祕魯酸漿抽出物及肉桂醛抗肝癌機制之研究

The apoptoticamechanism of Physalis peruviana extract and cinnamaldehyde from Cinnamomum cassia in human hepatoma cells

吳淑靜(Shu-Jing Wu)

指導教授 ： 林俊清

高雄醫學大學/藥學院/天然藥物研究所碩士班/博士(2006年)

摘要

第一部分:秘魯酸漿 (Physalis peruviana)是一種廣效性之民間藥物,已被應用於治療癌症、白血病、肝炎及其它疾患。秘魯酸漿之抗肝癌活性及造成細胞凋亡之分子機制目前並不清楚,因此值得進一步深入討論。本研究主要目的包括下列兩大方向: (1)評估秘魯酸漿抽出物,包括熱水及酒精抽出物對三種人類肝癌細胞: Hep G2、Hep 3B及 PLC/PRF/5之毒殺效應, (2)判別秘魯酸漿酒精抽出物對Hep G2細胞凋亡可能之作用機制。結果顯示,由XTT呈色分析結果發現, 秘魯酸漿酒精抽出物抑制 Hep G2細胞增生作用之IC50值表現最低。秘魯酸漿酒精抽出物抑制細胞增生呈現劑量與時間效應,並對鼠正常肝細胞(BALB/C)之增生作用無影響。高濃度之秘魯酸漿酒精抽出物處理,會明顯的提升sub-G1 峰的蓄積及Annexin V染色之凋亡細胞比率。秘魯酸漿酒精抽出物亦會造成細胞週期停滯在G0/G1期。以carbonyl cyanide m-chlorophenyl hydrazone (CCCP)處理,會造成膜電位(ΔΨm)去極化而導致降低ΔΨm。EEPP處理之Hep G2細胞會引起ΔΨm下降、產生ROS及glutathione (GSH)之損耗,呈現劑量效應。使用抗氧化劑(1.0 µg/ml Vit E)處理,可保護細胞免於秘魯酸漿酒精抽出物誘導而釋放ROS。秘魯酸漿酒精抽出物驅動細胞凋亡機制,係透過cytochrome c, Smac/DIABLO及Omi/HtrA2自粒線體釋放到細胞質並引起caspase-3活化之結果。預先以一種廣效性caspase抑制劑(z-VAD-fmk)處理,可避免cytochrome c之釋出。秘魯酸漿酒精抽出物處理48小時後,會導致Hep G2細胞凋亡跟促進p53、CD95、CD95L、Bax及Bad等凋亡訊息基因蛋白質的表現有關。然而,會顯著地降調節Bcl-2、Bcl-XL及XIAP基因蛋白質之表現。由上述的結果發現, 秘魯酸漿酒精抽出物誘導Hep G2細胞凋亡之機制,可能是透過CD95/CD95L系統及粒線體訊息傳遞途徑所致。
第二部份:本研究已證實肉桂醛(cinnamaldehyde, Cin)對人類肝癌細胞株 (PLC/PRF/5., 無CD95基因)展現抗細胞增生效應及細胞凋亡機制。由結果顯示,以肉桂醛處理,會藉由在細胞漿膜外面出現phosphatatidylserine、細胞形態之改變及sub-G1峰之蓄積作用而驅動細胞週期之perturbation(停滯於S期)及凋亡程序機制。由肉桂醛所主導之細胞凋亡機制,係因肉桂醛降調節Bcl-XL蛋白質之表現,升調節p53、 pSer15-p53及Bax蛋白質之表現並促使caspase-3活化及poly (ADP-ribose) polymerase (PARP)的裂解均呈時間-依賴性效應。經肉桂醛處理後,在細胞內會造成JNK、ERK及p38激酶活化與磷酸化。
同時發現抗氧化劑(維生素E及NAC)與mitogen-activated protein kinases (MAPKs)抑制劑包括JNK (SP600125)、p38 (SB203580)及ERK (PD98059)抑制劑,會藉由肉桂醛誘導而對細胞凋亡訊息基因傳遞機制造成影響。使用100 μM維生素E預處理很明顯地減低肉桂醛所誘導之細胞凋亡效應,而100 μM NAC效果較100 μM 維生素E差。肉桂醛在細胞中會造成活性氧自由基(ROS)之產生。然而, 維生素E也能顯著的抑制ROS之生成及不活化caspase-3。肉桂醛處理後,可自粒線體中釋放cytochrome c、Smac/Diablo及Omi/HtrA2到細胞質體中,進而活化caspase-3。維生素E可有效地阻擾肉桂醛處理之細胞的蛋白質如cytochrome c, Smac/Diablo及Omi/HtrA2從粒線體釋放到細胞質體中, 並降低caspase-3之活性。肉桂醛亦會升調節預凋亡蛋白質(Bax)及降調節抗細胞凋亡蛋白質如Bcl-2及IAP家族蛋白質(XIAP、 cIAP-1及cIAP-2)之表現。而抗細胞凋亡蛋白質如Bcl-2、凋亡抑制蛋白質(XIAP、cIAP-1及cIAP-2)及預凋亡蛋白質(Bax)之表現均會受維生素E預處理影響。因此使用維生素E預處理可防止肉桂醛誘導之細胞凋亡,與調控IAP家族及Bax蛋白質之活性具有相關性。使用JNK抑制劑(SP600125)及p38抑制劑(SB203580)可有效地抑制肉桂醛誘導之細胞凋亡, ERK抑制劑(PD98059)則無效應。JNK抑制劑(SP600125)及p38抑制劑(SB203580)兩者可顯著地防止JNK及p38蛋白質磷酸化,而非ERK抑制劑(PD98059)。

關鍵字

秘魯酸漿 ； 肉桂醛 ； CD95 ； p53 ； Cytochrome c ； 細胞凋亡 ； 肝癌細胞 ； 抗氧化劑 ； MAPKs抑制劑 ； 活性氧自由基 ； IAP家族 ； JNK ； p38 ； ERK ； 細胞凋亡

並列摘要

Part I:Physalis peruviana is a popular folk medicine used for treating cancer, leukemia, hepatitis and other diseases. However, the mechanism responsible for the anticancer effect remains unknown. The aims of this study were: (i) to evaluate the antihepatoma activity of aqueous and ethanol extracts of P. peruviana in three human hepatoma cell lines, namely Hep G2, Hep 3B and PLC/PRF/5; and (ii) to identify the mechanistic effects of ethanol extract of P. peruviana (EEPP) induced apoptosis in human Hep G2 cells. The XTT assay showed that ethanol extract of P. peruviana (EEPP) possessed the lowest IC50 value against the Hep G2 cells. The results confirmed that EEPP inhibited cell proliferation in a dose- and time-dependent manner. All extracts showed no cytotoxic effect on normal mouse BALB/C liver cells. At high concentrations, EEPP significantly increased the accumulation of the sub-G1 peak (hypoploid) and the portion of apoptotic annexin V positive cells. EEPP was also displayed G0/G1-phase arrest. Treatment with carbonyl cyanide m-chlorophenyl hydrazone, caused a reduction of membrane potential (Δψm) by mitochondrial membrane depolarization. EEPP induced the collapse of Δψm, the depletion of glutathione content and the formation of ROS in a dose-dependent pattern. Pretreatment with the antioxidant (1.0 ?慊/ml vitamin E) protected cells from EEPP-induced release of ROS. EEPP was found to trigger apoptosis through the release of cytochrome c, Smac/DIABLO and Omi/HtrA2 from mitochondria to cytosol and consequently resulted in caspase-3 activation. Pre-treatment with a general caspase inhibitor (z-VAD-fmk) prevented cytochrome c release. After 48 h of EEPP treatment, the apoptosis of Hep G2 cells was found to associate with an elevated p53, and CD95 and CD95L proteins expression. Furthermore, a marked down-regulation of the expression of the Bcl-2, Bcl-XL and XIAP, and upregulation of the Bax and Bad proteins were noted. Taken together, the results conclude that EEPP-induced Hep G2 cell apoptosis was possibly mediated through the CD95/CD95L system and the mitochondrial signaling transduction pathway.
Part II:This study has demonstrated that cinnamaldehyde (Cin) displayed the antiproliferative effect and apoptotic mechanism in human hepatoma PLC/PRF/5 (CD95-negative) cells. Treatment with Cin caused cell cycle perturbation (S-phase arrest) and triggered apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine, morphological changes and accumulation of sub-G1 peak. Cin down-regulated the expression of Bcl-XL, up-regulated p53, serine-15 phosphorylation of p53 (pSerine-15 p53) and Bax protein and promoted caspase-3 to active forms, as well as cleaving poly (ADP-ribose) polymerase (PARP) in a time-dependent pattern. JNK, ERK and p38 kinases in cells were activated and phosphorylated after Cin treatment. We have investigated the effect of the antioxidants (vitamin E; Vit E and N-acetyl-L-cysteine; NAC), and mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on the apoptotic signaling transduction mechanism induced by Cin. Following the pre-incubation of PLC/PRF/5 cells with antioxidants, it was found that 100 µM vitamin E significantly diminished the effect of Cin-induced apoptosis, whereas a lesser effect was seen with 100 µM NAC. Cin induces the generation of reactive oxygen species (ROS) in cells. Vitamin E also markedly blocked ROS formation and suppressed caspase-3 activation. The apoptotic effect induced by Cin could be further supported by the release of cytochrome c, Smac/Diablo and Omi/HtrA2 from mitochondria to the cytosol and activation of caspase-3. Cin also up-regulated the expression of pro-apoptotic protein (Bax) and down-regulated the levels of anti-apoptotic proteins such as Bcl-2 and IAP family (XIAP, cIAP-1 and cIAP-2). Vitamin E effectively blocked the release of cytochrome c, Smac/Diablo and Omi/HtrA2 from mitochondria to the cytosol in cells treated with Cin. The expression of apoptotic inhibitors (XIAP, cIAP-1, cIAP-2) and anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins was affected by vitamin E pretreatment. Pretreatment with vitamin E markedly prevented Cin-mediated apoptosis, which was associated with the inhibition of XIAP, cIAP-1, cIAP-2, Bcl-2 and Bax protein activity. Pre-incubation with SP600125 and SB203580 markedly suppressed the effect of Cin induced apoptosis, but not PD98059. Both SP600125 and SB203580 significantly prevented the phosphorylation of JNK and p38 proteins, but not ERK.

並列關鍵字

EEPP ； cinnamaldehyde ； CD95 ； p53 ； Cytochrome c ； Apoptosis ； MAPKs inhibitors ； ROS ； IAP family ； JNK ； Hepatoma cells ； Antioxidant ； p38 ； ERK ； Apoptosis

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