| 摘要: | 普來氏月桃(Alpinia pricei Hayata)是屬於台灣特有的薑科月桃屬植物。先前的研究發現月桃屬植物具有鎮靜、抗高血壓、抗氧化、抗菌及抗癌功能，但是對於抗口腔癌的研究卻很少探討，因此本研究將探討普來氏月桃萃取物(Alpinia pricei Hayata extract；APE)和其主要成分(Flavokawain B)對人類口腔鱗狀細胞癌細胞(Human oral squamous carcinoma cells；KB cells)及裸鼠異位移植腫瘤(nude mice xenograft tumor model)之治療模式，來探討細胞週期、細胞凋亡與轉移作用之機制。
在細胞實驗結果得知APE及flavokawain B皆隨著劑量及反應時間的增加，會抑制口腔癌細胞的Cyclin A、Cyclin B、Cdc 2與Cdc25c細胞週期的蛋白表現；增加Wee 1、p53、p-p53、p21的蛋白表現，使細胞週期停滯在G2/M期。兩種均會促進KB細胞內ROS釋放，導致粒線體膜電位(mitochondrial membrane potential)下降；APE會活化caspase-9、pro-caspase-3與poly(ADP)-ribosylpolymerase(PARP)表現，但flavokawain B則會活化procaspase-8、procaspase-9、procaspase-3及 PARP，再利用TUNEL assay確認普來氏月桃及flavokawain B對KB細胞均會造成細胞凋亡的情形。本研究同時發現APE和flavokawain B也會調控MMP-9、uPA、PAI-1、PAI-2與TIMP-1等癌細胞之轉移相關蛋白表現，並同時降低癌細胞轉移活性，因此普來氏月桃具有抑制腫瘤細胞轉移擴散的功能。在活體動物抗腫瘤試驗方面，給予注射APE及flavokawain B之實驗組其腫瘤生長顯著小於控制組，並利用TUNEL assay來確認腫瘤內部細胞凋亡有增加的現象。
總合上述細胞實驗與動物實驗之結果，發現APE及flavokawain B藉由調控細胞週期之蛋白表現，促使細胞停滯在G2/M期；並同時促使ROS釋放、粒線體膜電位下降、PARP裂解，進而造成細胞凋亡。在抗轉移方面，則會影響相關蛋白表現及活性來達到抑制癌細胞轉移的效果。因此普來氏月桃具有抗腫瘤之功效，在未來醫藥應用上可望成為有潛力的預防抗口腔癌、抗轉移藥物。
Alpinia pricei Hayata (A. pricei) is well known in Taiwan as a traditional Chinese medicine. It has been reported that Alpinia plants (family Zingiberaceae) possess antioxidant, anti-inflammatory, anticancer, immunostimulating, hepatoprotective and antinociceptive activities. In this study, the ability of extracts of A. pricei rhizome (AP extracts) and flavokawain B (A. pricei major compound) to induce cell cycle arrest, apoptosis, and metastasis inhibition in cultured human carcinoma KB cells was investigated through nude mice xenograft tumor model in vitro and in vivo.
Treatment of KB cells with various concentrations of AP extracts (25-200 μg/ml) and flavokawain B (5-20 μg/ml) resulted in sequences of events marked by apoptosis, such as loss of cell viability, morphology change, and internucleosomal DNA fragmentation. AP extracts and flavokawain B induced apoptotic cell death was associated with loss of mitochondrial membrane potential, cytochrome c translocation, caspase-3 and -9 activation, and poly ADP-ribose polymerase (PARP) degradation. Moreover flavokawain B was also associated with caspase 8 activation. This increase in AP extract-induced apoptosis was also associated with dysregulation of Bcl-2 and Bax. Furthermore, AP extracts induced a dose-dependent elevation of reactive oxygen species (ROS) in KB cells.
Flow cytometry analysis demonstrated that AP extracts and flavokawain B blocked cell cycle progress in the G2/M phase in KB cells. This cell cycle blockade was associated with reductions in cyclin A, cyclin B, Cdc 2, and cdc25c, and increased CDK inhibitor p53, p21 and Wee 1 in the AP extracts and flavokawain B-treated group in a dose and time-dependent manner relative to the untreated controls as evaluated using western blot analysis for cell cycle proteins, which corroborated the G2/M block.
The AP extracts and flavokawain B resulted in sequences of events marked by metastasis inhibition as shown by reductions in MMP-9 activation, down-regulation of uPA, and up-regulation of PAI-1, PAI-2, and TIMP-1 in KB cells. Our results revealed that treatment of AP extracts and flavokawain B inhibited KB tumor growth in experiment. Whereas, AP extracts and flavokawain B did not show any side effects in vivo studies. Therefore, A. pricei might have antitumor properties valuable for application in drug products. |
