Anti-angiogenic Effects of Terbinafine: Molecular Mechanisms Underlying

• Main Authors: Pei-Yin Ho, 何蓓茵
• Other Authors: Wen-Sen Lee
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/81935791310392070138

博士 === 臺北醫學大學 === 細胞及分子生物研究所 === 95 === Terbinafine, an oral antifungal agent used in the treatment of superficial mycosis, has been reported to have anti-cancer activity. In this study, we demonstrated that TB at a range of concentrations (0-120 ?嵱) dose-dependently decreased cell number in cultured human umbilical vascular endothelial cells (HUVEC). TB was not cytotoxic at a concentration of 120 ?嵱, indicating that it may have an inhibitory effect on the cell proliferation in HUVEC. The TB-induced inhibition of cell growth rate is reversible. [3H]thymidine incorporation assay revealed that TB reduced the [3H]thymindine incorporation into HUVEC during the S phase of the cell cycle. Western blot analysis demonstrated that the protein levels of cyclin A, but not cyclins B, D1, D3, and E, CDK2 and CDK4, decreased after TB treatment. The TB-induced cell cycle arrest in HUVEC occurred when the cyclin-dependent kinase 2 (CDK2) activity was inhibited just as the protein level of p21 was increased and cyclin A was decreased. Pretreatment of HUVEC with a p21 specific antisense oligonucleotide reversed the TB-induced inhibition of [3H]thymidine incorporation. These results suggest an involvement of the p21-associated signaling pathway in the TB-induced antiproliferation in HUVEC. We continued to investigate the underlying mechanisms for p21-mediated TB-induced growth arrest. Western blot analysis demonstrated that p53 protein level was increased in HUVEC with TB treatment. Promoter activation and expression increase of p21 induced by TB were prevented in p53-dominant negative cells, suggesting that p53 was involved in TB-induced p21 promoter activation. The level of phosphorylated extracellular signal-regular kinase (ERK) was also decreased by TB treatment. When MEK-1 was overexpressed, TB-induced increases of p53 and p21 protein levels were also abolished. These results indicated that TB stimulated the expression of p53 and p21 by suppressing the activation of Erk pathway. We further demonstrated that TB (0-120 ?嵱) dose-dependently inhibited the adhesion and migration of human umbilical vein endothelial cells (HUVEC). Western blot analysis demonstrated that TB decreased the levels of Ras protein and membrane bound Rho protein. Moreover, the TB-induced migration inhibition in HUVEC was prevented by pretreatment with farnesol (FOH) or geranylgeraniol (GGOH). Pretreatment of HUVEC with Ras inhibitor or a ROCK (a kinase associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the FOH or GGOH induced prevention effect on the TB-induced migration inhibition, respectively. These data suggest that the consuming or depletion of geranylgeranyl pyrophosphate and consequent suppression of the protein geranylgeranylation and farnesylation, which is essential for activation of Rho GTPases and Ras respectively, might account for the TB-induced inhibition of HUVEC migration. The levels of phosphorylated focal adhesion kinase (FAK) and paxillin protein and the mRNA levels of matrix metalloproteinase (MMP) 2 and 9 were also decreased by TB treatment. These results indicate that suppression of Rho-mediated pathway might be involved in the signal transduction leading to the inhibition of cell migration caused by TB in HUVEC. Furthermore, we also found that TB inhibited capillary-like tube formation, sprouting angiogenesis on CAM and angiogenesis in animal model. Taken together, our findings strongly suggest the potential applications of TB as an anti-angiogenic agent because it is capable of retarding the cell cycle of ECs, reducing the adhesion and migration of ECs, interrupting the tube formation, and inhibiting angiogenesis in vivo.