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

Abstract

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.