PO.ET02.12 · 实验与分子治疗
水飞蓟宾通过调节 JAK1/STAT3 信号通路和抑制 MMP 水平在三阴性乳腺癌细胞中的抗癌作用
The anti-cancer effects of silibinin through the modulation of JAK1/STAT3 signaling and inhibition of MMPs levels in triple-negative breast cancer cells
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
乳腺癌是美国女性中第二常见的癌症类型。最具侵袭性的亚型是三阴性乳腺癌，占比 10% 至 20%，并且不成比例地影响非裔美国女性。多种天然化合物已被鉴定为通过靶向肿瘤微环境在乳腺癌中具有潜在治疗应用。在这些化合物中，水飞蓟宾是一种存在于水飞蓟（Silybum marianum）中的天然化合物，已被证明对多种癌症类型表现出抗癌特性。本工作通过靶向 JAK/STAT 信号通路并测量基质金属蛋白酶（MMP）水平，检验了水飞蓟宾在 MDA-MB-231（高加索裔）和 MDA-MB-468（非裔美国裔）TNBC 细胞中的抗癌活性。所采用的方法包括细胞毒性分析、二维和三维细胞培养实验、RT-PCR、Western 分析以及 ELISA，以评估 IFN-gamma 和 TNF-alpha 刺激的 TNBC 细胞中 MMP-2 和 MMP-9 的蛋白水平。水飞蓟宾（0.78-400 μM）导致两种细胞的细胞活力下降，其中 MDA-MB-231 细胞的敏感性更高（IC₅₀ = 154.23 ± 4.15 μM），相比之下 MDA-MB-468 细胞为（IC₅₀ = 175.43 ± 5.87 μM）。两种细胞系均观察到抗增殖活性，MDA-MB-468 细胞随时间表现出更高的敏感性。此外，三维细胞实验表明水飞蓟宾在抑制球体生长和活力方面的效力，与二维培养中观察到的细胞毒性一致。RT-PCR 结果显示，在两种细胞系中，水飞蓟宾处理 6、12 或 24 小时后 JAK1 mRNA 水平降低。STAT3 mRNA 水平仅在 MDA-MB-231 细胞中降低（6 小时后），在 MDA-MB-468 细胞中无影响。两种细胞类型中均观察到磷酸化 JAK1 和 STAT3 蛋白水平降低。此外，使用 ELISA 实验，水飞蓟宾显著降低了 MDA-MB-231 和 MDA-MB-468 细胞中 TNF-alpha 诱导的 MMP-2 和 MMP-9 蛋白水平。总之，数据表明水飞蓟宾通过抑制 JAK/STAT 信号通路并降低在肿瘤生长、侵袭和转移中发挥关键作用的 MMP-2 和 MMP-9 水平，对 TNBC 细胞发挥抗癌活性。值得注意的是，本研究强调了具有不同遗传背景的不同 TNBC 细胞系之间的差异，进一步印证了水飞蓟宾作为侵袭性人乳腺癌有前景的辅助治疗药物的作用。
查看英文原文 English abstract
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.
利益披露 Disclosure
S. D. Mishra, None..
P. Mendonca, None..
S. Kaur, None..
K. F. Soliman, None.