| Summary: | 博士 === 高雄醫學大學 === 醫學研究所博士班 === 94 === Cooking oil fumes (COF) exposure was demonstrated to be associated with lung cancer development in Taiwanese nonsmoking women. Previous studies showed that Cox-2 overexpression and oxidative DNA damage were observed in CL-3 lung adenocarcinoma cells after exposed to COF. Involvement of COF in lung tumorigenesis might be mediated through induction of IAPs family protein expressions to cause cell survival and proliferation. To test the hypothesis, A549 lung adenocarcinoma cell line was used in this study. After the cells exposed to various concentrations of COF for 48 hr, the cell viability of A549 was unexpectedly increased in a concentration-dependent manner based on MTT assay. In addition, flow cytometery data showed that the proportion of A549 cells at S-phase was markedly increased after exposure of COF. To elucidate whether the antiapoptotic c-IAP2 (IAP2) was involved in COF-improved cell survival, Western blot data indicated that IAP2 protein level was significantly induced by COF in a concentration-dependent manner. Moreover, the effect of BAY, a NF-κB binding inhibitor, on the COF-induced IAP2 protein level was shown that NF-κB activation by COF may partly be involved in IAP2 induction. Thus, the positive impact of COF on cell survival and proliferation of A549 lung tumor cells may be result in induction of IAP2 overexpression at least in part through NF-κB signaling pathway. To verify whether other antiapoptotic proteins were also associated with lung cancer cell survival and proliferation, other IAP family proteins, including IAP1, XIAP and survivin expressions, were evaluated by Western blotting after the cells were treated with COF and its two components, BaP and 2,4-DDE. Our data show that IAP1 and survivin expression levels were increased by COF, BaP, and 2,4-DDE, but not like IAP2 induction in a dose-dependent manner. On the contrary, XIAP surprisingly was decreased by COF and 2,4-DDE, but not by BaP. Even though there were different effects of COF on IAP2 and XIAP protein expressions, the expression of apoptotic caspase-3 was diminished by COF. Therefore, to mimic the effects of exposure to a complex mixture of COF on Chinese female lung cancer development, the role of IAPs attenuated by COF on lung cancer cell proliferation was investigated. To verify which signaling pathway was responsible for IAP2 induction four inhibitors, SB203580 (p38 MAPK), PD98059 (ERK), BAY (NF-κB), and LY294002 (PI3K), were added to the A549 cells. IAP2 expressions induced by COF were abolished by the additions of BAY and LY294002, but not affected by SB203580 and PD98059 treatments, suggesting that IAP2 induction by COF may be mediated through the NF-κB and PI3K pathways. To further verify the role of both pathways in IAP2 induction by COF, the down-stream Akt and phospho-Akt (p-Akt) protein expressions were also determined by Western blotting after the cells were concomitantly treated with BAY and LY294002. Our data demonstrate that p-Akt expression increased with COF treatment and the expression level decreased with the addition of LY294002, but not by BAY. These results suggest that the PI3K pathway might play a more direct role in the IAP2 induction by COF. The effect of COF on the cell cycle of A549 cells was further investigated by flow cytometry. The proportion of the S-phase of A549 cells increased significantly with the COF treatment. Additionally, we also show that the cell cycle regulation involved in A549 cell proliferation is associated with induction of cyclin D1 and reduction of p21Cip1/Waf1 protein expression. Taken together, increases of IAP1, IAP2, survivin and cyclin D1 expressions and decreases of XIAP, caspase-3, and p21 expressions might partly contribute to lung cancer cell survival and proliferation after COF exposure.
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