牡荊素於細胞凋亡與脂肪生成之作用探討

類黃酮化合物(flavonoid)存在於日常食用的蔬果中，因此近年來深受研究者的注意與重視。牡荊素(vitexin)為天然的類黃酮化合物存在於某些特定草本植物中如山楂、牡荊、蔓荊。前人研究顯示其於醫藥上顯現非常廣泛的應用範圍包括抗發炎作用、抗氧化作用、抗肥胖作用、抗甲狀腺作用、抗細胞轉移作用及抗腫瘤生成作用。然而對於牡荊素誘發腫瘤細胞凋亡(apoptosis)與抑制脂肪生成(adipogenesis)有關的訊息傳導路徑著墨甚少。本研究之目的乃在探討牡荊素誘發的細胞凋亡與抗脂肪生成作用所可能存在的訊息傳導路徑。於細胞凋亡部分，牡荊素顯著降低口腔癌細胞(oral cancer cell 2, OC2)存活率同時顯著增加腫瘤抑制基因p53及其下游基因p21、Bax之表現。以p53抑制劑pifithrin-α (PFT-α)阻斷p53的訊息傳導，導致牡荊素喪失誘發口腔癌細胞凋亡的能力且抑制p53下游基因p21與Bax之表現。牡荊素具有抑制腫瘤細胞轉移之潛力乃伴隨增加plasminogen activator inhibitor-1 (PAI-1)積存與降低matrix metalloproteinase-2 (MMP-2)表現。使用PFT-α抑制p53活性的結果證實PAI-1及peroxisome proliferator-activated receptor γ (PPARγ)於牡荊素誘發的訊息傳導中為p53之下游調控基因。mitogen-activated protein kinases (MAPKs)抑制劑PD98059顯著降低OC2細胞的存活率。阻斷ERK 1/2 MAPK活性增加了牡荊素誘發的p53及其下游基因p21、Bax的表現。此外ERK 1/2 MAPK於p53誘發的腫瘤轉移與細胞凋亡中扮演負調控(negative regulation)的角色。於脂肪生成方面，牡荊素可抑制3T3-L1脂肪細胞的葡萄糖吸收、三酸甘油脂(triglyceride)合成與脂質堆積。牡荊素誘發ERK 1/2 MAPK表現顯著增加，然而脂肪生成的標的基因Akt與peroxisome proliferator-activated receptor γ (PPARγ)之表現則降低。PD98059 (10 μM)顯著增加脂肪堆積、三酸甘油脂生成與脂肪生成標的基因Akt、PPARγ表現。然而令人訝異的是PD98059對葡萄糖的吸收並無任何影響。此一結果顯示脂肪生成所需之大量原料可能並非源自於增加葡萄糖的吸收而是調控糖解作用(glycolysis)中葡萄糖的代謝路徑。於調控糖解作用方面，較高濃度(50 and 100 μM)的牡荊素可誘發甘油三磷酸去氫酶(glycerol-3-phosphate dehydrogenase, GPDH)表現。反之對於五碳糖磷酸路徑(pentose phosphate pathway)的速率限制酶葡萄糖六磷酸去氫酶(glucose-6-phosphate dehydrogenase, G6PDH)之表現則毫無作用。PD98059對於糖解作用則具完全相反的調控作用，增加G6PDH表現、降低GPDH表現。換言之，PD98059增加五碳糖磷酸路徑的代謝速率。伴隨牡荊素濃度增加誘發乳酸(lactic acid)生成增加至約3.8倍。然而PD98059則降低培養液中乳酸濃度至對照組的72%。此外PD98059導致牡荊素喪失抑制脂肪生成的能力。牡荊素藉由降低前脂肪細胞的轉移(migration)而影響脂肪組織擴展。此乃由於降低actMMP-2 (active MMP-2)表現所致。我們首度證實牡荊素藉由p53路徑誘發口腔癌細胞凋亡與抑制腫瘤細胞轉移。此外利用牡荊素抑制脂肪堆積係經由致活ERK 1/2 MAPK訊息傳導調控糖解作用。阻斷五碳糖磷酸代謝路徑可能對於肥胖(obesity)預防與治療為一嶄新的策略。

Vitexin, identified as apigenin-8-C-D-glucopyranoside, a natural flavonoid compound found in certain herbs such as hawthorn herb has been reported to exhibit anti-oxidative, anti-inflammatory, anti-metastatic and antitumor properties. The aim of this study was to investigate the possible existence of signaling pathways underlying vitexin-induced apoptosis and anti-adipogenesis. Vitexin decreased cell viability significantly. Meanwhile, the expression of tumor suppressor p53 and a small group of its downstream genes, p21 WAF1 and Bax, were upregulated. The p53 inhibitor pifithrin-α knockdown the signaling of p53 led vitexin lost its anti-tumor effect and inhibited the expression of p53 downstream genes, p21WAF1 and Bax. Vitexin had anti-metastatic potential accompanied with increasing plasminogen activator inhibitor 1 (PAI-1 ) accumulation and decreasing matrix metalloproteinase-2 (MMP-2) expression. Our present study evidenced, by using p53 inhibitor pifithrin-α, PAI-1 and PPARγ are downstream genes of p53 in vitexin-induced signaling. MAPK inhibitor PD98059 decreased the OC2 cells viability significantly. The expression of p53 and its downstream genes p21 WAF1 and Bax were enhanced by blocking the activation of ERK 1/2 MAPK in response to treatment with vitexin. Moreover, ERK 1/2 MAPK played a negative role in p53-dependent metastasis and apoptosis. Vitexin inhibited adipose accumulation, glucose consumption and triglyceride synthesis. The expression of ERK 1/2 MAPK greatly induced, whereas the expression of adipogenic markers Akt and peroxisome proliferator-activated receptor γ (PPARγ) diminished. ERK 1/2 MAPK inhibitor PD98059 (10 μM) significantly enhanced lipid accumulation, triglyceride synthesis and the expression of adipogenic markers. However, PD98059 had no effect on glucose consumption. In regulation of glycolysis, vitexin induced the expression of glycerol-3-phosphate dehydrogenase (GPDH) at higher dosage (50 and 100 μM) and without any effect on glucose-6-phosphate dehydrogenase (G6PDH) expression. PD98059 had an opposing effect that it significantly increased the expression of G6PDH, but decreased the expression of GPDH. Vitexin increased the lactic acid synthesis to about 3.8 folds in concentration dependent, whereas, PD98059 decreased the level of lactic acid in media to about 72% when compared with controls. Moreover, PD98059 abolished the anti-adipogenic effect of vitexin. Vitexin influence the expansion of adipose tissue through its ability to inhibit preadipocyte migration to about 80% via decreased the activity of active MMP-2. We demonstrated evidences for the first time thathe novel p53-dependent metastatic and apoptotic pathway induced by vitexin in human oral cancer OC2 cells and using of vitexin to against adipose accumulation. Moreover, blockade pentose phosphate pathway may be a novel strategy for obesity prevention and therapy.