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題名 | 探討靈芝免疫調節功能蛋白抑制終端酶活性進而限制肺癌細胞生長機制=Blocking Telomerase by Fungal Immunomodulatory Protein, FIP-gts, from Ganoderma Tsugae is a Major Mechanism for Limiting the Growth of Human Lung Cancer Cells |
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作者 | 柯俊良; | 書刊名 | 中醫藥年報 |
卷期 | 25:2 2007.10[民96.10] |
頁次 | 頁67-103 |
專輯 | 中醫藥基因體及免疫學研究 |
分類號 | 414.33 |
關鍵詞 | 松杉靈芝; 免疫調節蛋白; 染色體終端酶; Ganoderma tsugae; Immunomodulatory proteins; Telomerase; |
語文 | 中文(Chinese) |
中文摘要 | 靈芝一直以來被視為傳統中藥的翹楚,在細胞和動物實驗證實靈芝具有保護肝臟、抗腫瘤等功能。腫瘤的治療一直是生物醫學領域相當被重視的焦點,尤其近年來許多學者著重於促進腫瘤細胞死亡來達到減緩腫瘤形成的目的。由Hanahan和Weinberg等人指出癌症形成的六大特徵其中擁有無限次複製能力是癌化過程中必備的條件(Hanahan and Weinberg, 2000),所以終端酶在癌化過程中扮演著不可或缺的角色。最近有文獻指出在裸鼠模式中發現服用茶多酚類化合物epigallocatechin gallate(EGCG)後會明顯抑制腫瘤生長及染色體終端酶活性(Naasani et al, 2003),因此增加中草藥對於抗癌方面的科學證據。綜合以上觀點本計畫著重於FIP-gts抑制終端酶活性作用機制進而抑制癌細胞的生長,達到抗癌的作用。我們初步實驗證實松杉靈芝蛋白(FIP-gts)會造成A549肺癌細胞皺縮並抑制細胞生長,然而對人類正常肺纖維母細胞MRC-5毫無影響。接著進一步分析是否有抗腫瘤的特性,首先以染色體終端酶重覆增幅步驟(Telomeric Repeat Amplification Protocol, TRAP assay)分析有無處理FIP-gts後肺癌細胞株A549終端酶活性表現,結果發現隨著FIP-gts濃度增加其活性減少呈現劑量關係,以半定量和定量RT-PCR分析結果發現hTERT mRNA隨著FIP-gts濃度增加而減少轉錄表現量,接著以luceriferase活性分析,結果發現隨著FIP-gts濃度增加而減少hTERT promoter活性。接著並將其構築完成的hTERT-pGL3在經一系列的缺失短小突變決定其FIP-gts所影響的位置,結果發現在-196 to -177的區域為E-box site,主要是bHLHZ蛋白家族所結合的區域,其中c-Myc為轉錄因子會直接影響htert轉錄活化。然而我們發現重組FIP-gts並不會影響c-Myc蛋白的表現量但會降低磷酸化的表現。而我們利用EMSA證實c-Myc蛋白明顯降低與E-box結合能力。本實驗研究結果證實松杉靈芝菌絲體中純化的免疫調節功能蛋白具有抗癌的功效。此免疫調節蛋白可應用於抑制肺癌細胞的終端酶活性。所以將來有發展出治療或搭配其他抗癌藥物一起處理抵抗腫瘤並減少腫瘤細胞的再復發。 |
英文摘要 | Telomerase expression is the hallmark of tumor cells in which this ribonucleoprotein complex activation may therefore be a rate-limiting or critical step in cellular immortalization and oncogenesis. Fungal immunomodulatory proteins, FIP-gts, were found in Ganoderma tsugae. The protein has been implicated which activates human peripheral blood mononuclear cells. However, the effet of FIP-gts in cancer cells has not clearly been described. In the present study, we expressed and purified the recombinant fungal immunomodulatory protein (reFIP-gts) in E.coli. First, treatment of A549 cells with reFIP-gts significantly inhibited their growth but not affect in MRC-5, which is human normal lung fibroblast. We demonstrate that reFIP-gts suppressed telomerase activity in a concentration-dependent manner. Repression of telomerase activity preceded a decrease in expression of the telomerase catalytic subunit (hTERT) and at the mRNA level, suggesting that the reFIP-gts decrease in telomerase activity may be regulated telomerase subunit transcription. These results were also confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter sequences cloned upstream of a luciferase reporter gene with reFIP-gts. Further analysis by luciderase assays using a series of constructs containing unidrectionally delected fragments revealed that a 19-bp region (-196 to -177) was show clearly the FIP-gts transrepresses the hTERT promoter through an E-box located downstream of the hTERT transcription initiation site. Finally, electrophoretic mobility shift assays demonstrated that the binding activity of c-Myc transcriptional factor to the E-box sequence on the hTERT promoter was inhibited in response to reFIP-gts. These results show that FIP-gts could suppress telomerase activity and inhibited by transcriptional regulation of hTERT via a c-Myc responsive element-dependent mechanism. Our findings provide new insights into both the anticancer function of FIP-gts and the regulation of hTERT via a c-Myc responsive element-dependent mechanism. Our findings provide new insights into both the anticancer function of FIP-gts and the regulation of hTERT/telomerase expression, will be a very promising a chemopreventive agent candidate. |
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