Role of E-cadherin/ β-catenin complex in Chemopreventive and Anti-cancer Efficacy of Silibinin in colorectal cancer

Role of E-cadherin/ β-catenin complex in Chemopreventive and Anti-cancer Efficacy of Silibinin in colorectal cancer
Saba Sameri,¹ Massoud Saidijam,² Fatemeh Bahreini,³ Rezvan Najafi,^4,*
1. Hamadan University of Medical Sciences
2. Hamadan University of Medical Sciences
3. Hamadan University of Medical Sciences
4. Hamadan University of Medical Sciences
Introduction: Colorectal cancer (CRC) is among the top five world’s common cancers with high mortality. Silibinin is the most active flavonolignan constituent of Silymarin, the extract of milk thistle seeds. Epithelial-mesenchymal-transition (EMT) and Cancers stem cells (CSCs) are known to contribute to cancer initiation, progression and metastasis. In this study, we investigated the anti-cancer properties and molecular mechanisms of Silibinin on proliferation, apoptosis, the CSCs markers, EMT process and migration in vitro.
Methods: HCT-116 cells were treated with 50 μM of Silibinin for 24 and 48h to investigate its effects on proliferation, migration, EMT, CSCs, apoptosis and involved mechanisms by using MTT and colony formation assay, Acridine orange/propidium iodide double staining, sphere formation assay, RT-qPCR, Western blot and wound healing assay. One-way variance analysis (ANOVA), followed by Tukey–Kramer pairwise comparison, was performed to determine the significance of the difference between groups, using SPSS 25.0 software.
Results: : Silibinin significantly suppressed the proliferation of HCT-116 cells (p<0.001), while had no toxic effect on normal cells. Silibinin induced apoptosis by increasing the Bax/Bcl-2 ratio in cancer cells (p<0.001). Furthermore, the mRNA expression of cancer stemness markers; cluster of differentiation 133 (CD133), cluster of differentiation 44 (CD44), BMI1, Aldehyde dehydrogenase 1 (ALDH1), and doublecortin-like kinase 1 (DCLK1) was considerably decreased in treated HCT-116 cells with Silibinin (p<0.001). Moreover, Silibinin attenuated EMT by decreasing the mRNA expression of neural cadherin (N- cadherin) (p<0.001) and vimentin (p<0.001) and increasing the mRNA and protein expression of epithelial cadherin (E-cadherin) (p<0.001). The mRNA and protein expression of β-catenin was decreased in treated cells with Silibinin as well (p<0.001). Silibinin decreased the mRNA expression of MMP-2 (p<0.001) and MMP-9 (p<0.001) and inhibited cell migration in a time- and dose-dependent manner.
Conclusion: Our study revealed that the anti-cancer properties of Silibinin is mainly due to its ability to induce apoptosis, eliminate CSCs, attenuate EMT related markers and inhibit migration of CRC cells. It seems that Silibinin exhibits these anti-tumor mechanisms through blocking β-catenin molecule, which is a key component of the Wnt signaling pathway, one of the hallmarks of CRC development.
Keywords: Silibinin, Colorectal cancer, Epithelial-mesenchymal-transition, Cancer stem cell, β-catenin