Caffeic acid and metformin inhibit invasive phenotype induced by TGF-β1 in C-4I and HTB-35/SiHa human cervical squamous carcinoma cells by acting on different molecular targets

Abstract

During the progression of epithelial cancer, the cells may lose epithelial markers and gain mesenchymal phenotype via Epithelial-Mesenchymal Transition (EMT). Such transformation of epithelial cancer cells to mesenchymal-like characteristic benefits plasticity and supports their ability to migrate. The aim of this study was to evaluate the influence of natural compound Caffeic Acid (CA) alone and in combination with antidiabetic drug Metformin (Met) on metastatic progression of two human cervical squamous cell cancer lines, C-4I and HTB-35/SiHa cells. EMT program was triggered by exposition of both epithelial cell lines to TGF-β1. Gene expression patterns related to epithelial/mesenchymal phenotype were evaluated by Real-Time PCR analysis and the protein amount was detected by western blot. The treatment of human squamous cancer cells with CA and with Met, suppressed the motility of cells and the effect depended on a particular cell line. Both compounds regulated the EMT process in C4-I and HTB-35 cells by interfering with different molecular targets. In TGF-β1-stimulated C4-I cells, CA suppressed the expression of mesenchymal transcription factor SNAI1 which resulted in enhanced expression of epithelial markers E-cadherin, Occludin and Claudin. Additionally, CA blocked MMP-9 and upregulated TIMP-1 expression, a specific inhibitor of MMP-9. In HTB-35 cells stimulated with TGF-β1, Met decreased the expression of Vimentin. By suppressing hypoxia master regulator HIF-1α, Met caused downregulation of CAIX, an enzyme involved in metastasis of aggressive malignant cells. In this study we showed that CA and Met inhibited EMT process in cancer cells via different mechanisms. However, when applied together, compounds exerted the greater effect on EMT than each compound alone. This is the first report revealing that CA alone and co-treated with Met may reverse mesenchymal phenotype of TGF-β1-treated cervical tumor cells and we believe that the use of the two small molecules may be considered as a potential therapeutic approach for metastatic cervical cancer.