The anti-cancer effects of silibinin through the modulation of JAK1/STAT3 signaling and inhibition of MMPs levels in triple-negative breast cancer cells

Breast cancer is the second most common type of cancer in US women. The most aggressive subtype is triple-negative breast cancer, which corresponds to 10% and 20% and affects African American women disproportionally. Various natural compounds have been identified as having potential therapeutic applications in breast cancer by targeting the tumor microenvironment. Among these compounds, silibinin, a natural compound found in Silybum marianum, has been shown to exhibit anticancer properties against several cancer types. This work examined the anticancer activity of silibinin in MDA-MB-231 (Caucasian) and MDA-MB-468 (African American) TNBC cells by targeting the JAK/STAT signaling pathways and by measuring matrix metalloproteinase (MMP) levels. The approaches included cytotoxicity analyses, 2D and 3D cell culture assays, RT-PCR, Western analyses, and ELISA to evaluate MMP-2 and MMP-9 protein levels in IFN-gamma- and TNF-alpha-stimulated TNBC cells. Silibinin (0.78-400 μM) caused a decrease in cell viability in both cells, with higher sensitivity in MDA-MB-231 cells (IC₅₀ = 154.23 ± 4.15 μM) compared to MDA-MB-468 cells (IC₅₀ = 175.43 ± 5.87 μM). Antiproliferative activity was observed in both cell lines, with MDA-MB-468 cells exhibiting greater sensitivity over time. Additionally, 3D cellular assays demonstrated silibinin's potency in inhibiting spheroid growth and viability, consistent with the cytotoxicity observed in 2D culture. RT-PCR results show reduced levels of JAK1 mRNA after 6, 12, or 24 hours of silibinin treatment in both cell lines. STAT3 mRNA levels were decreased only in MDA-MB-231 cells (after 6 hours), with no effect in MDA-MB-468 cells. Reduced levels of phosphorylated JAK1 and STAT3 proteins were observed in both cell types. Moreover, using ELISA assays, silibinin significantly reduced TNF-alpha-induced MMP-2 and MMP-9 protein levels in MDA-MB-231 and MDA-MB-468 cells. In conclusion, the data demonstrate that silibinin exerts anticancer activity against TNBC cells by inhibiting the JAK/STAT signaling pathway and reducing MMP-2 and MMP-9 levels, which play pivotal roles in tumor growth, invasion, and metastasis. Notably, this study underscores differences among distinct TNBC cellular lines with distinct genetic backgrounds, reinforcing silibinin's role as a promising adjuvant treatment for aggressive forms of human breast cancer.