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Genetic Determinants of Pemetrexed Responsiveness and Nonresponsiveness in Non- small Cell Lung Cancer Cells  Ming-Fang Wu, MD, PhD, Yi-Min Hsiao, PhD,

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Presentation on theme: "Genetic Determinants of Pemetrexed Responsiveness and Nonresponsiveness in Non- small Cell Lung Cancer Cells  Ming-Fang Wu, MD, PhD, Yi-Min Hsiao, PhD,"— Presentation transcript:

1 Genetic Determinants of Pemetrexed Responsiveness and Nonresponsiveness in Non- small Cell Lung Cancer Cells  Ming-Fang Wu, MD, PhD, Yi-Min Hsiao, PhD, Chuan-Fu Huang, MD, PhD, Yu-Hsin Huang, MS, Wan-Jung Yang, BS, Hsiu-Wen Chan, MS, Jinghua Tsai Chang, PhD, Jiunn-Liang Ko, PhD  Journal of Thoracic Oncology  Volume 5, Issue 8, Pages (August 2010) DOI: /JTO.0b013e3181e0b954 Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

2 FIGURE 1 Cytotoxic effect of pemetrexed in H460, H1355, H1299, and A549 lung cancer cells and normal lung fibroblast cells MRC-5. Cells (5 × 103 cells/well) were treated with increasing doses of pemetrexed (0, 0.08, 0.4, and 2.0 μM) for 72 hours. Cell viability was measured by MTT assay and the results are presented as the calculated cell growth inhibitory ratio. Experiments were repeated three times. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e0b954) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

3 FIGURE 2 RT-PCR analysis of TS, DHFR, GARFT, RFC, and FPGS mRNA in A549 and H1355 cells. A549 and H1355 cells (8 × 105 cells/60 mm dish) were subjected to varying concentrations of pemetrexed (0, 0.08, 0.4, and 2.0 μM) for 48 hours, and mRNA of TS, DHFR, GARFT, RFC, and β-actin was determined. RT-PCR, reverse transcriptase polymerase chain reaction; TS, thymidylate synthase; DHFR, dihydrofolate reductase; RFC, reduced folate carrier; FPGS, folylpolyglutamate synthase. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e0b954) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

4 FIGURE 3 Cell cycle distribution by flow cytometry in A549 and H1355 cells. A549 and H1355 cells (8 × 105 cells/60 mm dish) were subjected to varying concentrations of pemetrexed (0, 0.08, 0.4, and 2.0 μM) for 48 hours. Cells were resuspended in dulbecco modified Eagle medium (DMEM) at 1 × 106 cells/ml. Cells were detected by flow cytometer and acquired by CellQuest. Data is shown as the percentage of nuclei in the sample and is representative of one of the three experiments performed. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e0b954) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

5 FIGURE 4 Western blot analysis of p16, p21, p27, P53, cyclin, and CDK in A549 and H1355 cells. A549 and H1355 cells (5 × 105 cells/60-mm dish) were subjected to varying concentrations of pemetrexed (0, 0.08, 0.4, and 2.0 μM) for 48 hours. Total amounts of protein expression were determined by immunoblot experiments with the corresponding antibodies. Expression of β-actin on the same immunoblot was used as a loading control. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e0b954) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

6 FIGURE 5 Genetic analysis by superarray. The oligo GEArray system was used to determine the regulation of 480 genes (for a gene table, see Briefly, a total of 3 μg were reverse transcribed into Biotin-16-dUTP-labled cDNA probes with the use of turelabling-AMP 2.0 method, according to the manufacturer's protocol. Cell cultures were incubated in 10 μM pemetrexed-containing or pemetrexed-free medium for 48 hours. A, A549. B, Pemetrexed-treated A549. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e0b954) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

7 FIGURE 6 RT-PCR analysis of Lcn-2 and nm23–H1 mRNA in A549 and H1355 cells after pemetrexed treatment. A549 and H1355 cells (8 × 105) were subjected to varying concentrations of pemetrexed (0, 0.08, 0.4, and 2.0 μM) for 48 hours, and mRNA of Lcn-2, nm23–H1, and β-actin was determined. RT-PCR, reverse transcriptase polymerase chain reaction. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e0b954) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

8 FIGURE 7 Western blot comparisons of Lcn-2, p53, and β-actin expressions in A549 and H1355 cells. A549 and H1355 (5 × 105 cells/60 mm dish) were treated with 0.08, 0.4, and 2 μM pemetrexed for 48 hours, and the total amount of protein expression was determined by immunoblot experiments with the appropriate antibodies. Expression analysis of β-actin served as a loading control. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e0b954) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

9 FIGURE 8 Cytotoxic effects of pemetrexed in A549 Shluc, A549 Shnm23–H1, and A549 ShLcn2 cells. A549 Shluc, A549 Shnm23–H1, and A549 ShLCN2 (5 × 103 cells/well) cells were treated with varying concentrations of pemetrexed (0.08, 0.4, 2, and 10 μM) and MTT assayed after 72 hours of treatment. Cell viability was measured and the results are presented as the calculated cell growth inhibitory ratio. Experiments were repeated three times. Journal of Thoracic Oncology 2010 5, DOI: ( /JTO.0b013e3181e0b954) Copyright © 2010 International Association for the Study of Lung Cancer Terms and Conditions

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