| Summary: | 碩士 === 中山醫學大學 === 醫學研究所 === 99 === Lung cancer is the leading cause of death worldwide from all cancers, and chemotherapy is the common treatment for lung cancer patients. The development of drug resistance is the single most important cause of treatment failure in patients with chemotherapy. When patients become resistant to chemotheraputic drugs, it is commonly developed cross-resistance to other anti-cancer drugs. In previous experiments, we have used A549 cells to select docetaxel resistant cell lines and two sublines of A549/D16 and A549/D32 were established. We have found these two sublines were more sensitive to Alimta than the parental A549 cells. Therefore, we further characterize Alimta sensitivity in these two A549/D16 and A549/D32 cells. We applied MTT assay, Clonogenic assay, Flow cytometry to determine the Alimta sensitivity in A549/D16 and A549/D32 cells. The results demonstrated that A549/D16 and A549/D32 are 4-fold more sensitive to Alimta when compared with the parental cells by IC50 of MTT assay. Furthermore, When A549/D16 cells were treated with Alimta, the thymidylate synthase (TS) protein was induced on the first day then decreased the expression with time-dependence; p53 and p21 proteins were induced on the first day and maintained the protein level until the fourth day, along with the increased apoptotic sub-G1 cells. The target genes of Alimta: TS, dihydrofolate reductase (DHFR), glycinaminde ribonucleotide formyl transferase (GARFT) and drug metabolism-related genes: reduced folate carrier (RFC), gamma-glutamyl hydrolase (GGH) expression were decreased in A549/D16 and A549/D32 cells. These results suggested that sensitivity of Alimta in A549/D16 and A549/D32 cells may be closely associated with these genes. Then we cloned the open reading frame of TS and GGH to p3XFlag-CMV-10 vector, and transfection to cell to examine their expression. Overexpression of TS and GGH in A549/D16 and A549/D32 cells to reduce Alimta sensitivity are required for research in the future. Moreover, we also found that Lipocalin-2 (LCN2) expression was increased in A549/D16 and A549/D32 cells, but inhibibted LCN2 expression by shRNA has no significantly effects in the sensitivity of Alimta in A549/D16 and A549/D32 cells.
Our results demonstrated that it is not cross-resistant, but more sensitive to Alimta in docetaxel resistant cell lines of A549/D16 and A549/D32.
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