| Abstract: | 類黃酮是一種天然的多酚類，廣泛存在於日常食用的蔬菜和水果之中，而諸多文獻證實flavonoids具有許多特殊的生物活性，在早期最被廣為研究的是抗氧化的效果，近年來也有文獻指出類黃酮中的許多成員也具有抗發炎、預防腫瘤生成、抑制癌細胞轉移、抑制細胞周邊血管增生、及誘導癌細胞凋亡的能力等。然而，臺灣最近幾年口腔癌的發生率有逐年上升的趨勢，當中有超過90%的口腔癌被診斷為鱗狀上皮細胞癌，其主要發生在舌部。因此，我們選用SCC-4這株高度惡化的口腔舌癌鱗狀上皮細胞，在體外實驗中，利用MTT assays、modified Boyden chamber assay、gelatin zymography與casein zymography assay篩選14種類黃酮化合物(包括7種黃酮醇類與7種黃酮類化合物)對SCC-4細胞的存活率、侵入和移動能力及其分泌proteinases能力的影響。其中有四種黃酮醇類與四種黃酮類化合物不會影響癌細胞的存活率，而當中只有kaempferol這一種黃酮醇類與7-hydroxyflavone、baicalein、apigenin這三種黃酮類能有效的抑制癌細胞的移動和侵入能力與MMP-2、u-PA的活性。我們進一步發現kaempferol會抑制MMP-2、u-PA蛋白及mRNA的表現並且抑制Erk1/2、Akt的磷酸化、AP-1在細胞核內的表現及其與DNA binding的活性。然而，我們利用人類臍帶血管內皮細胞 (human umbilical vein endothelial cells；HUVECs) 進行in vitro的抗血管新生實驗，發現7-hydroxyflavone、baicalein和apigenin這三個類黃酮成份能有效抑制HUVECs tube formation的能力。另外，由細胞存活率的實驗結果得知有6個類黃酮成份會抑制癌細胞的存活率，從中挑選效果最顯著的luteolin。在luteolin的處理之下，SCC-4細胞的生長率有明顯的下降且造成細胞的死亡，而此效果具有濃度及時間的效應。進一步的實驗證實G1 checkpoint 調控蛋白表達會受到抑制，造成細胞週期停滯在G1 phase；然而，在長時間的作用下，更發現SCC-4細胞有染色質聚集、DNA斷裂的產生，並伴隨著caspase的活化，證實luteolin誘導SCC-4細胞走向細胞凋亡。最後在動物實驗中，於BALB/c nu/nu老鼠皮下異種移殖SCC-4細胞，其結果與控制組相比，發現在有處理luteolin的組別中，其腫瘤大小有明顯被抑制的效果。綜合以上結果，kaempferol及luteolin或許可以應用在預防口腔癌的轉移或輔助口腔癌的治療上。
Flavonoids, a large group of natrual polyphenols existing in a wide range of daily fruits and vegetables, have been evidented to possess various prominent bio-activities. Previously, many studies have revealed an antioxidant ability of flavonoids, and more recently, members of flavonoids have found a variety of anticancer effect such as cell growth and kinase activity inhibition, apoptosis induction, suppression of the secretion of matrix metalloproteinases and of tumor invasive behavior in various studies. In Taiwan, the incidence of oral cancer shows an increased tendency during the last decade. More than 90% oral cancers have been identified as squamous epithelium cell carcinoma, especially in tongue. For this reason, we investigated that the inhibitory effects of 14 flavonoids (7 flavonols and 7 flavones) in SCC4 cells, a malignant oral squamous carcinoma cell line. We assayed the cell viability, cell invasion/motility, matrix metalloproteinases (MMPs) activity, and urokinase-plasminogen activator (u-PA) activity of SCC-4 cells with flavonoid treatments by MTT assays, invasion/motility assays, gelatin zymography, and casein zymography. We found that one flavonol (kaempferol) and three flavones (7-hydroxyflavone, baicalein, and apigenin) significantly inhibited the cell invasion/motility capacities and the activities of MMP-2 and u-PA in SCC-4 cells without affecting cell viability, while 4 flavonols and 4 flavones would not affect cell viability and other flavonols and flavones could markedly inhibit cell viability. Fouthermore, we showed that kaempferol downregulated the protein and RNA levels of MMP-2 and u-PA, attenuated the phosphorylation of extracellular-regulated kinase (ERK) 1/2 and Akt, as well as suppressed the DNA binding activity and the nuclear level of activator protein-1 (AP-1). Otherwise, we performed the in vitro angiogenesis analysis via human umbilical vein endothelial cells (HUVECs) culture system. The results showed that 7-hydroxyflavone, baicalein and apigenin significantly inhibited the tube formation of HUVECs. In addition, we generated that six flavonoids may have tumor growth-inhibitory potent based on the result of primary cell viability assay. We further investigated the tumor growth-inhibitory effect of luteolin, the most potential agent of those six flavonids, and the results indicated that luteolin inhibits the proliferation and induces cell death of SCC-4 cells in a dose- and time-dependent manner. The further analysis elucidated that luteolin induces G1 cell cycle arrest via suppressing the expression of G1 phase regulatory molecular. The results of chromatin condensation, DNA fragmentation, and activation of caspase cascade in SCC-4 cells with a long term treatment of luteolin revealed that luteolin exerts an apoptosis-induced acitivty. Finally, an in vivo anti-tumor study using nude mice (BALB/c nu/nu) xenograft model by a subcutaneous inoculation of SCC-4 cells was performed. The average tumor volume of treatment groups was statistically lower than that of control group. In conclusion, kaempferol and luteolin may be a powerful candidate for a preventive agent against oral cancer development and metastasis. |
