| 摘要: | 在全球與台灣，每年有很多肝癌病例發生，且發現時多為晚期而無有效的治療方法，因此積極找出抑制肝癌的機轉並開發藥物是重要的。肝癌的發生在於肝臟受到肝炎病毒或酒精侵犯下，使肝臟長期處於慢性發炎狀態，進而進展成肝硬化，再形成肝癌。在肝癌中可觀察到STAT3受到發炎細胞所釋出的細胞激素刺激而處於活化狀態。Sorafenib是目前FDA唯一核准用於晚期肝癌病患的治療藥物，近期研究顯示，可藉由調控活化SHP-1而抑制STAT3活性，而Sorafenib的衍生物SC-1也同樣可透過抑制STAT3而抑制腫瘤，因此STAT3可能為肝癌治療的重要標的。在本研究中將探討，利用Sorafenib衍生物SC-1所合成的一系列對稱性藥物中，篩選出ITRI-1525是否可透過抑制STAT3而有效抑制腫瘤。結果發現，ITRI-1525對於PLC/PRF/5與HuH-7可抑制細胞存活，並具有細胞凋亡的特徵，例如: 從顯微鏡觀察到凋亡小體，從propidium iodide (PI) 染色發現有DNA斷裂的hypodipoloid cells，由AnnexinV/PI 染色觀察到phosphotidylserine (PS) 外翻至細胞膜外，而caspase 3與PARP蛋白有增加cleavage form。其機轉為透過抑制STAT3而減少cyclin D1、Mcl-1及survivin的蛋白表現。Mcl-1與survivin為抑制細胞凋亡的蛋白，此蛋白的減少可促使細胞走向細胞凋亡。Mcl-1的減少可促使促細胞凋亡蛋白於粒線體膜上打洞，降低膜電位，釋出cytochome c與AIF而造成細胞凋亡。因此觀察粒線體膜電位發現，ITRI-1525可造成粒線體膜電位下降。另外在處理ITRI-1525後，也可觀察到reactive oxygen species (ROS) 的增加，並在ROS抑制劑N-acetyl-L-cysteine (NAC) 處理下，可反轉ITRI-1525所造成的細胞毒殺作用，代表ITRI-1525所造成的細胞毒殺作用有一部分來自於ROS的增加。
The incidence rate of liver cancer is high both worldwide and in Taiwan. However, there was no effective therapy developed for advanced hepatocellular carcinoma (HCC). Therefore, developing anti-cancer drug for HCC is important. Virus- or alcohol- induced chronic inflammation in liver leads to liver cirrhosis and the development of HCC. Inflammation induces cytokines in cells and results in the activation STAT3 in HCC. Sorafenib is the only FDA-approved drug for treating advanced HCC patients. Recent studies show that Sorafenib activates SHP-1 to inhibit STAT3 activation. In addition, SC-1, a Sorafenib derivative compound can also inhibit tumor growth through inhibiting STAT3 phosphorylation. Therefore, STAT3 can be an important therapeutic target for HCC. In this study, we research whether novel Sorafenib derivatives inhibit tumor growth through inhibiting STAT3 phosphorylation. Series of symmetrical compounds derived from SC-1 were screened and ITRI-1525 was identified as a lead candidate. ITRI-1525 significantly inhibited cell survival and induced the expression of several apoptotic markers in PLC/PRF/5 and HuH-7. For example, apoptotic bodies were observed under microscope, hypodipoloid cells with DNA fragmentation were observed by PI staining, plasma membrane phospholipid flip-flop was detected by AnnexinV/PI staining, and cleavage forms of caspase 3 and PARP were increased after ITRI-1525 treatment. Mechanistic studies showed ITRI-1525 suppressed STAT3 phosphorylation and reduced cyclin D1, Mcl-1 and survivin expression. Mcl-1 and survivin are anti-apoptotic proteins. Therefore, the reduction of these proteins can induce cell apoptosis. The reduction of Mcl-1 may promote the leakage of mitochondria membrane regulated by pro-apoptotic proteins, which leads to the release of cytochome c and AIF from mitochondria to cytosol and results in apoptosis. Decreased mitochondrial membrane potential was also observed after ITRI-1525 treatment. Furthermore, ROS induction was also observed after ITRI-1525 treatment. Pretreatment of N-acetyl-L-cysteine (NAC), a ROS inhibitor, reduced cytotoxicity of ITRI-1525, which suggested that the cytotoxicity of ITRI-1525 was partially due to the induction of ROS in cells. |
