Part I 探討薑黃素抑制肺癌細胞端粒酶活性及分子調控機轉;Part II ALIMTA抑制肺癌細胞機轉之探討

Part I Molecular mechanism of curcumin-mediated telomerase in the A549 lung carcinoma cells;Part II Study the tumor inhibition of ALIMTA in A549 lung carcinoma cells

詹琇雯(Hsiu-Wen Chan)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00075

摘要

Part I、探討薑黃素抑制肺癌細胞端粒酶活性及分子調控機轉

端粒酶負責合成位在染色體末端的端粒DNA，維持染色體穩定性。hTERT (human telomerase reverse transcriptase) 基因是主要催化端粒酶活性的一個基因，抑制hTERT基因表現會喪失端粒酶活性，所以調節hTERT基因的轉錄可以作為治療癌症的一個方向。本篇研究薑黃素抑制肺癌細胞端粒酶活性及分子調控機轉。本研究薑黃素明顯可抑制肺癌端粒酶活性及減少hTERT mRNA表現量，以西方點墨分析法觀察到hTERT蛋白質隨著薑黃素濃度的上升而明顯減少。我們短暫轉染及帶有不同端粒酶啟動子構築於pGL3載體到A549細胞，以Reporter gene assay活性分析，結果指出E-box參與端粒酶啟動子轉錄，也以EMSA來證實薑黃素會減少c-Myc與端粒酶啟動子結合的能力。除此之外，薑黃素會促使hTERT蛋白質從細胞核移至細胞質，造成端粒酶不穩定，這些結果指出端粒酶活性降低是因為hTERT蛋白質改變座落位置。本研究闡釋薑黃素可經由抑制hTERT基因表現量及造成hTERT蛋白位置改變等轉譯後修飾作用機轉，而達到抑制端粒酶活性。

Part II ALIMTA抑制肺癌細胞機轉之探討

Pemetrexed (ALIMTA) 上用於治療非小細胞肺癌和惡性肋膜間皮癌(Malignant Pleural Mesothelioma，MPM)。Pemetrexed除了抑制葉酸酵素活性 (TS、DHFR、GARFT)外，對於其他的作用機制仍不是很清楚。本篇主要研究Pemetrexed其他的抗癌機轉。Pemetrexed可效抑制癌細胞成長，細胞群落生成數目，並具有顯著性意義 (P<0.05)。我們使用Oligo GEArry 晶片來偵測480 個基因表現量改變，基因改變和細胞週期停滯有關的為p53、p21，和抗血管新生的Lipocalin 2基因。細胞處理高濃度Pemetrexed造成細胞凋亡。低濃度的Pemetrexed引起細胞週期停滯於G1期，可能是透過p21來調節。另外，Pemetrexed無法抑制端粒酶活性。並以Western blot證實Pemetrexed對肺癌A549細胞可誘發p53及p21的產生。另外以流氏細胞儀分析，無論有無p53表現的細胞株也可受Pemetrexed影響造成細胞週期停滯於G1期。本篇研究闡釋Pemetrexed另一抗癌機轉透過誘發p21表現量增加造成細胞週期停滯。

關鍵字

薑黃素 ； 愛寧達

並列摘要

Part I、探討薑黃素抑制肺癌細胞端粒酶活性及分子調控機轉
Telomerase maintains chromosomal length and stability thus leading to cellular immortalisation. The hTERT (human telomerase reverse transcriptase) subunit seems to be the rate-limiting determinant of telomerase and knowledge of factors controlling hTERT transcription may be useful in therapeutic strategies. This study was aimed at identifying the molecular mechanisms by which curcumin inhibits telomerase activity in the A549 lung cancer adenocarcinoma cell. Curcumin caused a significant reduction of telomerase activity as detected using the telomeric repeat amplification protocol, and inhibition correlated with decreased hTERT mRNA. Western blot analysis showed that curcumin significantly decreased hTERT protein in a dose-dependent manner. The results were also confirmed by transient transfection of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter sequences cloned upstream of luciferase reporter gene with curcumin. It indicated that E-box have the ability to regulation hTERT promoter activity. Electrophoretic mobility shift assays demonstrated that binding activity of c-Myc transcription factor to the E-box sequence on the hTERT promoter was inhibited in response to curcumin. Here, hTERT protein was found only in the cytoplasm and not detectably in the nucleus by curcumin. These observations suggest that curcumin enhances telomerase decrease is accompanied by change in hTERT protein location.

Part II ALIMTA抑制肺癌細胞機轉之探討
Pemetrexed (ALIMTATM) was approved as a second-line, single-agent treatment of locally advanced non-small cell lung cancer (NSCLC). In vitro studies have shown that Pemetrexed inhibits thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT), all folate-dependent enzymes involved in the de novo biosynthesis of thymidine and purine nucleotides. Here we have evaluated the potential anti-cancer action of Pemetrexed. Pemetrexed was able to inhibit the tumor cells growth as compared to the control with significant differences by colony formation (p<0.05). To study the difference of gene expression patterns by Pemetrexed in non-small cell lung cancer, the oligo GEArry system were used to determine the regulation corresponding to 480 genes. Our results showed different expression of p53、p21 and Lipocalin 2 genes associated with cell cycle and angiogenesis. The cell cycle distribution was analyzed in cells exposed to Pemetrexed by flow cytometry. Pemetrexed induced G1 phase arrest, probably p53-independent by p21 expression. We thus demonstrate a novel mechanism by Pemetrexed, leads directly to cell cycle arrest by p21, may be a useful approach to enhance pemetrexed-based chemotherapy.

並列關鍵字

Curcumin ； ALIMTA

參考文獻

Aggarwal, B.B., Shishodia, S., Takada, Y., Banerjee, S., Newman, R.A., Bueso-Ramos, C.E. and Price, J.E. (2005) Curcumin suppresses the paclitaxel-induced nuclear factor-kappaB pathway in breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice. Clin Cancer Res, 11, 7490-7498.

Araujo, C.C. and Leon, L.L. (2001) Biological activities of Curcuma longa L. Mem Inst Oswaldo Cruz, 96, 723-728.

Asaad, N.Y., Abd El-Wahed, M.M. and Mohammed, A.G. (2006) Human telomerase reverse transcriptase (hTERT) gene expression in thyroid carcinoma: diagnostic and prognostic role. J Egypt Natl Canc Inst, 18, 8-16.

Assaraf, Y.G. (2007) Molecular basis of antifolate resistance. Cancer Metastasis Rev, 26, 153-181.

Ayer, D.E., Kretzner, L. and Eisenman, R.N. (1993) Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell, 72, 211-222.