O tratamento com TKI-258 aumenta a sinalização RhoA/ROCKs e leva ao aumento de apoptose em carcinoma epidermoide oral in vitro

Abstract

Background: Recently therapies for oral squamous cell carcinoma targeting molecular pathways are the most useful. TKI-258 is a simultaneous inhibitor of tyrosine kinase receptors FGFRs, VEGFRs, and PDGFRs. These pathways activate proteins such as Rho GTPases that contribute to tumorigenesis, performing different biological processes, as apoptosis. Objectives: This study evaluated the effect of treatment with TKI-258 on apoptosis, involving Rho GTPases and their effectors ROCKs and PAKs in SCC-4 cells of oral squamous cell carcinoma. Materials and Methods: Markers of cell death and apoptosis were analyzed in control and TKI-258 treated SCC-4 cells by flow cytometry. The involvement of Rho GTPases and the effectors ROCKs in the induction of apoptosis by TKI-258 was evaluated by quantification of cleaved PARP. Also gene expression analysis of Rho GTPases and some of their effectors was performed. Results: The treatment with TKI-258 led to a significant increase in cell death (7-AAD) and apoptosis (Annexin V and cleaved PARP). When Rho GTPases were stimulated with LPA and inhibited with Toxin A Clostridium difficile, the percentage of apoptotic cells increased and decreased, respectively. The treatment with TKI-258 combined with LPA and Toxin A had a similar effect, increasing and decreasing apoptosis, respectively. Treatment with TKI-258 significantly increased RhoA gene expression, while RhoB, RhoC, Rac1 and Cdc42 decreased significantly. ROCKs inhibitors (Y-27632 and HA-1077) reduced apoptosis compared with control. TKI-258 combined with Y-27632 or HA-1077 led to an increase in apoptosis compared with inhibitors only. Treatment with TKI-258 led to an increase in ROCK1 and ROCK2 gene expression, and a decrease in PAK1 and PAK2 gene expression. Conclusion: TKI-258 stimulates apoptosis in SCC-4 cells of oral squamous cell carcinoma. Possibly, Rho GTPases and their effectors ROCKs participate in the signaling pathway inhibited by TKI-258. Therapies with multi-target inhibitors, such as TKI-258, may be promising alternatives for the clinical treatment of oral squamous cell carcinoma.