- 學位論文
抗凋亡蛋白在廚房油煙誘發肺腺癌細胞存活與增生之角色研究
The Role of Inhibitory of Apoptosis Proteins in Lung Adenocarcinoma Cell Survival and Proliferation Induced by Cooking Oil Fumes
摘要
文獻指出廚房油煙 (cooking oil fumes, COF) 暴露與台灣女性肺癌發生有關。本研究室先前之研究結果指出，COF會經由活化NF-κB路徑，誘發肺腺癌細胞Cox-2基因過度表現，而NF-κB路徑活化能影響腫瘤細胞之存活，因此假設長時間暴露COF可能會促進肺腫瘤細胞的增生。本研究以肺腺癌 A549細胞為研究模式，探討COF 是否確實會誘發肺癌細胞存活與生長，並了解其可能之分子機轉。首先於MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide) 之細胞存活實驗中發現，當細胞處理COF 48小時後會增加細胞之存活率，而流式細胞細胞儀之結果亦顯示COF暴露會增加細胞 S phase之分佈比率。西方點墨法之結果顯示，IAP2蛋白會隨著COF濃度增高，增強IAP2 (inhibitory of antiapoptosis protein 2) 蛋白的表現量。當同時處理NF-κB抑制劑 BAY，則會阻斷COF誘導IAP2蛋白之能力。這顯示NF-κB 路徑可能參與 COF 誘導IAP2蛋白之表現。COF除了會誘導IAP2表現外，是否亦會誘發其他三種IAPs 蛋白的表現（IAP1、survivin以及XIAP）？這些蛋白之表現與COF所誘導肺腺癌細胞增生是否有關？結果顯示，IAP1，IAP2及 survivin蛋白會分別被 COF, BaP，及2,4-DDE誘導而表現，其中以IAP2蛋白之誘導表現較強。而XIAP的表現卻會被 COF及2,4-DDE抑制，但不會被BaP影響。雖然COF對IAP2及XIAP之表現有相反之作用，但是COF仍然會抑制凋亡蛋白 caspase-3 之活性，而使細胞存活與增生。因此爲了反應實際生活中，台灣婦女暴露COF引起女性肺癌之可能機轉，本研究即以COF混合物來探討誘發IAP2的可能參與之訊號路徑。首先將三種不同訊息路徑之抑制劑（SB203580，PD98059，LY294002）分別與COF同時處理細胞，IAP2 蛋白表現可被PI3K 之抑制劑LY294002 抑制，而SB203580 (p38) 及PD98059 (ERK) 則無此能力。在細胞週期 sub-G1 phase 與 TUNEL assay 之結果顯示，LY294002 會促進肺癌細胞之凋亡，因此除了 NF-κB路徑之外，PI3K在COF所誘導肺腺癌細胞增生與存活可能亦扮演重要之角色。爲了解COF誘導A549細胞增生，是否與細胞週期相關之調控蛋白有關，本研究同樣以西方墨點法得知，COF確實會促進cyclin D1的表現，卻會顯著抑制p21蛋白的表現。由以上之結果得知，活化 IAP1, IAP2, survivin 以及cyclin D1蛋白之表現與抑制 XIAP，caspase-3，以及p21蛋白之表現，可部份解釋COF誘導肺腺癌細胞之存活與增生的分子機轉。至於COF引起肺癌細胞增生是否與ROS產生有關？本研究以DCF-dA分析ROS之形成，發現COF處理肺腺癌細胞會產生ROS，並發現COF所誘導ROS之產生可能是經由PI3K活化，進而誘導p-Akt之表現，促進肺腺細胞之增生。總結，本研究結果發現COF暴露會促進肺腺癌細胞之存活與增生，有助於了解廚房油煙暴露與台灣女性肺癌的形成之分子機轉，在台灣不抽菸女性肺癌之預防與治療策略上提供學理之參考及助益。
並列摘要
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.