Novel strategies for inhibiting β-catenin signaling pathways in cervical cancer
Introduction
Cervical cancer (CxC) is a significant cause of illness and death among women worldwide. The current chemotherapeutic drugs used for CxC treatment have been associated with systemic toxicity in patients. ORM is a non-toxic and non-steroidal drug with well-established pharmacokinetic and pharmacodynamic properties in humans. Previous studies have demonstrated its anti-cancer potential in various pre-clinical mouse models. In this study, we have successfully synthesized and characterized a novel analogue of ormeloxifene called Bromo-ormeloxifene (Br-ORM). Our findings indicate that Br-ORM exhibits enhanced therapeutic efficacy against CxC in both in vitro and in vivo model systems when compared to its parent compound. This suggests that Br-ORM holds promise as a potential anti-cancer agent for the treatment of cervical cancer.
Methods
To investigate the impact of Br-ORM on the growth and proliferation of CxC cells (specifically CaSki and SiHa cells), we employed colony formation and MTS assays. We also employed the AutoDock4 software for molecular docking analysis to examine the interaction of Br-ORM with β-catenin. Furthermore, we assessed the effect of Br-ORM on the expression of epithelial-to-mesenchymal (EMT) markers, such as N-cadherin, slug, snail, as well as MMPs (MMP2 and MMP9), and miR-200a using Western blot and qPCR analyses, respectively. Apoptosis analysis was carried out using an Annexin-V staining kit. Additionally, we investigated the effect of Br-ORM on the cellular localization of β-catenin in CxC cells through Western blotting and immunofluorescence analysis. To further evaluate the anti-tumor efficacy of Br-ORM, we conducted experiments in an orthotopic xenograft mouse model of CxC.
Results
The results indicate that Br-ORM (10-20 μM) effectively suppressed the growth and proliferation of CxC cells in a dose manner. Additionally, Br-ORM exhibited significant inhibition (P<0.05) of the invasion and migration potential of CxC cells, leading to a decrease in metastatic phenotypes. The enhanced Annexin-V staining and PARP protein cleavage observed indicated an increased level of apoptosis in response to Br-ORM treatment. Furthermore, Br-ORM demonstrated suppression of the EMT process, as evidenced by the repression of N-cadherin, slug, snail, MMPs (MMP2 and MMP9), and β-catenin/TCF-4 transcriptional activity. It also greatly reduced the translocation of β-catenin to the nucleus. Bioinformatic analysis revealed that Br-ORM binds proficiently to the active site of β-catenin with a minimum energy of -7.6 kcal/mol. In CxC cells, Br-ORM treatment resulted in the restoration of miR-200a expression, which directly targets β-catenin. In an orthotopic xenograft mouse model, Br-ORM treatment significantly (P<0.01) regressed cervical tumor growth. Similar molecular effects of Br-ORM were also observed in excised tumor tissues.
Conclusion
In conclusion, the findings of this study strongly indicate that Br-ORM effectively inhibits the metastatic phenotypes of CxC cells by targeting the β-catenin signaling pathway. These results suggest that Br-ORM holds promise as a novel therapeutic approach for the treatment of CxC.