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| 題 名 | 以臺灣產茄科中藥調控肝細胞腫瘤中p53及Rb抑癌基因表現與調節修補DNA變異之酵素活性及其在抗腫瘤機轉上的研究 |
|---|---|
| 作 者 | 郭國華; | 書刊名 | 中醫藥年報 |
| 卷 期 | 15:2 1997.05[民86.05] |
| 頁 次 | 頁259-288 |
| 分類號 | 414.34 |
| 關鍵詞 | 茄科中藥; 抗癌機轉; 抑癌基因調控; 肝細胞腫瘤; Solanaceae herbs; Anti-tumorogenecity; Regulation of tumor supressor gene; Hepatocellular carcinoma; |
| 語 文 | 中文(Chinese) |
| 中文摘要 | 世界各國致力於癌症機轉的所究已獲得顯著的成果，但對於癌症的治療除了藉助 外科手術與化學藥物外，基因療法目前僅於臨床實驗性階段，同時僅限於致癌機轉較明白之 部份特定癌症，因此尚未有廣泛與顯著的治療效果。由於空氣與環境污染或長久曝露於致癌 物質下，癌症遂成為台灣第一大死亡原因，其發生率、有逐年增加的驅勢。尤其肝細胞腫瘤 (HCC) 則是國人最主要之癌症， 目前對於 HCC 尚無理想治療藥物， 採用一般化學療法對 HCC 治療並無法降低其死亡率。對於如何啟動體內正常細胞形成腫瘤的機轉不明，但腫瘤的 發生已知係導因於細胞基因的改變，因此僅利用化學藥物毒殺癌細胞同時對正常細胞亦造成 傷害並不是理想的治療方式。p53 及 Rb 抑癌基因在調節細胞正常生長其有重要地位，因此 具有抑制癌細胞生長的功能， 但是 p53 及 Rb 抑癌基因若發生點變異 (Point mutation) 或異對偶染色體失落 (loss of heterozygosity)，則轉變為具有致癌性。大部份的 HCC 或 其他癌症均伴隨 p53 及 Rb 基因變異的現象，顯示 p53 及 Rb 抑癌基因變異與癌症形成有 關。因此本計畫乃應用 RT-PCR 等分子生物學技術探討已被報告具有保肝及抗腫瘤作用之臺 灣產茄科 (Solanaceae) 中藥 - 黃水茄 (Solanum incanum) 、 冬珊瑚 (Solanum capsicastrum)、 印度茄 (Solanum indicum)、 龍葵 (Solanum nigrum) 及白英 (Solanum lyratum) 等中藥成份對調控 p53 及 Rb 抑癌基因表現的影響， 以及調控細胞內修補 DNA 酵素 (例如 DNA glycosylase，polymerase 及 ligase) 之活性。期望能由抗腫瘤中藥中獲 得具有上述細胞調節作用之中藥成份，則此中藥將不僅不會影響正常細胞的功能，同時藉著 提高 DNA 修補能力以矯正癌細胞基因，對感染 B 型肝炎病毒之高危險群患者有預防癌化作 用，亦對 HCC 將提供一新的治療方向。因此本計畫以 MacDNASIS 等軟體分析具最低自由能 之 p53 與 Rb mRNA 結構，設計其特定 primers 後，進行 RT-PCR。 所萃取的 RNA 為由經 或未經中藥處理之人類肝癌細胞株 (Hep3B) 與正常皮膚纖維母細胞， 結果發現上述茄科中 藥中藥對肝癌細胞及正常細胞之抑癌基因 (p53 及 Rb) 表現 (expression) 並無顯著差異 ，顯示茄科中藥之抗腫瘤機轉可能並非經由調控抑癌基因表現而達成。為進一步了解其真正 抗腫瘤機轉，本計畫以 [ �� H]thymidine incorporation 探討其門胞之毒殺作用，結果顯 示以茄科中藥之粗萃取液在 900 μ g/ml/l X 10 �� cells 下， 以白英具有顯著肝癌細胞 毒殺效果，但對正常細胞並無毒殺作用，而民間常用於治療肝炎之黃水茄在此濃度下並無作 用。經分別由白英及黃水茄分離其成份一 Capsimine 與 Solamargine 並測定其細胞毒性結 果， Capsimine 在 2 μ g/ml/l X 1O �� cells 下即具有特異性毒殺癌細胞的作用，而在 15 μ g/ml/l X 1O �� cells 可達到 60% 以上之癌細胞死亡之活性，但此濃度對正常細胞 並不影響， 此結果與利用白英粗萃取物所得實驗結果一致， 顯示控制 Capsimine 濃度於 2-15 μ g 可選擇性作用於肝癌細胞而不影響正常細胞之功能，因此進一步分析 Capsimine 對其它癌症如肺、 乳癌等是否其有相同之藥理作用及共體內之藥效動力學後， Capsimine 可能具有臨床應用之潛力。另一方面，Solamargine 對肝癌細胞與正常細胞均具有很強的致 死毒性 (IC5O=1.5 μ g/ml) ， 同時對肝癌細胞與正常細胞之抑制曲線相重疊， 顯示 Solamargine 對兩者細胞之作用機轉一致，為了解其機轉，肝癌與正常細胞受藥物作用後之 細胞形狀與其 DNA 之改變情形分別以螢光顯微鏡及細胞分析儀 (Flowcytometry) 分析之， 其 Sub-G �螻茩M周期在 Solamargine 作用 3 小時後顯著增加，顯示 Solamargine 係經由 Apoptosis 機轉造成細胞死亡， 同時細胞癌壞死因子受器 I (Tumor necrosis factor receptor I) 之基因表現明顯的提高， 因此 Solamargine 可能係藉由調控細胞中 TNFR I 的基因表現，進而引發細胞進行自殺性死亡 Apoptosis) 的作用。以上結果正分別撰寫成論 文。 另外對於研究中藥在調節修補 DNA 酵素活性之偵測以應用於中藥抗腫瘤之研究上，目 前正進行中，其結果將於成果發表會中提出。 |
| 英文摘要 | Advancement has been made in the study of mechanism of tumorgenecity. To date, treatment of tumors in patients remains predominantly by surgery and chemotherapy. Although gene therapy has been shown an effective method in the treatment of cancers, it is not widely employed and restricted only in some particular tumors. Hepatocellular carcinoma (HCC) is a predominant malignancy in Taiwan. The clinical treatments for HCC by chemotherapy did not improve the mortality of HCC patients. Administration of medicines can inhibit the growth of HCC cell, but it also injures the normal cells. The mechanism for the tumor formation is not yet clear. However, it goes almost without saying that it is necessary to make changes in gene expression in order to convert a cell to the transformed phenotype. The tumor suppresser genes (p53 and Rb) were important in controlling cell-cycles and could prevent the development of tumors. They become oncogenic regulators when p53 and Rb were mutated or lost heterozygosity. The p53 and Rb mutations might be correlate with the tumorgenecity of HCC. The DNA reparative enzymes exhibit in cells for the automatic reconstruction of mutated sequences. Therefore, studies of the regulation of tumor suppresser genes expression and DNA reparative enzymes activities by Chinese herbs are essential. It can prevent a tumorgenecity by increasing the DNA reparative enzymes activities and the expression of tumor suppresser genes without interfering the function of normal cells. The Solanaceae Chinese herbs in Taiwan such as S. incanum, S~ capsicastrum, S, indicum, S. nigrum and S. lyratum have been reported as antihepatotoxic and antineoplastic agents. In this project, we studied the regulation of p53 and Rb gene expressions with Solanaceae herbs by RT- PCR. The mRNA secondary structures of p53 and Rb genes were predicted by MacDNASIS software. Primers designed according to the lowest free eneragy were synthesized. It shew that the gene expressions of p53 and Rb genes were not affected by the extracts of solanaceae herbs, suggesting another pathways for the anti-tumorogenecity of herbs. In order to realize the mechanism of cytotoxicity of solanaceae herb, the activity was determined by [ �� H]thymidine incoporation. The results indicated that S. lyratum possessed a potent cytotoxicity in Hep3B cells in the concentration of 900 μ g/ml/l x 10 �� cells. The S. incanum which has been widely used in the treatment of hepatitis exhibits no cytotoxicity to the cells. In order to further investigation of the activity of solanaceae herbs, capsimine and solamargine were then purified from S. lyratum and S. incanum, respectively. Capsimine selectively inhibited the growth of Hep3B cell in 2 μ g/ml/l x 10 �� cells, and 60% of Hep3B cells died in the concentration of 15 μ g/ml. However, the normal cell was not affected in the concentration of 15 μ g/ml. However, the normal cell was not affected in the concentration (2-15 μ g) of capsimine. Thus, it suggests that manipulation of the dose of capsimine would selectively inhibite the Hep3B cells. Once the further investigation of the pharmacokinetics and effects of capsimine in another tumors was studied, capsimine migh be potentially used in clinical trail. On the other hand, solamargine possessed a potent cytotoxicity to human hepatocyte (Hep3B) and normal skin fibroblast. The inhibition curves of solamargine to the both cells wer essentially overlapped, suggesting a parallel effect for the cell death. To define mechanism of cytotoxicity of solamargine, the changes of morphology and DNA content in cells were studied. A sub-G �� cell stage was drastically increased after 3-h incubation with solamargine. The results evidence that solamargine arises cell death by apoptosis. In addition, the gene expression of TNFR I and II wer up- regulated within 30 min of solamargine treatment. Since TNF Receptors have been involved in apoptosis, the overexpression of TNF receptors may be related with the mechanism of cytotoxicity of solamargine. In addition, regulation of the activity of DNA reparative enzymes by Chinese herbs is currently studied. This study illustrate the antitumor mechanism of Solanaceae herbs, and will provides a new direction in the treatment of HCC by Chinese herbs. |
本系統中英文摘要資訊取自各篇刊載內容。