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.

Alchemilla vulgaris agg. (Lady's mantle) from central Balkan: antioxidant, anticancer and enzyme inhibition properties

“…Namely, it is well recognized that both caffeic and gallic acid, that are present in EA extract, possess strong anti-proliferative and cytotoxic properties in wide range of tumor types such as those used in this study (breast, cervical, ovarian, prostate carcinoma). [51][52][53] These phenolic acids affected main signaling pathways involved in cell cycle progression and cell death. Moreover, they interfered with migrative and invasive properties, epithelial/ mesenchimal transition and even angiogenesis.…”

Metformin in Cervical Cancer: Metabolic Reprogramming

“…Recently, it was reported by Laskov et al [39] that Met downregulated the expression of vimentin in endometrial cancers in vitro and in vivo in diabetic patients. The incubation of cervical cancer cell lines with Met reduced cells' ability to move, as shown using functional scratch test in C4-I and HTB-35 cells stimulated with TGF-β1 [17]. Mechanistic study revealed that Met inhibited the expression of transcription factors Snail-1, ZEB-1, and Twist-1.…”

“…The regulation of apoptosis pathways in HTB-35 (SiHa) cells highly reflects the specificity of cervical tumor in vivo [16]. HTB-35 cells, even unstimulated with cytokines, have mesenchymal-like characteristics, especially high vimentin expression, along with enhancement of cell scattering and ability to move [17]. Another cell line, C-4I cells (ATCC, designation CRL1594) with epithelial phenotype, was derived from primary in situ tumor [18].…”

“…In our study, EMT process was induced upon 48 h incubation of cervical cancer cells with 10 ng/mL of cytokine TGF-β1, as described in detail in [17]. HTB-35 cells, even unstimulated, expressed mesenchymal-like characteristics, and the incubation with TGF-β further enforced expression of mesenchymal marker, vimentin, along with enhancement of cell scattering and ability to move [17]. The study showed that Met was an effective suppressor of mesenchymal phenotype and, in particular, downregulated vimentin in HTB-35 cells (Figure 3).…”

“…Due to its relevant role in cell invasion, CAIX was proposed as a potential therapeutic target, also in cervical cancers [41,42]. We showed that the exposition of HTB-35 cells to Met under hypoxia suppressed HIF-1α, which resulted in decreased transcription of CAIX gene, thereby alleviating invasive properties of cervical malignant cells [17].…”