| Abstract: | 癌症居國內十大死因之首位，其中子宮頸癌對女性而言，更是位居癌症死亡率的第一名，平均約有百分之八十的癌症病人因癌細胞之轉移而致命，故若能有效抑制癌細胞的轉移及入侵，將可大幅減低癌症的死亡率。因此發展新穎天然藥物來當作癌症預防或是治療藥物是目前最重要的課題。我們每天飲食中都會攝取植物和蔬果類，此類都包含大量的類黃酮成分且人體每天都會消耗1 公克以上的類黃酮。漆黃素(Fisetin)是一種類黃酮化合物(flavonoids)家族成員之ㄧ，它廣泛地存在各種水果和蔬菜。目前漆黃素被發現的生物功能中包括：抗轉移、抗癌、抗發炎、抗氧化和細胞凋亡等多重功用。但是，目前漆黃素對於人類子宮頸癌細胞的作用機制，至今仍然未知。最近我們的初步結果以MTT 方式測試不同濃度fisetin 處理人類子宮頸癌細胞 (C33A、HeLa、CaSki 和SiHa)分別作用24 和48 小時，我們實驗證實fisetin 會抑制HeLa 細胞的生長，但不影響CaSki 和SiHa 細胞生長，因此我們認為fisetin 會誘導細胞凋亡。另外，我們利用傷口癒合和細胞移動實驗，作用48 小時證實fisetin 會抑制CaSki 和SiHa 的侵襲和移動。未來我們將會利用fisetin 以細胞和動物實驗證實fisetin 誘導細胞凋亡和抑制細胞侵襲的分子機制。因此本實驗計畫構想分為三年進行：【第一年】以fisetin 處理子宮頸癌HeLa 細胞分別以DAPI 染色、流式細胞儀和Annexin V/PI 染色證明fisetin 誘導細胞凋亡，進一步利用訊息傳遞路徑的抑制劑和caspase 抑制劑，分別探討fisetin 對HeLa 細胞的相關訊息傳遞路徑及其蛋白變化，如MAPK family、PI3K/Akt 途徑和細胞凋亡路徑。此外我們採用西方墨點法和RT-PCR 方法初步證實fisetin 會誘導DR5 的mRNA 和蛋白質表現，因此我們要釐清fisetin 是否會加強DR5 的轉錄作用，透過何種轉錄因子調控。【第二年】我們將會證實fisetin 是否抑制CaSki 和SiHa 細胞侵襲能力，此種現象是否會透過抑制EMT 基因或侵襲相關蛋白的轉錄和轉譯機制，因此我們採用Luciferase 方法、 ChIP、EMSA 方法和RNAi 技術釐清fisetin 影響EMT 基因或侵襲相關蛋白是否透過其它轉錄因子的調控。【第三年】接下來，我們將建立動物模式證明fisetin 對於腫瘤生長和侵襲能力。另外，我們也利用不同訊息傳遞的生物晶片來尋找fisetin 誘導細胞凋亡和抑制侵襲的相關基因。如果找到特定基因，利用RNAi 和過度表現的方法來證實fisetin 影響特定基因的功能。最後，利用fisetin 與一些臨床抗癌藥物或化療方式一起結合作用來達到延緩子宮頸癌細胞生長和侵襲能力。綜合以上的三年的研究將證實fisetin 在子宮頸癌方面當作抗癌藥物和抗侵襲藥物的發展上深具潛力。
Cancer is the top of ten causes of death in Taiwan, especially cervical cancer threatens all of female. The metastasis (invasion) of cancer cells leads 80% patients died, and inhibition of cancer cells metastasis will markly decrease the death rate. Therefore, development of novel nature drug or drug therapy was important topic. The flavonoids are present in almost all higher plants in the normal diet they were consumed more than one gram per day. Fisetin is a naturally occurring flavonoid found in many fruits and vegetables and display a wide range of pharmacological properties including anti-metastatic, anti-cacinogenic, anti-inflammatory, apoptosis and anti-oxidant effects. However, fisetin effect molecular mechanism of human cervical cancer is presently unknown. Recently, we have obtained preliminary data from MTT assay studies on test the different concentration of fisetin to treatment with four cervical cancer cells (C33A、HeLa、 CaSki and SiHa) for 24 or 48 hrs. We found that the growth inhibition of fisetin on HeLa cells were observed by MTT assay, not effect the growth of C33A, SiHa and CaSki cells. Upon our preliminary data showed that the inhibition by fisetin might be through apoptosis. In addition, we found that the fisetin inhibited the migrate and invasive inhibition of fisetin on SiHa and CaSki cells were observed by wound healing assay and migration assay after treatment for 48 h. Further we will investigation the molecular mechanism of fisetin induces cell apoptosis and inhibition of invasion on human cervical cancer cell line in vitro and in vivo. Therefore, three years grant proposal aims to study whether treatment of fisetin would induce cell apoptosis and inhibit cell invasion via mechanism(s) involved in vitro and in vivo models. In the first year study, to determine the fisetin induced apoptosis by DAPI stain, Annexin V/PI stain and flow cytometry in HeLa cells; and investigation the signal pathways for fisetin affect human cervical cancer cell by MAPK or caspase inhibitors, including MAPK family, PI3K/Akt pathway, and extrinsic/intrinsic pathway. In addition, our preliminary data from western blotting and RT-PCR assay suggest that fisetin induced the mRNA and protein levels of DR5. Therefore we will focus on studying whether fisetin induces transcription activity of DR5, and what kinds of transcription factor to regulate DR5 transcription activity. In the second year study, we will focus on studying whether fisetin inhibited cell migration and invasion in CaSki and SiHa cells, and whether fisetin suppresses EMT gene or invasion-related protein via transcription-dependent and translational mechanisms. Using Luciferase assay, ChIP, EMSA assay, and RNA interference techniques to clarify which mechanisms of fisetin effect EMT genes or invasion-related protein in association with transcriptional factors or repressors in vivo and in vitro. In the third year study, we will establish animal model to confirm that effect of fisetin on growth and invasion of cervical cancer cell inoculated mice. In addition, we found that fisetin induce apoptosis and inhibit cell invasion related target gene by different signaling cDNA microarray. If any candidate genes were identified, RNAi and gene overexpression will be applied on these genes for studying the involvement of these genes on fisetin effects. Finally, to evaluate some clinical anti-cancer drugs or IR was combined fisetin to alleviate growth and invasion in human cervical cancer cells. This study supports that fisetin may have potential as anti-cancer and anti-invasion agents in human cervical cancer. |
