| 摘要: | 大黃素（Emodin）為蒽醌類（anthraquinone）類的化合物。過去的研究已指出大黃素於使用於肝癌與乳癌細胞上，具有抗腫瘤的效果。然而，關於大黃素對人類肺腺癌細胞株（A549）的效應未曾有文獻報導，且大黃素抗腫瘤之分子作用機轉亦不十分清楚。在本研究中，藉由探討大黃素（emodin）對於人類肺腺癌細胞株（A549）抑制細胞增生（Anti-proliferation）、細胞週期（cell cycle）及細胞凋亡（apoptosis）等機制，評估抗癌其抗癌活性。研究發現大黃素呈現劑量及時間依存性使細胞進行死亡。由細胞形態的改變、DAPI及TUNEL螢光染色結果得知，大黃素明顯誘導DNA斷片產生並造成細胞凋亡。經實驗數據得知，大黃素對人類肺腺癌細胞株會抑制細胞增生、細胞週期停止在S 期（S phase arrest）及誘導細胞凋亡。利用西方點墨法（immunobloting） 分析cyclin A、cyclin B及Cdk1、Cdk2，結果顯示蘆薈大黃素對人類肺腺癌細胞株造成細胞週期停止在S 期可能是與減少細胞內cyclin A、B與Cdk1、2蛋白量的表現有關以及增加p53、p21 CIP1/WAF1/sdi1 and和p27 KIP1的表現量有關。大黃素處理肺癌細胞會使細胞內ROS產生並且造成粒腺體膜電位下降。此外，將細胞前處理抗氧劑Ascorbic acid時，可以阻斷大黃素所誘導之ROS產生以及細胞凋亡。另一方面， Bcl-2在粒腺體膜上表現量減少及Bax蛋白表現增加。另外，細胞利用腺病毒感染方式大量表現Bcl-2蛋白時，可保護細胞對抗大黃素所誘發之細胞死亡。處理大黃素之肺癌細胞A549，會引起caspase -2、-3、-8 以及 -9的活化。若處理caspases-2、-3、-8 或是 -9抑制劑，可明顯抑制大黃素所引起細胞的凋亡。此外，我們也發現大黃素會抑制ERK、JNK和AKT的蛋白活性。當處理Erk 的活化劑ATA（Aurintricarboxylic acid）時，可以抑制大黃素所誘導之細胞凋亡。綜合以上結果顯示，大黃素所造成細胞停滯在S期是經由調節cyclin A 和 B、 Cdk1和2、p53、 p21CIP/WAF1/sdil及 p27KIP1蛋白表現量。另外，ROS之產生、ERK和AKT蛋白活性降低以及Bcl-2家族所調控下游caspases活化都會引起大黃素對肺癌細胞A549所造成之細胞凋亡。; Emodin, an anthraquinone compound isolated from Rheum palmatum L., has been reported to possess anticancer effect on several human cancers such as liver cancers and breast cancers. However, the molecular mechanisms of emodin-mediated tumor regression are not fully defined. In this study, we found that treatment of human lung adenocarcinoma A549 cells with emodin resulted in dose- and time-dependent decrease in cell viability. Moreover, emodin significantly induced apoptosis evidenced by morphological changes, DAPI staining, and TUNEL assay. We also found that emodin resulted in cell cycle S-phase arrest and subsequently progressed to apoptotic cell death. The emodin-induced S-phase arrest was accompanied by downregulation of cyclin A, B and Cdk1, 2 and upregulation of p53, p21CIP1/WAF1/sdi1, and p27KIP1 proteins. Moreover, emodin stimulated the generation of reactive oxygen species (ROS) generation and the Membrance permibility transition （MPT）change. Pretreatment with ascorbic acid significantly blocked the emodin-induced production and apoptosis. In emodin-treated cells, a marked increase of Bax and decrease of Bcl-2 protein in mitochondrial membrance were detected. Overexpression of Bcl-2 by adeno-Bcl-2 virus infection dramatically protected A549 cells against emodin-triggered apoptosis. Furthermore, administration of emodin resulted in the activation of caspases -2,-3, -8, and -9. Cotreatment with the inhibitor of caspases -2, -3, -8, or -9 significantly prevented emodin-mediated apoptotic cell death. We also observed that the ERK, JNK, and AKT kinase activities are decreased. Preincubation with Aurintricarboxylic acid (ATA), an ERK activator, also markedly blocked the emodin-induced apoptosis. Taken together, our observations demonstrated that regulation of the expression levels of cyclin A and B, Cdk1 and 2, p53, p21CIP/WAF1/sdil , and p27KIP1 proteins might involve in emodin-induced S-phase arrest. In addition generation of ROS, down regulation of the ERK and AKT and activation of Bcl-2 family-dependent mitochondrial downstream caspases contributed to apoptosis in emodin-treated adenocarcinoma A549 cells. |
