抗腫瘤之紫蘇、霍香、防風、刺五加葉和石蓮花萃出物對人類肝腫瘤細胞HepG2基因表現樣態之影響

Abstract

近幾年微陣列技術以應用獨特的基因表現樣態，並結合特定細胞內的反應來提供一個快速的檢測方法。許多天然植物來源的產品已經被證實具有抗癌能力。因此傳統中草藥的應用可以增加對於預防與治療疾病的研究。處理癌症主要治療的方法，包括誘發不正常細胞產生凋亡作用。所以我們嘗試作細胞層面的探討以及cDNA微陣列技術，來篩選可以抑制癌症細胞增生與致使癌細胞產生凋亡作用的中草藥萃出物。 製備紫蘇、霍香、防風、刺五加葉、石蓮花及七層塔的水萃出物，並用來評估對於人類肝癌細胞株HepG2抑制生長的影響。應用MTT細胞存活分析來測量六種中草藥萃出物對於抵抗癌細胞的增生能力。並以Annexin V-FITC與PI螢光染色分析偵測細胞凋亡作用。分離細胞total mRNA後，分別使用cyanine 3-dUTP標定PBS處理的細胞mRNA，而cyanine 5-dUTP標定經中草藥萃出物處理後的細胞mRNA。應用含有7,680個spots的晶片，研究經中草藥萃出物處理HepG2細胞的基因調節。晶片掃瞄後，以GeneCluster軟體作資料分析。 經多種濃度的中草藥萃出物處理72小時後，確立可抑制HepG2細胞生長的中草藥濃度。紫蘇、霍香、防風、刺五加葉及石蓮花抑制細胞生長50%的濃度依序為100、300、400、400及400 mg/mL。刺五加葉與防風對於誘發HepG2細胞產生凋亡作用經流式細胞儀分析，凋亡細胞族群（PI－/AnnexinV＋）的百分比依序為15.8 ± 0.8%與21.4 ± 0.9%（P < 0.001）。以Tukey公正顯著差異法、鄧氏新多變測驗法及最小顯著差異法的變異數分析結果，顯示霍香與刺五加葉對於HepG2的處理，具有相似的基因表現。Spearman’s等級相關係數（r）分析結果也指出經中草藥水萃出物處理後所誘發的基因表現樣態中，以霍香與刺五加葉具有高相關性（r = 0.8）。cDNA微陣列的群集分析結果為多數基因表現與細胞凋亡、細胞增生及Cytochrome P450 superfamily有關。許多基因具有低表現量，尤其是與細胞增生相關的基因。比較逐步叢集分析法，自組圖分析法對於五種中草藥萃出物所誘發的基因表現樣態更具備良好的分群能力。分析的結果可以讓我們增加對於中草藥抗癌能力的瞭解。此外細胞毒性與微陣列的結果可以定義經防風、刺五加葉、紫蘇、霍香及石蓮花萃出物處理HepG2細胞後，抵抗癌細胞增生的能力與細胞凋亡相關的基因。因此在治療肝癌上，這些關於中草藥萃出物的研究發現，使其具有純化為試劑的價值。 中草藥萃出物對於HepG2細胞的抗腫瘤效用，於某種程度上可抑制癌細胞的增生。在cDNA微陣列中，此研究提供一個在cytochrome P450 superfamily、細胞增生及細胞凋亡作用上，可能是抑制與肝臟方面相關的基因表現樣態之開端。根據樣態而比較群集分析的結果，自組圖分析更適合用來運算及分類與抗腫瘤有關的基因表現樣態。研究cDNA微陣列可幫助瞭解中草藥萃出物在生理活動層面上的意義。

In recent years, microarray technology provides a rapid method to associate specific cellular responses with unique gene expression patterns. Many natural plant source products have demonstrated that including anticancer ability. Moreover, traditional herbs application is increasing in investigation into prevention or treatment in the disease. The main therapeutic approach of cancer treatments is inducing abnormal cells apoptosis. Therefore, we attempt to screen herbal extracts that can inhibit cancer proliferation and lead cancer cells to apoptosis by the cellular studies and cDNA microarray technology. Herbal extracts, Perilla frutescens, Agastache rugosa, Saposhnikovia divaricatae, Acanthopanax senticosus L., Echeveria elegan, and Salvia Plebeia R. Brown were prepared and used to evaluate the effect of growth inhibition on human hepatoma HepG2 cells. MTT cell viability assay was used to measure the antiproliferative effects of six herbal extracts. Apoptosis was detected by Annexin V-FITC and PI fluorescent staining assay. After total mRNA isolation, mRNAs from PBS-treated cells were labeled with cyanine 3-dUTP and mRNAs from the herbal extracts treated cells were labeled separately with cyanine 5-dUTP. The chip comtains 7,680 spots were used for studying the gene regulation in HepG2 cells treated with herbal extracts. The slides were scanned and the data were analysed by GeneCluster software. The growth of HepG2 cells in the presence of various concentrations of herbal extracts for 72 h were determined. The growth inhibition (IC50) of P. frutescens, A. rugosa, S. divaricatae, A. senticosus L. and E. elegans were about 100, 300, 400, 400 and 400 mg/mL, respectively. Significant induction of apoptosis was observed in the HepG2 cells treated with A. senticosus L., and S. divaricatae extract by flow cytometric analysis, and the fraction of apoptosis cells (PI－/AnnexinV＋) were as following 15.8 ± 0.8% and 21.4 ± 0.9%, respectively (P < 0.001). ANOVA analysis of Tukey’s honest significant difference, Duncan’s new multiple range test and least significant difference test, A. rugosa and A. senticosus L. had similar gene expression in HepG2 cells. The spearman correlation coefficients (r) were calculated for the gene expression levels of these herbal extracts treatment, and high correlation value was between A. rugosa and A. senticosus L. (r = 0.8). The clustering results of cDNA microarray indicated that the expression of numerous genes associated with the apoptosis, proliferation, and Cytochrome P450 superfamily. Most gene expression levels were down-regulation, especially in proliferation-related genes. Compared with the k-mean analysis, the SOM analysis in five herbal extracts had good ability of grouping genes. The result may improve our understanding of the actions of herbs with anti-tumor activities. Additionally, the cytotoxicity and microarray results enabled the apoptosis-associated genes that are responsible for the anti-proliferative activities of S. divaricatae, A. senticosus L., P. frutescens, A. rugosa and E. elegans extracts in HepG2 cells to be identified. Moreover, these findings revealed that herbal extracts are the material with potential to be purified as an agent for treating hepatocellular carcinoma. The anti-tumor effects of herbal extracts on HepG2 cells are due in part to inhibition of cancer cells proliferation. Our study provided a preliminary profile of gene expression may be related to suppress hepatic expression of cytochrome P450 superfamily, proliferation and apoptosis in cDNA microarray. According to the profile, we compared with cluster analysis, and SOM was more suitable to use in assaying the gene expression profiles associated with the anti-tumor. Such cDNA microarray studies can help to identifying herbal extracts in physiological activities.