- 學位論文
探討漆黃素結合Sorafenib誘導人類子宮頸癌細胞凋亡之分子機制
The Molecular Mechanisms of Fisetin Combination of Sorafenib Induces Apoptosis in Human Cervical Cancer
摘要
癌症是目前造成人類致死率最高的死因之一，因此許多科學家致力於分析有效的天然物來對抗癌症，如果能夠找到專一且低毒性的標靶治療方式，對於臨床病患的助益將會更大。漆黃素（Fisetin）屬於類黃酮化合物（flavonoid）家族成員之一，它廣泛的存在於各種水果和蔬菜中。目前漆黃素已被證實的生物功能包括：抗菌、抗發炎、抗氧化等多重功用，更有文獻指出漆黃素能有效促使癌細胞凋亡以及抑制癌細胞轉移。近年來有許多文獻指出，藉由降低標靶藥物劑量之使用並與天然物合併，能達到加乘抗癌之效果。因此本實驗擬以漆黃素合併標靶藥物對人類子宮頸癌HeLa細胞作用，探討合併處理之凋亡機制及加成效應。 首先，本實驗以MTT方式證實，隨著濃度的增加，Fisetin以及Sorafenib皆明顯抑制細胞生長，且2.5、5.0 μM之Sorafenib合併40μM的Fisetin處理，則具有加乘效應來抑制人類子宮頸癌HeLa細胞的生長。接著利用細胞週期以及Annexin-V染色分析證明，Fisetin合併Sorafenib處理下相較於單獨作用，有更明顯促進促使細胞凋亡（apoptosis）現象發生。另外，西方墨點法也證實Fisetin合併Sorafenib處理可以增加活化caspase-8、caspase-3、DR5和Bax蛋白表現以及減少Bcl-2蛋白表現量，並以JC-1染色觀察到粒線體膜電位的改變，因而誘導細胞凋亡。而加入caspase抑制劑後則減緩caspase-8及caspase-3活化路徑。接著利用siRNA干擾系統抑制DR5蛋白表現，也相同減緩細胞凋亡現象。綜合以上結果證實Fisetin合併Sorafenib處理可加成性誘導人類子宮頸癌細胞凋亡。本研究結果說明Fisetin合併使用抗癌藥物Sorafenib具有發展為子宮頸癌治療方式的潛在價值，值得進一步探討。
並列摘要
Cancer is the top of ten cause of death in Taiwan, in the past especially cervica cancer threatens all of female. According the government policy to support Pap smear to prevent cervical cancer it have markly decrease the death rate now. Therefore, development of novel nature drug combination targeted therapy was important topic. Fisetin is a nature occurring flavonid 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. Sorafenib is a relatively new multi-kinase inhibitor which targets receptor tyrosine and serine/threonine kinases involved in tumor progression and tumor angiogenesis,it used to treat a wide range of cancers. Sorafenib is recommended treatment for patients with RCC and advanced HCC in Taiwan now. We expect that combinatorial treatment HeLa cells with Fisetin and Sorafenib can enhances the treat efficiency. In this study, MTT assay suggested that combinatorial treatment HeLa cells with fisetin and Sorafenib can enhance inhibit human cervical cancer HeLa cell viability. To determine the Fisetin combinatorial with Sorafenib induced apoptosis by AnnexinV/PI stain and flow cytometry. Furthermore, western blot showed that fisetin combinatorial with Sorafenib trigged the activation of caspase-3, -8 and DR5, Bax protein expression and decrease Bcl-2 protein expression, then confirm the mitochondrial membrane potential by JC-1 stain, resulting in apoptosis induction. Moreover, inhibition of activation of caspase-8,-3 and DR5 expression by Z-VAD-FMK, Z-IETD-FMK and siDR5 separate. The result can prove Fisetin combination with Sorafenib can enhance inhibit human cervical cancer HeLa cell growth through trigged apoptosis. Using a combination of fisetin and Sorafenib to increase their cytotoxic effects against the viability of cancer cells deserves further investigation.