抑制人類非小細胞肺癌細胞生長之模式

Establishment of tumor suppressing models of human non-small cell lung cancer cells

邱建智(chien-chih chiu)

指導教授 ： 方剛

國立臺灣師範大學/理學院/生命科學研究所/博士(2003年)

摘要

無資料

關鍵字

GCV ； 細胞凋亡 ； 人類非小細胞肺癌 ； VP-16 ； 老化 ； 延滯性細胞死亡 ； p53

並列摘要

Lung cancer is the leading cause of death in Taiwan, and the non-small cell lung cancer (NSCLC) represents 80% of the total lung cancer cases. New therapeutic paradigms are constantly pursued and improved. Thus, in this thesis, gene therapy using suicide gene HSV-tk/GCV strategy was evaluated in NSCLC cells. On the other hand, the molecular pathway in NSCLC cells induced by chemotherapeutic agent, VP-16 was also discussed in-depth in this study.
In the part I of thesis, NSCLC cells were transfected with a recombinant prodrug herpes simplex virus type I thymidine kinase (HSV-tk) cDNA, and the selected clones underwent apoptosis in response to induction by antiviral ganciclovir (GCV). The efficiency of GCV-induced growth inhibition and the extension of the bystander effect were associated with the expression level of HSV-tk in stable transfectants. Western blot was used to analyze the release of the apoptosis initiator cytochrome c and activation of the downstream effectors caspase-9, caspase-3 and poly(ADP-ribose) polymerase 16 hr after GCV sensitization, followed by transient escalation of tumor-suppressor p53 and cell-cycle modulators cyclin A and B1 before committing to apoptosis cell death. These findings demonstrated that the HSV-tk/GCV system effectively inhibited the proliferation of NSCLC cells in vitro and in vivo through induction of apoptosis, therefore providing a rationale for further development.
In the part II of thesis, we found that the dynamic changes occurring in NSCLC, H1299 (null-p53) cell populations during protracted VP-16 (DNA-topoisomerase II inhibitor) exposure. Flowcytometry was used to analyze changes in cell cycle progression in H1299 cells, and showed a significant decrease in G1 phase cells with a concurrent increase in G2/M phase cells following VP-16 treatment. Result of western blotting showed cytochrome c releasing from mitochrondria occurred in early stage of apoptosis. Activation of caspase-7 was detected, while the cleavage of caspase-3 was not seen. This work also showed that an immediate accumulation of cell cycle modulators, cyclin A and B1 was visualized during VP-16-sensitized cell arrest at G2/M-phase. Whereas upregulation of p21waf/cip prevented cells to overcome mitotic arrest before entering programmed cell death. In addition, the presence of caspase specific inhibitor abrogated caspase activation as well as VP-16-induced apoptotic phenotype. The finding of apoptosis mediated by VP-16 in subsets of human NSCLC cells provides an opportunity for targeted therapy for cancers caused by p53 deletion.
In the part III of thesis, we have identified a pathway leading to VP-16-induced dose-dependent cell death in highly metastatic and tumorigenic NSCLC, H1437 cells with mutant p53. It was found that H1437 cells exposing to VP-16 was susceptible to cell proliferation and the effect proved irreversible both in vivo and in vitro. The inhibition of cell proliferation was triggered by senescence events exerted by low-dosage VP-16. In addition, H1437 cells underwent growth arrest at G2/M-phases prior to emergence of sub-G0/G1 cells. To further address the pathway in VP-16-induced H1437 cells, we have observed transient elevation of cyclin-dependent kinase inhibitor, p21CIPI/WAF1 and tumor suppressor, p16INK. At the meaning time, persistent escalation of endogenous cell cycle modulators, cyclin A and B1 signifantly induced DNA repair upon DNA damage before cell death. H1437 cells underwent apoptosis triggered by VP-16 at day 6 of exposure and were detected by annexin-V assay. Thereby, low-dosage VP-16 can be an effective agent in treating human NSCLC cells with mutated p53 genotype through a delayed-apoptotic pathway

並列關鍵字

GCV ； apoptosis ； human non-small-cell-lung-cancer ； VP-16 ； senescence ； delayed-apoptosis ； p53

參考文獻

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