| 摘要: | 石榴（pomegranate）學名為Punica granatum是石榴樹的果實。目前已知石榴可以造成細胞凋亡來抑制許多惡性癌細胞的生長，本論文利用從石榴萃取出的多酚物（polyphenol）— TMU023，來探討TMU023是否會造成U87MG神經膠質瘤細胞的生長抑制和細胞凋亡。實驗結果發現，TMU023會抑制U87MG神經膠質瘤細胞的生長，使細胞的cyclin E表現增加，抑制cyclin A和cyclin B的表現，因而使細胞週期停留在S期。我們也發現TMU023會改變細胞中Bax和Bcl-2的比例，使的細胞色素c（cytochrome c）離開粒線體，活化caspase-9造成PARP的斷裂而使細胞走向細胞凋亡。將U87MG神經膠質瘤細胞以TMU023處理之後，會活化AMPK（AMP activated protein kinase）造成細胞自噬的細胞死亡，我們利用chloroquine來抑制細胞自噬，結果發現chloroquine可以保護U87MG神經膠質瘤細胞，避免因TMU023造成的細胞死亡。TMU023、AICAR（5-aminoimidazole-4-carboxamide riboside）和metformin都能活化AMPK，增加autophagosome的形成使LC3被切割。我們結果證實TMU023會造成細胞的死亡是透過細胞凋亡和細胞自噬的途徑。
The extract of Pomegranate, fruit derived from the tree Punica granatum, has been shown to inhibit cell growth followed by apoptosis in many highly aggressive caner cells. In the present study, we demonstrated that treatment of human glioma U87MG cells with TMU023, a polyphenol isolated from Punica granatum, resulted in inhibition of cell growth [as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide assay], arrest of cells in S phase of the cell cycle (as assessed by DNA cell cycle analysis), and induction of apoptosis (as evaluated by DNA ladder formation and flow cytometry). These events were associated an increase protein levels of cyclin E, down regulation of cyclin B and cyclin A and an alteration of the Bax:Bcl-2 ratio in favor of apoptosis. Indeed, TMU023 induced cleavage of poly(ADP-ribose) polymerase, release of cytochrome c from mitochondria, and activation of caspase-9 in U87MG cells. Incubation of U87MG cells with TMU023 activated the AMP activated protein kinase (AMPK), which may lead to autophagic cell death. Inhibition of autophagy by treatment with chloroquine (10 μM) protected U87MG cells from TMU023-induced cell death. Both TMU023, AICAR and metformin, another AMPK stimulator, increased autophagosome formation and LC-3 II cleavage (as demonstrated by immunoblotting). Our results suggest that in addition to promoting death via apoptotic pathway, pomegranate may induce autophagic cell death. |
