基于mTOR/HIF-1<i>α</i>/VEGF信号通路探讨菟丝子醇提物对4T1乳腺癌细胞增殖的抑制作用

宋辉; 黄晓俏; 孙向明; 胡扬; 单文猜; 汤静; 李文兰

doi:10.13386/j.issn1002-0306.2024050320

基于mTOR/HIF-1α/VEGF信号通路探讨菟丝子醇提物对4T1乳腺癌细胞增殖的抑制作用

Inhibition of Proliferation of 4T1 Breast Cancer Cells by Cuscuta Alcohol Extract Based on mTOR/HIF-1α/VEGF Signaling Pathway

摘要

摘要: 目的：通过体内外实验，探讨菟丝子醇提物对4T1乳腺癌细胞增殖的抑制作用及机制。方法：体外采用MTT、划痕、Transwell、流式细胞术实验分别研究菟丝子醇提物对4T1乳腺癌细胞增殖、迁移、侵袭、凋亡的影响；体内建立4T1荷瘤小鼠模型，菟丝子醇提物灌胃给药14 d后，ELISA试剂盒检测小鼠血清中肿瘤坏死因子-alpha（Tumor necrosis factor-alpha，TNF-α）、白介素2（Interleukin-2，IL-2）、白介素10（Interleukin-10，IL-10）的浓度。同时将模型组、菟丝子醇提物高剂量组瘤组织进行转录组测序分析，筛选差异基因，进行GO和KEGG富集分析，采用Western Blot法对mTOR/HIF-1α/VEGF信号通路进行实验验证。结果：与空白组相比，菟丝子醇提物（400、800、1200 μg/mL）能够显著抑制4T1乳腺癌细胞增殖、迁移、侵袭（P<0.05，P<0.01），促进细胞凋亡（P<0.01）。体内实验显示，与模型组相比，菟丝子醇提物（6.24、12.48 g/kg）可显著提高小鼠血清中TNF-α、IL-2、IL-10（P<0.05，P<0.01）的含量；转录组筛选出差异基因512个，其中上调154个，下调358个；GO富集分析共富集到1870个条目；KEGG富集分析富集到Cytokine-cytokine receptor interaction、HIF-1 signaling pathway、IL-17 signaling pathway等信号通路；菟丝子醇提物能够降低瘤组织中p-mTOR、HIF-1α、p-RPS6K、p-P70S6K、VEGF、VEGFR2蛋白表达（P<0.05，P<0.01），具有剂量依赖性。结论：菟丝子醇提物可抑制4T1乳腺癌细胞增殖，升高小鼠血清中炎症因子表达，其机制与调控mTOR/HIF-1α/VEGF信号通路有关。

Abstract: Objective: To examine the inhibitory effects and mechanisms of Cuscuta alcohol extract on the proliferation of 4T1 breast cancer cells, both in vitro and in vivo. Methods: MTT, scratch, transwell, and flow cytometric assays were used to examine the effects of Cuscuta alcohol extract on the proliferation, migration, invasion, and apoptosis of 4T1 breast cancer cells in vitro. Additionally, an in vivo 4T1 tumor-bearing mouse model was established. ELISA kits measured TNF-α, IL-2, and IL-10 levels in 4T1 tumor-bearing mice after 14 days of intragastric Cuscuta alcohol extract administration. Concurrently, tumor tissues from both the model mice and the high-dose Cuscuta alcohol extract group were analyzed using transcriptome sequencing to identify differentially expressed genes. GO and KEGG enrichment analyses were conducted, and the Western Blot assays were performed to verify the expression of the mTOR/HIF-1α/VEGF signaling pathway. Results: Cuscuta alcohol extract (400, 800 and 1200 μg/mL) significantly reduced the proliferation, migration, and invasion of 4T1 breast cancer cells (P<0.05, P<0.01), and enhanced their apoptosis (P<0.01), compared to blank control. In the in vivo model, Cuscuta alcohol extract (6.24 and 12.48 g/kg) significantly increased the serum levels of TNF-α, IL-2, and IL-10 in mice relative to model group (P<0.05, P<0.01). Transcriptome sequencing identified 512 differentially expressed genes, including 154 upregulated and 358 downregulated genes. GO enrichment analysis identified 1870 items, while KEGG enrichment analysis enriched for cytokine receptor interactions, HIF-1 signalling pathway, IL-17 signalling pathway and other signalling pathways. The extract reduced the p-mTOR, HIF-1α, p-RPS6K, p-P70S6K, VEGF, and VEGFR2 protein expression levels in tumor tissues in a dose-dependent manner (P<0.05, P<0.01). Conclusions: The alcohol extract of Cuscuta inhibits the proliferation of 4T1 breast cancer cells and increase the serum expression levels of inflammatory factors in mice. Its antitumor mechanism is related to the modulation of the mTOR/HIF-1α/VEGF signaling pathway.