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

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.