黃烷酮類化合物導致骨肉瘤細胞死亡的探討及其2’-羥基黃烷酮引起骨肉瘤細胞株143 B產生細胞凋亡的機制與探討

類黃酮為多酚類化合物中的一個族群，廣泛存在於蔬菜與水果之中，而類黃酮具有許多生物活性，包含抗氧化、抗微生物與抗發炎，近來越來越多的研究中發現，類黃酮具有抗癌與抗癌化的作用。因此，首先分析幾個黃烷酮類藥物（黃烷酮、2''-羥基黃烷酮、3''-羥基黃烷酮、4''-羥基黃烷酮、5''-羥基黃烷酮、6''-羥基黃烷酮、7''-羥基黃烷酮）對骨肉瘤細胞株（U-2 OS、143 B、MG-63、SaoS2）的影響，MTT分析細胞存活率的結果發現，黃烷酮、2''-羥基黃烷酮與3''-羥基黃烷酮對骨肉瘤細胞株具有明顯的影響，其中2''-羥基黃烷酮對所有的骨肉瘤細胞株最具有明顯的影響力。在死細胞與活細胞數目計算的實驗結果中也指出2''-羥基黃烷酮導致所有的骨肉瘤細胞株的死亡細胞數目增加。更進一步，我們發現在加藥之後，143 B細胞產生染色質聚集、粒線體膜電位改變與二倍體細胞（sub-G1期）等現象出現。接著以西方墨點法分析細胞凋亡相關的蛋白表現量，結果顯示出半胱胺酸蛋白酶-3、半胱胺酸蛋白酶-8與半胱胺酸蛋白酶-9產生活化的現象，因此，半胱氨酸蛋白酶的活化證明了細胞凋亡產生。實驗結果顯示2''-羥基黃烷酮會增加TRAIL與DR5的表現量。另一方面，2''-羥基黃烷酮會誘導細胞色素c從粒線體中釋放出來，我們也發現到抗凋亡蛋白 （Bcl-2、Bcl-xL）會受到抑制，以及促凋亡蛋白（Bid、Bax、Bcl-xS）會被活化。因此，2''-羥基黃烷酮會藉由TRAIL-DR5訊息路徑誘導143 B細胞凋亡。最後，我們進行in vivo實驗（143 B-BALB/c nude mice），結果顯示腫瘤的生長會受到2''-羥基黃烷酮的影響與抑制。這些結果顯示2''-羥基黃烷酮會經由TRAIL-DR5路徑方式去活化細胞內的外在路徑與內在路徑，並且在in vivo實驗中對腫瘤的生長呈現有效的影響，因此2’-羥基黃烷酮為一個在癌症化學預防發展上呈現有效生物活性的化合物。

Flavonoids are a group of polyphenol compounds that exist extensively in dietary foods of vegetables and fruits, and the biological effects of flavonoids are anti-oxidant, anti-microbial, and anti-inflammatory activities. Recently, more and more studies have showed that flavonoids have the anti-tumor and anti-carcinogenesis effects. Therefore, the anti-tumor effects of flavanone （flavanone、2''-OH flavanone、3''-OH flavanone、4''-OH flavanone、5''-OH flavanone、6''-OH flavanone、7''-OH flavanone） in the osteosarcoma cell line （U-2 OS、143 B、MG-63、SaOS2） were analyzed. The result of MTT assay indicated that effect of flavanone、2''-OH flavanone and 3''-OH flavanone showed the most potent cytotoxic effect in the osteosarcoma cell line, and we also found that 2''-OH flavanoneare was the most potent cytotoxic compound. The result of cell’s live and death number calculation showed that 2''-OH flavanone induced the death number increasing of osteosarcoma cells. The further way, we found the phenomenon of chromatin condensation、mitochondria membrane potential change and diploid cell （sub-G1 phase） appearance after treatment to 143 B cell. Then, we analyzed the expression of apoptotic protein molecule by western blotting. The result showed the activation of caspase-3、caspase-8 and caspase-9. Therefore, the activation of caspase evidenced the occurrence of apoptosis. The result showed that 2''-OH flavanone increased the expression of TRAIL（TNF-released apoptosis-inducing ligand） and DR5 （Death receptor 5）. On the other hand, the result also showed that 2''-OH flavanone induced cytochrome c was released from mitochondria. We also found the anti-apoptotic proteins （Bcl-2、Bcl-xL）were inhibited, and the pro-apoptotic proteins（Bid、Bax、Bcl-xS）were activated. Therefore, the 2''-OH flavanone induced the 143 B cell apoptosis through TRAIL-DR5 pathway.Finally, we conducted the in vivo experiment（143 B-BALB/c nude mice）. The result showed that tumor growth were suppressed by 2''-OH flavanone. These results indicated that 2''-OH flavanone activated the extrinsic pathway and the intrinsic pathway through TRAIL-DR5 pathway, and it had potent effect to tumor growth in in vivo model. Therefore, 2''-hydroxyflavanone is a potent biological activity compound for the development of cancer chemotherapy.