Terbinafine抗血管增生及其分子機轉探討

Anti-angiogenic Effects of Terbinafine: Molecular Mechanisms Underlying

何蓓茵(Pei-Yin Ho)

指導教授 ： 李文森

臺北醫學大學/醫學院/細胞及分子生物研究所/博士(2007年)

https://doi.org/10.6831/TMU.2007.00071

摘要

Terbinafine (TB)在臨床上作為抗表皮黴菌感染的口服藥物，過去的研究指出TB具有有抗腫瘤生成的活性。在本研究中，我們發現隨著TB可以抑制人類臍帶血管內皮細胞(HUVEC)細胞生長，且其抑制作用隨這TB濃度的增加而有逐漸增強的趨勢。在實驗中所使用的TB最高濃度(120 ?嵱)，對細胞不造成死亡，這表示TB對於人類臍帶血管內皮細胞數目的減少不是因為造成細胞死亡的結果，而是因為抑制細胞週期的進行；同時，TB所引發的生長抑制作用是具有可逆性的。利用[3H]thymidine incorporation的實驗結果顯示，在TB的影響下進入細胞週期S期的細胞數目有顯著的下降情形。而利用西方墨點法的實驗分析，我們發現TB能夠減少人類臍帶血管內皮細胞中cyclin A蛋白質的表現量，但是對於cyclin B、D1、D3、E或CDK2、CDK4則無顯著影響；另一方面TB對於能抑制細胞週期進行的蛋白質P21則具有抑制作用。利用p21 antisense oligonucleotide則可使TB對於細胞增生的抑制作用消失。這些結果暗示TB可能藉由增加p21的表現進而抑制了HUVEC的增生作用。我們認為TB能藉由增加p21蛋白質表現與減少cyclin A表現而抑制cyclin-dependent kinase 2(CDK2)活性，最終導致細胞週期停滯在G0/G1期。

我們進一步研究TB引起細胞內p21表現增加的分子機制。實驗結果顯示TB可造成細胞內p53蛋白質表現量增加。若細胞投予p53-dominant negative，則原本受到TB引發的p21 promoter活化作用及隨之增加的p21表現量增加的現象均會消失。這實驗結果表示TB活化P21 promoter的作用是藉由增加細胞內P53的表現量而引起的。另外，TB會抑制extracellular signal-regular kinase (ERK)蛋白質的磷酸化。然而，當在細胞內大量表現MEK-1時，TB處理下細胞內的p53及p21表現量並無增加的現象。這些結果暗示TB可能是藉由抑制Erk活化路徑而造成p53及p21在細胞內的表現量增加。

除了對於細胞增生能力的影響，我們發現TB對HUVEC的貼附及移動能力也具有抑制作用。以西方墨點法分析，結果顯示TB能減少Ras及連結於細胞膜上的Rho蛋白質含量。以farnesol(FOH)或geranylgeraniol(GGOH)作預處理則可以避免TB對於細胞移動能力的抑制作用。另外，以Ras抑制劑或ROCK(藉由與RhoA接觸而傳遞相關訊息的下游分子)抑制劑(Y27632)進行預處理則能使原本FOH或GGOH對於TB作用的預防效果消失。我們認為其可能是由於geranylgeranyl pyrophosphate耗用或減少，進而抑制蛋白質(例如：Rho與Ras)進行與活性相關的geranylgeranylation及farnesylation修飾作用，是造成TB抑制細胞移動能力的原因。TB也會減少focal adhesion kinase (FAK)與paxillin蛋白質的磷酸化，同時降低細胞內MMP-2與MMP-9的mRNA表現。藉由這些實驗結果，我們推測TB對於HUVEC移動能力的抑制作用可能經由與Rho相關的訊息傳遞路徑。此外，我們發現TB能夠抑制活體外類血管結構的形成、活體內雞胚胎絨毛膜上與動物體內的血管增生作用。

綜觀以上所有的實驗結果，我們發現TB能夠阻滯血管內皮細胞週期的進行、抑制細胞貼附與移動的能力、干擾類血管的形成及活體內血管增生的現象。因此，我們認為TB應具有可開發成為抗血管增生藥物的潛力。

關鍵字

抗血管增生 ； 細胞週期 ； 移動 ； 訊息傳遞 ； 烯化作用

並列摘要

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.

並列關鍵字

terbinafine ； anti-angiogenesis ； cell cycle ； migration ； signal transduction ； prenylation

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