机构地区：[1]暨南大学药学院,广东广州510632 [2]暨南大学附属第一医院(广州华侨医院),广东广州510630 [3]暨南大学中医学院,广东广州510632 [4]暨南大学肿瘤研究所,广东广州510632 [5]广东省中医药信息化重点实验室,广东广州510632
出 处：《中国病理生理杂志》2024年第5期796-805,共10页Chinese Journal of Pathophysiology
基 金：Supported by the National Natural Science Foundation of China (No. 81603342);the Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Informatization (No. 2021B1212040007);the Guangdong Basic and Applied Basic Re-search Foundation (No. 2022A1515012641;No. 2024A1515012948);the Guangdong Provincial Bureau of Traditional Chinese Medi-cine Research Project (No. 20221107);the Guangzhou Science and Technology Projects (No. 2024A03J0154;No. 2023B01J1004);the Foshan “Summit Plan” of Building High-Level Hospitals。
摘 要：目的:探讨原人参三醇(PPT)对耐紫杉醇(PTX)人乳腺癌MDA-MB-231细胞(MB231-PR细胞)耐药性的影响。方法:构建MB231-PR耐药细胞作为细胞模型。以不同浓度的PPT作用于MB231-PR细胞一定时间。用Cell Titer-Glo试剂和集落形成实验测定MB231-PR细胞和MDA-MB-231亲本细胞(MB231-PT细胞)的活力。用流式细胞术测定PPT和PTX联合使用后细胞sub-G1期的变化。Western blot法用于评估细胞凋亡相关蛋白cleaved caspase-3、cleaved多腺苷二磷酸核糖聚合酶(PARP)、survivin、B细胞淋巴瘤2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平。萤光素酶报告基因实验和免疫荧光染色用于检测核因子κB(NF-κB)活性。ELISA检测白细胞介素6(IL-6)和IL-8蛋白表达水平。q PCR检测IL-6、IL-8、CXC趋化因子配体1(CXCL1)、CC趋化因子配体2(CCL2)、CD44、NANOG、八聚体结合转录因子4(OCT4)、性别决定区Y框蛋白2(SOX2)和醛脱氢酶1(ALDH1)的m RNA表达水平。肿瘤球形成实验用于评估干细胞特性。结果:(1)PPT以剂量依赖性方式显著降低MB231-PR细胞活力(P<0.01),半数抑制浓度(IC50)为18.17μmol/L。PPT和PTX联合治疗后,MB231-PR细胞活力显著降低(P<0.01),诱导sub-G1期的积累(P<0.01),Bax/Bcl-2的比值上调(P<0.01),cleaved caspase-3和cleaved PARP蛋白水平升高(P<0.05),survivin蛋白表达水平下降(P<0.01)。(2)PPT与PTX联合治疗后,炎症细胞因子(IL-6、IL-8、CXCL1和CCL2)和肿瘤干细胞标志物(OCT4、SOX2、NANOG、ALDH1和CD44)的m RNA表达水平下调(P<0.05),IL-6和IL-8的蛋白表达水平降低,显著抑制MB231-PR细胞NF-κB的活性(P<0.05),并损害MB231-PR细胞的肿瘤球体生长(P<0.05)。(3)PTX处理诱导了核p-p65的表达,这种作用可以被PPT减弱。结论:PPT联合PTX可通过抑制炎症细胞因子和肿瘤干细胞来降低MB231-PR细胞对紫杉醇的耐药性。AIM:To investigate the effect of protopanaxatriol(PPT)on the drug resistance of paclitaxel(PTX)-resistant human breast cancer MDA-MB-231 cells(MB231-PR cells).METHODS:The MB231-PR cells were constructed as cell models.They were treated with PPT,and incubated for a certain period of time according to the experi-mental settings.CellTiter-Glo was used to determine the viability of MB231-PR cells and MDA-MB-231 parental cells(MB231-PT cells).The change of sub-G1 phase was detected by flow cytometry.Western blot was used to evaluate the apoptosis-related proteins,such as cleaved caspase-3,cleaved poly(ADP-ribose)polymerase(PARP),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax)and survivin.The activity of nuclear factor-κB(NF-κB)was detected by lu-ciferase reporter assay and immunofluorescence assay.The mRNA expression levels of interleukin-6(IL-6),IL-8,chemo-kine CXC motif ligand 1(CXCL1),chemokine CC motif ligand 2(CCL2),CD44,NANOG,octamer-binding transcrip-tion factor 4(OCT4),sex-determining region Y-box 2(SOX2)and aldehyde dehydrogenase 1(ALDH1)were detected by qPCR.The protein levels of IL-6 and IL-8 were measured by ELISA.Tumor sphere formation assay was used to evaluate the characteristics of stem cells.RESULTS:(1)The viability of MB231-PR cells was suppressed by PPT treatment in a dose-dependent manner compared with MB231-PT cells(P<0.01).Besides,the viability of MB231-PR cells was de-creased after combined treatment with PPT and PTX(P<0.01),the accumulation of sub-G1 phase was induced(P<0.01),the ratio of Bax/Bcl-2 was elevated(P<0.01),and the protein levels of survivin,cleaved PARP and cleaved cas-pase-3 were increased(P<0.05).(2)After PPT treatment combined with PTX,the mRNA expression of inflammatory cy-tokines(IL-6,IL-8,CXCL1 and CCL2)and cancer stem cell-related markers(OCT4,SOX2,NANOG,ALDH1 and CD44)was reduced(P<0.05),and the protein levels of IL-6 and IL-8 were decreased(P<0.01).The activity of NF-κB in MB231-PR cells was suppressed(P<0.05),and the growth of tumor spheres from MB231-PR cells
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