摘要目的：三阴性乳腺癌（triple-negative breast cancer,TNBC）具有耐药性强的特点,因此,患者会对临床上最常用的化疗药物紫杉醇敏感性降低,从而导致治疗效果不佳。谷氨酰胺合成酶（glutamine synthetase,GS）是合成谷氨酰胺所需的关键代谢酶,其高表达可诱导卵巢癌细胞对化疗药物降低敏感性。但是,GS是否影响TNBC细胞对紫杉醇的敏感性及其可能机制仍不清楚。本研究的目的是探讨GS是否影响TNBC细胞对紫杉醇的敏感性及其机制。方法：使用质粒转染和慢病毒感染分别构建过表达和敲低GS的TNBC细胞系。通过Western blot、细胞活力检测、克隆形成实验和细胞凋亡检测分析GS在TNBC细胞中对紫杉醇敏感性的影响。使用qRT-PCR检测过表达GS的细胞中药物代谢酶、药物转运蛋白、抗凋亡蛋白的表达。结果：GS在乳腺癌细胞中高表达,GSlow（MDA-MB-231）细胞对紫杉醇的敏感性高于GShigh（MDA-MB-468）细胞。过表达GS使TNBC细胞对紫杉醇的敏感性降低、克隆能力增强、抗凋亡能力增加,敲低GS可以增加紫杉醇对TNBC细胞的杀伤效果;过表达GS的TNBC细胞中,抗凋亡蛋白BAG3的表达显著升高。结论：在TNBC细胞中,高表达GS促进细胞增殖,使TNBC细胞凋亡减少,克隆形成能力增强;GS可能是通过上调BAG3的表达增强抗凋亡能力从而降低TNBC细胞对紫杉醇的敏感性。
Abstract：Objective Triple-negative breast cancer (TNBC) is characterized by strong drug resistance. So patients will be less sensitive to paclitaxel, which is the most commonly used chemotherapeutic drug in the clinic, resulting in poor treatment efficacy. Glutamine Synthetase (GS) is a key metabolic enzyme needed to synthesize glutamine, and its high expression can induce ovarian cancer cells to reduce their sensitivity to chemotherapeutic drugs. However, whether GS affects the sensitivity of TNBC cells to paclitaxel and its possible mechanism are still unclear. The purpose of this study was to investigate whether GS affects the sensitivity of TNBC cells to paclitaxel and its mechanism. Methods TNBC cell lines overexpressing and knocking down GS were constructed by plasmid transfection and lentivirus infection, respectively. The effect of GS on paclitaxel sensitivity in TNBC cells was analyzed by Western blot analysis, cell viability assay, colony formation assay and apoptosis detection. qRT-PCR was used to detect the expression of drug metabolic enzymes, drug transporters, and anti-apoptotic proteins in cells overexpressing GS. Results GS was highly expressed in breast cancer cells. The sensitivity of GSlow cells (MDA-MB-231) to paclitaxel was higher than that of GShigh cells (MDA-MB-468). Overexpression of GS decreased the sensitivity of TNBC cells to paclitaxel, enhanced the ability of clone formation and anti-apoptosis, knocking down GS could increase the killing effect of paclitaxel on TNBC cells, and the expression of anti-apoptotic protein BAG3 was significantly increased in TNBC cells overexpressing GS. Conclusions In TNBC cells, the overexpression of GS promotes cell proliferation, reduces apoptosis, and enhances the ability of clone formation in TNBC cells. The anti-apoptotic effect of GS was mediated by up-regulating the expression of BAG3, thus reducing the sensitivity of TNBC cells to paclitaxel.
[1] Ferlay J, Colombet M, Soerjomataram I, et al.Cancer statistics for the year 2020: An overview[J]. Int J Cancer, 2021. [2] Li Y, Zhan Z, Yin X, et al.Targeted Therapeutic Strategies for Triple-Negative Breast Cancer[J]. Front Oncol, 2021, 11: 731535. [3] Jiang Y Z, Ma D, Suo C, et al. Genomic and Transcriptomic Landscape of Triple-Negative Breast Cancers: Subtypes and Treatment Strategies [J]. Cancer Cell, 2019, 35(3): 428-440. e5. [4] Choupani E, Mahmoudi Gomari M, Zanganeh S, et al.Newly Developed Targeted Therapies Against the Androgen Receptor in Triple-Negative Breast Cancer: A Review[J]. Pharmacol Rev, 2023, 75(2): 309-327. [5] Chang A, Botteri E, Gillis R D, et al. Beta-blockade enhances anthracycline control of metastasis in triple-negative breast cancer [J]. Sci Transl Med, 2023, 15(693): eadf1147. [6] Denkert C, Liedtke C, Tutt A, et al.Molecular alterations in triple-negative breast cancer-the road to new treatment strategies[J]. Lancet, 2017, 389(10087): 2430-2442. [7] Derakhshan F, Reis-Filho J S. Pathogenesis of Triple-Negative Breast Cancer[J]. Annu Rev Pathol, 2022, 17: 181-204. [8] Krajewski W W, Collins R, Holmberg-Schiavone L, et al.Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design[J]. J Mol Biol, 2008, 375(1): 217-228. [9] Marquez J, Alonso F J, Mates J M, et al.Glutamine Addiction In Gliomas[J]. Neurochem Res, 2017, 42(6): 1735-1746. [10] Wise D R, Thompson C B.Glutamine addiction: a new therapeutic target in cancer[J]. Trends Biochem Sci, 2010, 35(8): 427-433. [11] Xuan D T M, Wu C C, Wang W J, et al. Glutamine synthetase regulates the immune microenvironment and cancer development through the inflammatory pathway[J]. Int J Med Sci, 2023, 20(1): 35-49. [12] Wang Y, Fan S, Lu J, et al.GLUL Promotes Cell Proliferation in Breast Cancer[J]. J Cell Biochem, 2017, 118(8): 2018-2025. [13] Guo J, Satoh K, Tabata S, et al.Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells[J]. BMC Cancer, 2021, 21(1): 174. [14] Modi A, Roy D, Sharma S, et al.ABC transporters in breast cancer: their roles in multidrug resistance and beyond[J]. J Drug Target, 2022, 30(9): 927-947. [15] Das C K, Linder B, Bonn F, et al.BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells[J]. Neoplasia, 2018, 20(3): 263-279. [16] Wein L, Loi S. Mechanisms of resistance of chemotherapy in early-stage triple negative breast cancer (TNBC) [J]. Breast, 2017, 34 Suppl 1: S27-S30. [17] Fu S, Li Z, Xiao L, et al. Glutamine Synthetase Promotes Radiation Resistance via Facilitating Nucleotide Metabolism and Subsequent DNA Damage Repair [J]. Cell Rep, 2019, 28(5): 1136-1143. e4. [18] Zhang J, Pavlova N N, Thompson C B.Cancer cell metabolism: the essential role of the nonessential amino acid, glutamine[J]. EMBO J, 2017, 36(10): 1302-1315. [19] Morotti M, Zois C E, El-Ansari R, et al.Increased expression of glutamine transporter SNAT2/SLC38A2 promotes glutamine dependence and oxidative stress resistance, and is associated with worse prognosis in triple-negative breast cancer[J]. Br J Cancer, 2021, 124(2): 494-505. [20] Muthu M, Kumar R, Syed Khaja A S, et al. GLUL Ablation Can Confer Drug Resistance to Cancer Cells via a Malate-Aspartate Shuttle-Mediated Mechanism[J]. Cancers (Basel), 2019, 11(12): 1945. [21] Dan V M, Raveendran R S, Baby S.Resistance to Intervention: Paclitaxel in Breast Cancer[J]. Mini Rev Med Chem, 2021, 21(10): 1237-1268. [22] Rajput S, Volk-Draper L D, Ran S. TLR4 is a novel determinant of the response to paclitaxel in breast cancer[J]. Mol Cancer Ther, 2013, 12(8): 1676-1687.
2023, Vol. 20 
