丝胶靶向Akt调控PI3K/Akt/mTOR信号通路诱导三阴乳腺癌细胞自噬

Sericin targets akt to regulate the PI3K/Akt/mTOR signaling pathway to induce autophagy in triple-negative breast cancer cells

摘要

目的 探讨丝胶对PI3K/Akt/mTOR信号通路的调控及其对三阴性乳腺癌（TNBC) MDA-MB-468细胞自噬的诱导作用,并阐明作用靶点。方法 将MDA-MB-468细胞随机分为对照组（不含药物）、实验组（8 mg·mL^-1丝胶）、Akt激活剂组（8 mg·mL^-1丝胶+10 μmol·L^-1 SC79）,分别给予相应药物作用24 h后,采用RT-qPCR法检测各组细胞PI3K、Akt、mTOR、自噬效应蛋白（Beclin1）、自噬相关蛋白5（ATG5）、微管相关轻链蛋白3（LC3）、螯合体1（P62）mRNA表达水平;Western blotting法检测各组细胞PI3K、Akt、p-Akt、mTOR、p-mTOR、Beclin1、ATG5、LC3、P62蛋白表达水平;免疫荧光染色法检测各组细胞LC3、P62蛋白的定位及荧光强度。结果 与对照组相比,实验组MDA-MB-468细胞PI3K、Akt、mTOR、P62 mRNA及PI3K、Akt、p-Akt、mTOR、p-mTOR、P62蛋白水平均显著降低（P<0.05）,Beclin1、ATG5、LC3 mRNA水平及Beclin1、ATG5、LC3Ⅱ/LC3Ⅰ蛋白水平均显著升高（P<0.05）;与实验组相比,Akt激活剂组MDA-MB-468细胞PI3K、Akt、mTOR、P62 mRNA及PI3K、Akt、p-Akt、mTOR、p-mTOR、P62蛋白水平均显著升高（P<0.05）,Beclin1、ATG5、LC3 mRNA水平及Beclin1、ATG5、LC3Ⅱ/LC3Ⅰ蛋白水平均显著降低（P<0.05）。结论 丝胶对MDA-MB-468细胞自噬的诱导作用可通过以Akt为靶点调控PI3K/Akt/mTOR信号通路的转导来实现。

Abstract

Objective To investigate the induction of autophagy of MDA-MB-468 cells in triple-negative breast cancer (TNBC) by sericin through regulating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, and to clarify the target of action. Methods MDA-MB-468 cells were randomly divided into a control group (without drugs), an experimental group (8 mg·mL^-1 sericin), and an Akt activator group (8 mg·mL^-1 sericin + 10 μmol·L^-1 SC79). After 24 hours treatment with corresponding drugs, the mRNA expression levels of PI3K, Akt, mTOR, autophagy effector protein (Beclin1), autophagy associated protein 5 (ATG5), microtubule associated light chain protein3 (LC3) and chelator 1 (P62) in cells of each group were detected by RT qPCR. The protein expression levels of PI3K, Akt, p-Akt, mTOR, p-mTOR, Beclin1, ATG5, LC3 and P62 in cells of each group were detected by Western blotting. The localization and fluorescence intensity of LC3 and P62 proteins in cells of each group were detected by immunofluorescence staining. Results Compared with the control group, the mRNA levels of PI3K, Akt, mTOR, and P62 and the protein levels of PI3K, Akt, p-Akt, mTOR, p-mTOR, and P62 in MDA-MB-468 cells of the experimental group were significantly decreased (P<0.05), while the mRNA levels of Beclin1, ATG5, LC3 and the protein levels of Beclin1, ATG5, LC3Ⅱ/LC3Ⅰ were significantly increased (P<0.05). Compared with the experimental group, the mRNA levels of PI3K, Akt, mTOR, P62 and the protein levels of PI3K, Akt, p-Akt, mTOR, p-mTOR, P62 in MDA-MB-468 cells of the Akt activator group were significantly increased (P<0.05), while the mRNA levels of Beclin1, ATG5, LC3 and the protein levels of Beclin1, ATG5, LC3Ⅱ/LC3Ⅰ were significantly decreased (P<0.05). Conclusion The induction effect of sericin on autophagy of MDA-MB-468 cells can be achieved by targeting Akt to regulate the transduction of the PI3K/Akt/mTOR signaling pathway.

关键词

丝胶 / 三阴性乳腺癌 / PI3K/Akt/mTOR信号通路 / 自噬 / MDA-MB-468细胞

Key words

sericin / triple-negative breast cancer / PI3K/Akt/mTOR signaling pathway / autophagy / MDA-MB-468 cells

[1] Nan J, Hu X, Guo B, et al.Inhibition of endoplasmic reticulum stress alleviates triple-negative breast cancer cell viability, migration, and invasion by Syntenin/SOX4/Wnt/β-catenin pathway via regulation of heat shock protein A4[J]. Bioengineered, 2022, 13(4): 10564-10577.
[2] 李粤泷,蔡茂坪,卢燕. 铜死亡及其在乳腺癌中的作用研究进展[J]. 中国医药生物技术,2024,19(4):353-356,369.
[3] 王冠瑛. 分数微积分模型在乳腺疾病诊断中的应用价值[D]. 郑州:河南大学,2023.
[4] Chen Y, Li WX, Wu JH, et al.Does the dose of standard adjuvant chemotherapy affect the triple-negative breast cancer benefit from extended capecitabine metronomic therapy? an exploratory analysis of the SYSUCC-001 trial[J]. Breast Cancer (Dove Med Press), 2024, (16): 223-231.
[5] 陈安莉,沈浩元,舒王. 三阴性乳腺癌新辅助治疗的临床研究进展[J]. 现代肿瘤医学,2023,31(3):566-571.
[6] 张芳. 三阴性乳腺癌新辅助化疗的研究进展[J]. 中国冶金工业医学杂志,2024,41(2):137-138.
[7] Leon-Ferre RA, Goetz MP.Advances in systemic therapies for triple negative breast cancer[J]. BMJ, 2023, 381: e071674.
[8] Devis-Jauregui L, Eritja N, Davis ML, et al.Autophagy in the physiological endometrium and cancer[J]. Autophagy, 2021, 17(5): 1077-1095.
[9] 蒋琦炜,张德宇,石烟祝,等. 细胞自噬与肿瘤发生发展[J]. 军事医学,2021,45(3):234-241.
[10] Zhou J, Jiang Y, Chen H, et al.Tanshinone I attenuates the malignant biological properties of ovarian cancer by inducing apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway[J]. Cell Prolif, 2020, 53(2): e12739.
[11] Xu Z, Han X, Ou D, et al.Targeting PI3K/AKT/mTOR-mediated autophagy for tumor therapy[J]. Appl Microbiol Biotechnol, 2020, 104(2): 575-587.
[12] 张亚亨,李慧,刘雪乐,等. 中药蚕茧壳的药理及临床研究进展[J]. 上海中医药杂志,2020,54(4):102-105.
[13] Niu L, Yang S, Zhao XY, et al.Sericin inhibits MDAMB468 cell proliferation via the PI3K/Akt pathway in triplenegative breast cancer[J]. Mol Med Rep, 2021, 23(2): 140.
[14] Deretic V.Autophagy in inflammation, infection, and immunometabolism[J]. Immunity, 2021, 54(3): 437-453.
[15] Orhon I, Rocchi C, Villarejo-Zori B, et al.Autophagy induction during stem cell activation plays a key role in salivary gland self-renewal[J]. Autophagy, 2022, 18(2): 293-308.
[16] Fleming A, Bourdenx M, Fujimaki M, et al.The different autophagy degradation pathways and neurodegeneration[J]. Neuron, 2022, 110(6): 935-966.
[17] Kocak M, Ezazi Erdi S, Jorba G, et al.Targeting autophagy in disease: established and new strategies[J]. Autophagy, 2022, 18(3): 473-495.
[18] Liu S,Yao S,Yang H, et al.Autophagy: regulator of cell death[J]. Cell Death Dis, 2023, 14(10): 648.
[19] Wang B, Mao J, Wang B, et al.Exosomal miR-1910-3p promotes proliferation,metastasis,and autophagy of breast cancer cells by targeting MTMR3 and activating the NF-κB signaling pathway[J]. Cancer Lett, 2020, 489: 87-99.
[20] Li PP, LI RG, Huang YQ, et al.LncRNA OTUD6B-AS1 promotes paclitaxel resistance in triple negative breast cancer by regulation of miR-26a-5p/MTDH pathway-mediated autophagy and genomic instability[J]. Aging (Albany NY), 2021, 13(21): 24171-24191.
[21] Pareek G, Kundu M.Physiological functions of ULK1/2[J]. J Mol Biol, 2024, 436(15): 168472.
[22] 朱可芯,吴敏,卞芝琳,等. 基于自噬调控探讨平衡阴阳治疗慢性肾脏病理论创新和临床实践[J]. 环球中医药,2024,17(8):1573-1579.
[23] Popelka H, Lahiri V, Hawkins WD, et al.The intrinsically disordered N terminus in Atg12 from yeast is necessary for the functional structure of the protein[J]. Int J Mol Sci, 2023, 24(20): 15036.
[24] 田华,郭连军,刘富,等. 葛根素下调Atg5、LC3Ⅰ/Ⅱ蛋白对缺血再灌注脑损伤小鼠的神经保护作用[J]. 中国临床药理学杂志,2023,39(4):508-512.
[25] Lee Y, Kim J, Kim MS, et al.Coordinate regulation of the senescent state by selective autophagy[J]. Dev Cell, 2021, 56(10): 1512-1525.
[26] 周岚. 益气活血法调控自噬延缓失神经肌萎缩的理论[J]. 光明中医,2024,39(16):3344-3346.
[27] Kageyama S, Gudmundsson SR, Sou YS, et al.p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response[J]. Nat Commun, 2021, 12(1): 16.
[28] Nowosad A, Jeannot P, Callot C, et al.p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth[J]. Nat Cell Biol, 2020, 22(9): 1076-1090.
[29] 刘蒙,黄晓东,韩峥,等. 钙黏蛋白17对结直肠癌细胞增殖和凋亡的影响及其PI3K/AKT/mTOR信号通路调节机制[J]. 吉林大学学报(医学版),2023,49(4):1008-1017.
[30] Barra F, Evangelisti G, Ferro Desideri L, et al.Investigational PI3K/AKT/mTOR inhibitors in development for endometrial cancer[J]. Expert Opin Investig Drugs, 2019, 28(2): 131-142.
[31] 武强,赵冉,葛中宽,等. 细胞自噬治疗肿瘤的研究进展[J]. 中华肿瘤防治杂志,2024,31(16):1029-1036.

基金资助

国家自然科学基金项目(81441133); 2025年承德医学院硕士在读研究生创新能力培养资助项目（CYCXZZ202501）