Investigating Tangeretin Targeted Pathway by Chemical-Genetic Interaction in Yeast

多氧甲基黃酮 (polymethoxyflavones, PMFs) 為柑橘類果皮中含多氧甲基的酚類物質，目前已知其具有廣泛的生理活性，包括抗癌與抗發炎等。先前有許多研究探討PMFs與水解PMFs的抗癌活性，唯目前為止，其在細胞中的作用目標仍不明確。因此，我們利用化合物-基因協調作用的概念，以酵母菌作為研究平台，發掘PMFs在細胞中的可能作用目標。研究顯示，PMFs維持基因穩定上無顯著作用，顯示PMFs不是透過提升細胞的DNA修復因子來達到其生理活性。利用化合物對酵母菌DNA修復基因缺失的突變株作篩選，我們發現sgs1基因突變株在色胺酸被限制的情況下對PMFs成員之一- 橘皮素 (tangeretin) 敏感，無論是在生長速度或是存活率被顯著被抑制。另外，sgs1突變菌株在橘皮素的介入下，其細胞週期會停滯在G1 / S phase，並提升了γH2A的量。透過基因晶片，我們發現橘皮素會抑制sgs1突變株之RNA合成相關的基因的表現量，並提高其含氮化合物與胺基酸的合成基因之表現。透過sgs1突變株對放射菌酮的測試，發現橘皮素並無直接抑制蛋白質合成的效果，反而卻拯救了sgs1與slx4為主的雙股修復相關基因突變株在色氨酸酸剔除或橘皮素介入下的敏感性。結果推測，橘皮素可能是透過抑制sgs1突變株的rRNA的形成，在色胺酸被限制的情況下，間接導致蛋白質合成不利，進而促使菌株死亡。此研究顯示橘皮素在抗癌途徑的可能機制，並發現胺基酸與DNA雙股斷裂修復過程可能存在某種互動關係，唯需進一步實驗驗證。Polymethoxyflavones (PMFs) is a dietary phytochemical and they have been widely investigated in the field of inflammatory and anti-carcinogenic with proven bioactivities but they actual functioning target in cells remain unclear. Here, we used the model of chemical-genetic interaction model, with budding yeast as platform, to screen the potential targets or pathways involved by PMFs. We found out that SGS1 gene deleted yeast is sensitive to tangeretin, one of a member of PMFs. sgs1 defective yeast showed lower growth rate, survival rate, delayed G1 and S phase and elevated γH2A level in cell cycle under tryptophan drop-out condition. Through microarray, we found out that tangeretin is potential to inhibit expression of RNA progression related genes and up-regulated nitrogen compounds and amino acid synthesis pathways of sgs1 defective cells. Data also showed that tangeretin is incapable to inhibit protein synthesis directly as cycloheximide do. Interestingly, cycloheximide rescued the sensitive- phenotype of sgs1 and slx4 defective cells under tryptophan depletion and tangeretin involved condition. Our results showed tangeretin is potential to suppress rRNA precession of sgs1 defective cells and resulting improper protein synthesis under tryptophan depletion condition. This result demonstrated the potential pathway of anti-carcinogenic effect played by tangeretin. Additionally, we also observed the potential of availability of relationship between amino acid balances and DNA double strains break repair pathways. However, further investigation is required for this observation