大黃粗萃物與其主成分大黃素對於人類大腸直腸癌細胞株細胞凋亡之研究

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Title:	大黃粗萃物與其主成分大黃素對於人類大腸直腸癌細胞株細胞凋亡之研究 The Study of Apoptosis Induction by Rheum Palmatum L and Emodin on Human Colorectal Cancer Cell Line
Authors:	馬易世;Yi-Shih Ma
Contributors:	中醫學系博士班
Keywords:	大黃素;大黃;人類大腸直腸癌LS1034細胞株;細胞凋亡;粒線體;異體轉殖;轉移;Emodin;Rheum palmatum L;Human colon cancer LS1034 cells;Apoptosis;Mitochondrial;Xenograft tumor;Metastasis
Date:	2013-05-29
Issue Date:	2013-10-02 09:45:44 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	根據統計，大腸直腸癌是臺灣的主要死亡癌症之一，而數千年來大黃一直是中醫用於治療消化系統疾病的常用藥物。本研究藉由流式細胞儀與DAPI染色法證實大黃粗萃物可誘導LS1034人類大腸直腸癌細胞株產生細胞凋亡與G0/G1期休止。大黃粗萃物可增加LS1034癌細胞株之細胞內鈣離子濃度，並刺激caspase-9與-3之活性。其可增加Bcl-XS促凋亡蛋白表現，增加Bax/Bcl-2比例，抑制Bcl-2與 Bcl-XL抗凋亡蛋白表現。其亦可促進caspase-9與-3、cytochrome c、AIF、Endo G等蛋白之表現，進而誘導LS1034癌細胞株產生粒線體路徑之細胞凋亡。大黃素為大黃主要之活性成分，具有抑制多種人類癌細胞株的作用。本研究證實大黃素可造成LS1034細胞株型態改變、存活率下降、DNA損傷與產生G0/G1期休止等。其可誘導LS1034細胞產生活性氧物質、增加鈣離子濃度並降低粒線體膜電位。並可增加caspase-3和-9之活性、促進caspase-9、細胞質cytochrome c與AIF蛋白質表現、增加Bax/Bcl-2比例，促進caspase-3與-9 mRNA表現。結果說明大黃素誘導LS1034癌細胞株之凋亡亦可能與粒線體路徑有關。此外，LS1034癌細胞株異體轉殖體內試驗亦證實大黃素在腫瘤重量或體積上都具有顯著之抑制作用。最後，轉移侵犯是影響大腸直腸癌患者預後與存活的重要因素。本研究顯示大黃粗萃物可抑制LS1034細胞株之轉移與侵犯，可抑制核內與胞質中MMP-2與-9蛋白轉位與表現，而大黃粗萃物抑制LS1034癌細胞株轉移侵犯的作用則可能經由抑制ERK與PI3K/AKT路徑。 Colorectal cancer is one of the major cancer causes death each year in Taiwan. The crude extract of Rheum palmatum L (CERP) has been used to treat digestive diseases in the Chinese population for thousand years. In this study, we found that CERP induced apoptosis and G0/G1 phase arrest that were seen by DAPI staining and flowcytometric assay on LS1034 human colorectal cancer cells. The Ca2+ level was increased by CERP which also promoted the activities of caspase-9 and -3 in LS1034 cells. CERP treatment also indicated that an increase in the Bcl-XS level and Bax/Bcl-2 protein ratio but decrease in the levels of Bcl-2 and Bcl-XL. Data also confirmed that CERP promoted the expressions of caspase-9, caspase-3, cytochrome c, AIF, and Endo G that led to induce apoptosis in LS1034 cells through mitochondrial-dependent pathway. Emodin, an active natural anthraquinone derivative of Rheum palmatum L and exhibits anticancer effects on many types of human cancer cell lines. In vitro study, emodin induced cell morphological changes, decreased the percentage of viability, promoted the DNA damage, induced G0/G1 phase arrest and increased ROS and Ca2+ productions as well as loss of ?ψm in LS1034 cells. Emodin also promoted the activities of caspase-9 and -3 in LS1034 cells. Western blot analysis indicated that the protein levels of cytochrome c, caspase-9, AIF and the ratio of Bax/Bcl-2 were increased in LS1034 cells after emodin exposure. In conclusion, emodin induced mitochondrial dysfunction for causing LS1034 cell apoptosis. In in vivo study, emodin effectively suppressed tumor growth in xenografts tumor nude mice bearing by LS1034 cells. Finally, we investigated the anti-metastasis effects of CERP on LS1034 cells in vitro and also examined possible mechanisms. Data suggested that the CERP significant inhibit the cell migration and invasion of LS1034 cells. We also found that CERP inhibited the protein of matrix MMP-2 and MMP-9 by using immune-staining assay. Furthermore, we found CERP inhibited ERK and PI3K/AKT pathways in LS1034 cells by using Western blotting assay.
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