Sirtuin抑制劑sirtinol對於肺癌細胞轉移能力機制探討

The inhibitory effects of sirtinol, a sirtuin inhibitor, on invasiveness of lung cancer cells

林頴婕(Yin-Chieh Lin)

指導教授 ： 邱建智

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2012年)

https://doi.org/10.6832/KMU.2012.00128

摘要

Sirtuin (Silent information regulator)是一種煙醯胺腺嘌呤二核苷酸 (NAD+)依賴型的去乙醯化酵素，在哺乳類發現共有7種homologues，分別為SIRT1∼7，統稱為sirtuin，皆屬class III histone deacetylase (HDAC)，其特徵為可對組蛋白及組蛋白以外的蛋白質進行去乙醯化，例如轉錄因子p53、NF-κB、FOXO等，並藉此調控細胞凋亡或存活的機制。近幾年研究指出，在癌細胞中SIRT1會大量表現，我們初步研究也發現，SIRT1在肺癌細胞中，表現量較正常細胞高，其中又以具高侵略性、高轉移能力的細胞株H1299和CL-1表現最多。有鑑於此，我們推測SIRT1對於肺癌細胞的侵略和轉移能力，應扮演相當重要的角色。
我們利用常見的SIRT1 抑制劑Sirtinol發現，Sirtinol對於肺癌細胞具有較小的毒性，但抑制SIRT1後都能明顯抑制細胞轉移、侵略和非貼附性生長的能力。此外，抑制SIRT1會導致細胞生存相關蛋白FOXO3a及抑制細胞轉移相關蛋白RYK、Tyk2、FAK/Pyk2大量表現，而促進細胞轉移相關蛋白Vimentin的表現量和Src的磷酸化被抑制。因此，我們推測sirtinol抑制SIRT1，進而抑制vimentin和Src信息傳遞路徑，並抑制肺癌細胞轉移、侵略及非貼附性生長的能力。在研究中也發現，抑制SIRT1會使其下游目標蛋白SP1下降，進而降低下游的MMP-9活性，及細胞轉移能力，推測sirtinol抑制SIRT1也可能直接影響SP1表現，並抑制MMP-9的活性，以達到抑制細胞轉移的目的。

關鍵字

肺癌 ； 轉移 ； sirt1

並列摘要

Sirtuin (SIRT) family is a class III histone deacetylase (HDAC). In mammalian cells, SIRTs were reported to exert the activities of NAD+-dependent deacetylase, which target histones and a variety of non-histone proteins in different subcellular compartments. Among SIRT proteins, SIRT1 involves in a variety of cellular processes that determines longevity, such as anti-apoptosis, cellular senescence, ageing, and certain metabolism pathways. Previous studies showed the physiological role of mammalian SIRT1 in tumorigenesis and cellular longevity. However, the precise effect of SIRT1 is highly dependent on the genetics of the cell and the presence or absence of p53. Our preliminary results showed the overexpression of SIRT1 in non-small-cell lung cancer cells (NSCLC), especially the two most invasive NSCLC H1299 and CL-1 cells. Accordingly, we proposed the high correlation of metastasis and SIRT1 overexpression and the critical role of SIRT1 in promoting tumorigenesis of NSCLC.
To test the hypothesis, the specific inhibitor of sirtuin, sirtinol was used in our study. Proliferation assay and annexin-V staining showed the low cytotoxicity of sirtinol towards NSCLC H1299 cells. On the contrary, the hallmarks of metastasis, the ability of cellular migration, invasion and anchorage-independent growth were effectively attenuated by sirtinol. Furthermore, the Western blot result showed that sirtinol inhibited the phosphorylation of Src, an important tyrosine kinase associated with cellular metastasis. The result suggests that sirtinol may inhibit migration or anchorage independent growth through Src signaling pathway. Our study indicates the correlation of SIRT1 and Src signaling in modulating the metastasis of NSCLC cells. The Western blot result also showed that the protein level of FOXO3a, RYK, Tyk2, FAK/Pyk2 were increased after sirtinol treatment, suggested that sirtinol may inhibit the cell invasiveness ability through these proteins up-regulation. According to gelatin zymography and Western blot results, we also found that sirtinol-induced inhibition of SIRT down-regulates the protein level of SP1 and MMP-9 activity. Overall, this study demonstrates that sirtinol-induced SIRT1 inhibition potently attenuates the metastasis ability of NSCLC cells, suggesting the promising therapy of lung cancer by targeting SIRT1 in the future.

並列關鍵字

SIRT1 ； sirtinol ； metastasis

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