| Summary: | 碩士 === 中山醫學大學 === 毒理學研究所 === 92 === 1. p53 is a tumor suppressor gene, which plays a central role in the native control of growth, survival and migration of abnormal cells. The protein level of p53 in normal cells is extremely low. The aim of the study was invastigate the complex consequences on the cellular responses of RNA interference of p53 in A549 cells to anticancer drug, cisplatin. When p53 expression was inhibited by RNA interference in an adenocarcinoma cell line A549 (p53 RNAi/A549 cells), cells exhibited a faster growth rate as a greater percentage of cells in S phase than A549 cells with wild type p53 (pcDNA/A549 cells). But, point mutation p53 overexpression in A549 cells (H179Y/A549 cells) reduced growth rate as a lower percentage of cells in S phase than pcDNA/A549 cells by flow cytometry assay. However, the effect of cisplatin to cells depleting of p53 reduced p21 expression that caused to G1 arrest. Bax and COX-2 had less expression and more sensitive in p53 RNAi/A549 cells after treated with cisplatin. Cells lacking of p53 were also more sensitive to cisplatin treatment by MTS assay, counting cell numbers and flow cytometry measuring after different days. In contrast to cell cycle and cell mortality analysis, p53 RNAi/A549 cells had a better colony formation ability after cisplatin treatment. It was suggest that cisplatin had more cytotoxicity to pcDNA/A549 cells than p53 RNAi/A549 cells. In wound migration assay, the motility of p53 RNAi/A549 cells was lower than pcDNA/A549 cells but H179Y/A549 cells had a highest motility. It was due to the changes in MMP-2 activity secreted by A549 cells. We also observed different proteins that changed between p53 RNAi/A549 cells and pcDNA/A549 cells using 2-DE gel analysis. We are going to identify these proteins in the future.
2.Fungal immunomodulatory proteins, FIP-gts, were found in Ganoderma tsugae. The protein has been implicated which activates human peripheral blood mononuclear cells. However, the effect of FIP-gts in cancer cells has not clearly been described. Metastasis was one of the most difficult problems in lung cancer therapy. We observed the A549 cells turn into round shape and were not changed cell shapes in normal cell line-BEAS-2B after FIP-gts treatment. MTS anlysis and counting cell numbers decreased the viability of A549 cells. In colony formation assay, FIP-gts showed cytotoxicity to A549 cells. FIP-gts induced p53 expression that in terms induced p21 activity, which caused in G1 arrest. In addition, procaspase-3 by western blotting was decreased. Similarly, there was a small amount of subG1 increased after FIP-gts treatment. But, COX-2 that has the function of anti-apoptosis, increased after treated with FIP-gts. In wound healing assay, the level of motility were decreased by the increasing concentration of FIP-gts. MMP-2 activity was also decreased measured by gelatin-zymography. In RT-PCR, the level of MMP-2 decreased but TIMP-2 had no effect and the MMP inhibitor TIMP-1 and PA inhibitor PAI were increased after treated with FIP-gts. Alltogether, FIP-gts could not only cause cytotoxicity but also potentially inhibit metastasis.
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