438

肿瘤相关巨噬细胞通过激活IGF-1R信号通路诱导三阴性乳腺癌细胞对白蛋白紫杉醇耐药的研究

刘淑娟¹^,²(), 刘梦莹¹, 苏乌云³, 窦佳³, 王薇³^,^△()

1.内蒙古医科大学第一临床医学院（邮编010059）
2.内蒙古阿荣旗人民医院内科
3.内蒙古医科大学附属医院肿瘤内科

收稿日期:2023-03-27 修回日期:2023-05-23 出版日期:2023-11-15 发布日期:2023-11-07
通讯作者: ^△E-mail：656225284@qq.com
作者简介:刘淑娟（1984），女，硕士在读，主要从事肿瘤诊治及机制方面研究。E-mail：33562569@qq.com
基金资助:
北京科创医学发展基金会项目(KC2021-JX-0044-9);内蒙古自治区卫生健康科技计划项目(202202191)

Investigation on the determination of tumor-associated macrophages inducing the drug resistance of albumin-bound paclitaxel in triple-negative breast cancer cells by activating IGF-1R signaling pathway

LIU Shujuan¹^,²(), LIU Mengying¹, SU Wuyun³, DOU Jia³, WANG Wei³^,^△()

1. The First Clinical College of Inner Mongolia Medical University, Hohhot 010059, China
2. Department of Internal Medicine, Inner Mongolia A Rong Qi People's Hospital
3. Department of Oncology, Affiliated Hospital of Inner Mongolia Medical University

Received:2023-03-27 Revised:2023-05-23 Published:2023-11-15 Online:2023-11-07
Contact: ^△E-mail：656225284@qq.com

刘淑娟, 刘梦莹, 苏乌云, 窦佳, 王薇. 肿瘤相关巨噬细胞通过激活IGF-1R信号通路诱导三阴性乳腺癌细胞对白蛋白紫杉醇耐药的研究[J]. 天津医药, 2023, 51(11): 1158-1163.

LIU Shujuan, LIU Mengying, SU Wuyun, DOU Jia, WANG Wei. Investigation on the determination of tumor-associated macrophages inducing the drug resistance of albumin-bound paclitaxel in triple-negative breast cancer cells by activating IGF-1R signaling pathway[J]. Tianjin Medical Journal, 2023, 51(11): 1158-1163.

摘要/Abstract

摘要：

目的探讨肿瘤相关巨噬细胞（TAM）对三阴性乳腺癌（TNBC）细胞白蛋白紫杉醇（Nab-PTX）化疗敏感性的影响及作用机制。方法构建并鉴定TAM模型；通过Transwell小室共培养法建立TAM与TNBC细胞系MDA-MB-231细胞共培养模式，分为对照组（MDA-MB-231细胞及空白小室）、Nab-PTX组（MDA-MB-231细胞、空白小室及0.5 nmol/L Nab-PTX）、TAM组（MDA-MB-231细胞、含M2型巨噬细胞的小室）、TAM+Nab-PTX组（MDA-MB-231细胞、含M2型巨噬细胞的小室及0.5 nmol/L Nab-PTX）、胰岛素样生长因子1受体（IGF-1R）抑制剂组（MDA-MB-231细胞、含M2型巨噬细胞的小室、0.5 nmol/L Nab-PTX及4 nmol/L IGF-1R抑制剂Linsitinib）；CCK-8法检测各组细胞增殖情况；流式细胞术检测细胞凋亡；实时荧光定量PCR（qPCR）检测多药耐药蛋白（MDR）1和胱天蛋白酶（Caspase）-3 mRNA水平；Western blot检测IGF-1R信号通路关键蛋白表达。结果 THP-1细胞经诱导分化为M2型巨噬细胞；与Nab-PTX组相比，TAM+Nab-PTX组细胞增殖水平升高，凋亡率降低（P<0.01），MDR1 mRNA表达升高，Caspase-3 mRNA表达降低（P<0.05），IGF-1R信号通路关键蛋白激活（P<0.01）；与TAM+Nab-PTX组相比，IGF-1R抑制剂组细胞增殖水平降低，凋亡率升高，MDR1 mRNA表达降低，Caspase-3 mRNA的表达增高，IGF-1R信号通路关键蛋白的表达降低（P<0.01）。结论 TAM可能通过激活IGF-1R信号通路诱导TNBC细胞对Nab-PTX耐药。

Abstract:

Objective To investigate the effect of tumor-associated macrophages (TAM) on the chemosensitivity of triple-negative breast cancer (TNBC) cells to albumin-bound paclitaxel (Nab-PTX). Methods TAM model was constructed and identified. TNBC cell line MDA-MB-231 was established by Transwell cell co-culture method. They were divided into the control group (MDA-MB-231 cells and blank chamber), the Nab-PTX group (MDA-MB-231 cells, blank chamber and 0.5 nmol/L Nab-PTX), the TAM group (MDA-MB-231 cells, containing M2-type macrophages), the TAM+Nab-PTX group (MDA-MB-231 cells, cells containing M2-type macrophages and 0.5 nmol/L Nab-PTX) and the insulin-like growth factor 1 receptor (IGF-1R) inhibitor group (MD-MB-231 cells, macrophage compartment containing M2-type, 0.5 nmol/L Nab-PTX and 4 nmol/L IGF-1R inhibitor Linsitinib). The cell survival rate of each group was determined by CCK-8 method. Flow cytometry was used to detect cell apoptosis. The mRNA levels of multidrug resistant protein (MDR) 1 and Caspase-3 were detected by real-time quantitative PCR (qPCR). The key proteins of IGF-1R signaling pathway were detected by Western blot assay. Results THP-1 cells were induced to differentiate into M2 macrophages. Compared with the Nab-PTX group, the cell proliferation rate was increased and apoptosis rate was decreased in the TAM+Nab-PTX group (P<0.01). The mRNA expression of MDR1was increased and Caspase-3 mRNA was decreased (P<0.05). The key protein of IGF-1R signaling pathway was activated (P<0.01). Compared with the TAM+Nab-PTX group, the proliferation rate was decreased and apoptosis rate of cells was increased in the IGF-1R inhibitor group. The mRNA expression of MDR1 was decreased, the mRNA expression of Caspase3 was increased, and the expression of key proteins in IGF-1R signaling pathway was decreased (P<0.01). Conclusion TAM may induce resistance of TNBC cells to Nab-PTX by activating IGF-1R signaling pathway.

Key words: tumor-associated macrophages, triple negative breast neoplasms, albumin-bound paclitaxel, drug tolerance, receptor, IGF type 1

中图分类号:

R737.9

图/表 10

图1 TAM表面标志物CD14、CD206的表达特征

Fig.1 The characteristics of cell surface markers CD14 and CD206 in TAM

表1 4组MDA-MB-231细胞不同时间点增殖情况比较（n=3，OD490，$\bar{x}±s$）

Tab.1 Comparison of proliferation of MDA-MB-231 cells at different time points between four groups

组别	24 h	48 h	72 h
对照组	0.470±0.002	0.630±0.001	0.770±0.004
Nab-PTX组	0.440±0.002^a	0.540±0.004^a	0.620±0.003^a
TAM组	0.510±0.002^b	0.720±0.001^b	0.930±0.006^b
TAM+Nab-PTX组	0.460±0.002^b	0.610±0.003^b	0.760±0.006^b
F	716.879^＊＊	2 090.309^＊＊	2 276.647^＊＊

图2 TAM对Nab-PTX干预MDA-MB-231细胞凋亡的影响

Fig.2 Effects of TAM on apoptosis of MDA-MB-231 cells induced by Nab-PTX

表2 2组MDA-MB-231细胞不同时间点增殖情况比较（n=3，OD490，$\bar{x}±s$）

Tab.2 Comparison of proliferation of MDA-MB-231 cells at different time points between two groups

组别	24 h	48 h	72 h
TAM+Nab-PTX组	0.460±0.002	0.610±0.003	0.760±0.006
IGF-1R抑制剂组	0.420±0.001	0.500±0.003	0.560±0.003
t	19.290^＊＊	42.832^＊＊	45.987^＊＊

图3 IGF-1R抑制剂对TAM共培养MDA-MB-231细胞凋亡的影响

Fig.3 Effects of IGF-1R inhibitors on apoptosis of MDA-MB-231 cells co-cultured with TAM

表3 4组MDA-MB-231细胞MDR1和Caspase-3 mRNA表达的比较（n=3，$\bar{x}±s$）

Tab.3 Comparison of MDR1 mRNA and Caspase-3 mRNA expression between four groups of MDA-MB-231 cells

组别	MDR1	Caspase-3
对照组	1.00±0.03	1.00±0.02
Nab-PTX组	0.29±0.01^a	2.03±0.04^a
TAM组	1.72±0.61	0.58±0.40
TAM+Nab-PTX组	0.83±0.03^b	1.20±0.02^b
F	934.279^＊＊	21.360^＊＊

表4 各组MDA-MB-231细胞内p-IGF-1R、p-AKT、p-ERK蛋白表达水平比较（n=6，$\bar{x}±s$）

Tab.4 Comparison of protein expression levels of p-IGF-1R, p-AKT and p-ERK between MDA-MB-231 cells of each group

组别	p-IGF-1R/ IGF-1R	p-AKT/AKT	p-ERK/ERK
对照组	1.030±0.001	0.780±0.001	0.980±0.007
Nab-PTX组	0.400±0.002^a	0.460±0.003^a	0.570±0.002^a
TAM组	1.300±0.004^ab	0.960±0.002^ab	1.320±0.002^ab
TAM+Nab-PTX组	0.980±0.008^b	0.740±0.004^b	0.770±0.013^b
F	17 108.381^＊＊	13 656.675^＊＊	5 499.251^＊＊

图4 Western blot检测各组MDA-MB-231细胞内IGF-1R、p-IGF-1R、AKT、p-AKT、ERK、p-ERK蛋白表达

Fig.4 The protein expression levels of IGF-1R, p-IGF-1R, AKT, p-AKT, ERK and p-ERK in each group

表5 2组细胞内p-IGF-1R、p-AKT、p-ERK比较（n=6，$\bar{x}±s$）

Tab.5 Comparison of p-IGF-1R, p-AKT and p-ERK rations between the two groups of cells

组别	p-IGF-1R/IGF-1R	p-AKT/AKT	p-ERK/ERK
TAM+Nab-PTX组	1.280±0.014	1.120±0.020	0.860±0.006
IGF-1R抑制剂组	0.500±0.009	0.450±0.002	0.420±0.001
t	77.249^＊＊	57.151^＊＊	119.965^＊＊

图5 TAM+Nab-PTX组和IGF-1R抑制剂组蛋白表达印迹图

Fig.5 Western blot assay of the TAM+Nab-PTX group and the IGF-1R inhibitor group

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肿瘤相关巨噬细胞通过激活IGF-1R信号通路诱导三阴性乳腺癌细胞对白蛋白紫杉醇耐药的研究

Investigation on the determination of tumor-associated macrophages inducing the drug resistance of albumin-bound paclitaxel in triple-negative breast cancer cells by activating IGF-1R signaling pathway

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