Notoginsenoside R1 Attenuates Breast Cancer Progression by Targeting CCND2 and YBX3
Breast cancer (BC) remains one of the most prevalent malignancies among women worldwide, with an increasing incidence rate and significant challenges in treatment, especially for metastatic or relapsed cases. Despite advancements in therapeutic strategies, the survival rates for BC patients, particularly those with advanced stages, remain suboptimal. The complexity of BC carcinogenesis necessitates the exploration of novel therapeutic agents and the elucidation of their underlying mechanisms. Notoginsenoside R1 (NGR1), a bioactive compound extracted from Panax notoginseng, has shown potential in various pharmacological activities, including anti-inflammatory, antioxidant, and anti-tumor effects. This study investigates the role of NGR1 in BC progression, focusing on its impact on cell proliferation, migration, invasion, angiogenesis, apoptosis, and the cell cycle, as well as its molecular mechanisms involving CCND2 and YBX3.
NGR1 Inhibits Proliferation, Migration, Invasion, and Angiogenesis in BC Cells
The study begins by determining the optimal concentration and treatment duration of NGR1 using the cell counting kit-8 (CCK-8) assay. The results reveal that NGR1 significantly inhibits the proliferation of Michigan Cancer Foundation-7 (MCF-7) cells in a dose- and time-dependent manner. The 50% growth inhibitory concentration (IC50) for MCF-7 cells at 24 hours is found to be 148.9 mmol/L. Colony formation assays further confirm that NGR1 treatment reduces the long-term proliferation ability of both MCF-7 and MDA-MB-231 cells, two commonly used BC cell lines.
To assess the effect of NGR1 on cell migration and invasion, Transwell assays are performed. The results demonstrate that NGR1 treatment markedly reduces the migration and invasion capabilities of BC cells. Additionally, capillary tube formation assays are conducted to evaluate the impact of NGR1 on angiogenesis, a critical process in tumor growth and metastasis. The findings indicate that NGR1 significantly inhibits the formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs), suggesting its anti-angiogenic potential.
NGR1 Induces Cell Cycle Arrest and Apoptosis in BC Cells
Flow cytometry analysis is employed to investigate the effect of NGR1 on the cell cycle. The results show that NGR1 treatment leads to a dose-dependent increase in the proportion of MCF-7 cells arrested in the G0/G1 phase. Specifically, the proportions of cells in the G0/G1 phase are 36.94% ± 6.78%, 45.06% ± 5.60%, and 59.46% ± 5.60% in the control group, 75 mmol/L, and 150 mmol/L NGR1-treated groups, respectively. This suggests that NGR1 induces cell cycle arrest, thereby inhibiting BC cell proliferation.
The study also examines the effect of NGR1 on apoptosis using the TUNEL assay and Western blotting. TUNEL staining reveals a significant increase in apoptotic cells in NGR1-treated groups compared to the control group. Western blotting analysis further confirms that NGR1 treatment upregulates the expression of pro-apoptotic proteins, including Bax, cleaved caspase-3, and cleaved caspase-9, indicating that NGR1 induces apoptosis through the mitochondrial pathway.
NGR1 Downregulates CCND2 and YBX3 Expression in BC Cells
To explore the molecular mechanisms underlying the anti-cancer effects of NGR1, bioinformatics analysis is performed using microarray datasets from the Gene Expression Omnibus (GEO) database. The analysis identifies 141 downregulated and 114 upregulated genes in NGR1-treated BC cells compared to the control group. Among the top ten downregulated genes, CCND2 and YBX3 are of particular interest due to their established roles as oncogenes in various cancers.
Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) are used to validate the downregulation of CCND2 and YBX3 by NGR1 treatment. The results demonstrate that NGR1 significantly reduces the expression of CCND2 and YBX3 in a dose- and time-dependent manner in MCF-7 cells. This suggests that NGR1 exerts its anti-cancer effects, at least in part, by targeting these oncogenes.
YBX3 Activates the KRAS/PI3K/Akt Signaling Pathway in BC Cells
To further elucidate the role of YBX3 in BC progression, chromatin immunoprecipitation sequencing (ChIP-seq) is performed to identify the genomic regions bound by YBX3 in MCF-7 cells. The analysis reveals that YBX3 binds to the promoter region of KRAS, a well-known activator of the phosphatidylinositol 3-phosphate kinase (PI3K)/protein kinase B (Akt) signaling pathway. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis confirms that the PI3K/Akt pathway is the most enriched pathway associated with YBX3.
ChIP-qPCR and qRT-PCR assays further validate that YBX3 regulates the expression of KRAS by binding to its promoter region. Western blotting analysis shows that NGR1 treatment downregulates the expression of KRAS, phosphorylated PI3K (p-PI3K), and phosphorylated Akt (p-Akt) in a dose-dependent manner, indicating that NGR1 inhibits the KRAS/PI3K/Akt signaling pathway by targeting YBX3.
Functional assays, including colony formation and Transwell assays, are conducted to assess the impact of NGR1 on BC cells after YBX3 knockdown. The results show that NGR1 has minimal effect on BC cell proliferation, migration, and invasion following YBX3 knockdown, suggesting that NGR1 primarily exerts its anti-cancer effects through the regulation of YBX3.
Discussion and Conclusion
This study provides comprehensive evidence that NGR1 attenuates BC progression by targeting CCND2 and YBX3. NGR1 treatment inhibits the proliferation, migration, invasion, and angiogenesis of BC cells, induces cell cycle arrest and apoptosis, and downregulates the expression of oncogenes CCND2 and YBX3. Furthermore, the study reveals that YBX3 activates the KRAS/PI3K/Akt signaling pathway by binding to the KRAS promoter, and NGR1 inhibits this pathway by downregulating YBX3.
The findings highlight the potential of NGR1 as a novel therapeutic agent for BC. By targeting multiple oncogenic pathways, NGR1 offers a multifaceted approach to inhibiting BC progression. Further studies are warranted to explore the clinical efficacy of NGR1 in BC patients and to investigate its potential synergistic effects with existing therapies.
In conclusion, this study elucidates the molecular mechanisms by which NGR1 exerts its anti-cancer effects in BC, providing a strong rationale for its development as a therapeutic agent. The identification of CCND2 and YBX3 as key targets of NGR1 opens new avenues for the development of targeted therapies for BC.
doi.org/10.1097/CM9.0000000000001328
Was this helpful?
0 / 0