Speakers - CIRWC 2024

Abstract

Oral cancer incidence and mortality are rising worldwide, with squamous cell carcinoma accounting for more than 90% of the cases and tongue carcinoma being considered among the most aggressive forms (1,2). Often diagnosed at a late stage, the prognosis of oral tumours remains bleak despite therapeutic management with chemotherapy agents alone or in combination with radiotherapy or surgery (3). Thus, the current focus is leaning towards plant-derived compounds as they are shown to be cheaper, more effective, and convenient to use while exhibiting less toxicity and reduced adverse effects (4). Eugenol, found in plants including cloves, nutmeg, basil and cinnamon, has been reported to induce apoptosis in multiple types of cancer (5). However, very few studies investigated its clinical relevance for oral cancer. In our study, we ought to explore the effect of Eugenol on oral cancer cell lines with different levels of aggressiveness. For this purpose, non-oncogenic human oral epithelial cells (GMSM-K) were used together with the Tongue (SCC-9) and Gingiva (Ca9-22) carcinoma cell lines. Key tumorigenesis processes, including cell proliferation, cytotoxicity, colony formation, cell cycle progression, apoptosis, migration, inflammation, and cell signalling, were assessed in vitro using the MTT, LDH, crystal violet staining, flow cytometry, QPCR, western blot and gelatin zymography assays. Moreover, the effect of Eugenol on cell cycle and apoptosis gene expression was evaluated by QPCR-array. Overall, our results showed that Eugenol inhibits cell proliferation and colony formation while inducing cytotoxicity in oral cancer cells as compared to normal counterparts. Tumor-specific IC50 were recorded around 200µM for Ca9-22 and 300µM for the SCC-9 line. As revealed by western blot and flow cytometry assays, Eugenol exhibits its effects on proliferation through p21/p27/cyclin D1 modulation and subsequent Ca9-22 cell cycle arrest at the G0/G1 phase in a p53-independent manner. QPCR array results confirmed SKP2, CDK4/6, CDK5RAP1, TFDP1 and p15 modulation-among, others-thus supporting this hypothesis. Moreover, Eugenol was proven to significantly induce gingival carcinoma apoptosis via the downregulation of pro-PARP protein expression. Distinct apoptotic profiles with a total number of 56 genes were uncovered for both cell lines by QPCR array. On the other hand, it seems that our active component limited Ca9-22 cell migration through MMP1/3 downregulation. Stimulation of inactive MMPs complex formation is expected to restrict SCC-9 migration. Finally, Ca9-22 behaviour appears to be mainly modulated by IL1-ß suppression, p-P38/p-STAT3 upregulation and p-NFKB downregulation. In this context, a less potent effect on IL1ß cytokine expression and elevated p-STAT3 were detected in SCC-9. In Summary, we can disclose that Eugenol-mediated anti-cancer mechanisms vary according to the cell type, with gingiva carcinoma being more sensitive to this phytotherapy agent.