Mechanism of Iodoacetic acid-Induced Apoptosis in Human Lung Cancer Cells

肺癌目前乃是臨床上相當棘手之癌症，目前除使用外科手術及cytotoxic chemotherapy之外，即使用最新二種之EGFR tyrosine kinase inhibitors (Iressa或 Tarceva)，77% NSCLC病人短期內，有明顯腫瘤縮小反應，特別是對東亞非吸煙之女性有明顯療效。雖然立即藥效明顯，可惜目前臨床試驗用上述二種EGFR-TKI之最新結果，相當令人失望，因在使用約6-12月後，原來對TKI inhibitors有反應的病患會產生抗藥性。由以上簡述得知目前國內外對NSCLC以TKI inhibitors之最新治療仍陷入困境(Sharma et al., 2007)。本研究乃探討以iodoacetic acid (IAA)處理人類肺癌細胞株，探討IAA對肺癌細胞之生長影響及其作用機轉。目前已知，肺癌病人中之Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)基因表現有明顯上升，而IAA是GAPDH的抑制劑，而GAPDH除了扮演醣解作用裡的重要角色，GAPDH似乎扮演其他較少被了解的功能。本研究結果顯示，IAA可經由抑制人類肺癌(A549，NCI-HOP62，NCI-H23， NCI-EKVX，H522及H1299)細胞生長及促進凋亡之方式，造成對細胞的傷害。起先利用MTT與Hoechst螢光染色觀察，研究的結果顯示IAA對人類肺癌細胞的生長及分化會產生dose-dependent的毒性，然而利用免疫化學染色技術,我們可觀察到人類肺癌細胞在經過IAA處裡後，發生cytochrome c釋出及caspase活化及凋亡的比例增加。進一步實驗結果顯示經Hoechst染色後發現染色質有縮濃的現象、Annexin V(+)的螢光強度及細胞數目增加，由此可知，細胞死亡很可能是經由細胞凋亡所形成。為了進一步的研究IAA造成細胞凋亡的機制，由MCB螢光染色發現IAA會引起細胞內Glutathione( GSH )減少，並且IAA會引起細胞內ATP含量下降。另外，我們發現IAA會下降細胞的生長及細胞群落形成的能力。綜合我們的實驗結果，我們發現IAA會造成人類肺癌細胞的凋亡。對於IAA造成人類肺癌細胞的凋亡途徑的了解，將有助於提供治療目標，進而治療人類肺癌提升治療效果。Lung cancer is very difficult to be cured even using target gene therapy. In East Asia’s non-smoking women Iressa and Tarceva, two newly developed EGRF tyrosine kinase inhibitor (EGFR TKIs), largely reduce the tumor size in 77% patients in a few weeks time. In most of the lung patients who treated with EGFR TKIs, however, it is very disappointing that the cancer is recurrent within 6-12 months with drug resistance (Sharma et al., 2007). Iodoacetic acid (IAA) is not only an inhibitor of GAPDH but also plays a crucial role in variety novel cellular functions. Using human lung cancer cell lines, I investigated possible mechanism(s) of IAA-induced cell death. My results showed that IAA induced dose-dependent cell death, including A549, NCI HOP62, NCI H23 NCI EKVX, H522 and H1299. I also found that IAA induced cytochrome c release, caspase-3 activation, and chromatin condensation in A549. Furthermore, IAA induced intracellular ATP and GSH depletion, and pretreatment with either NAC or DTT prevented IAA-induced cell death. In conclusion, my data demonstrated that IAA effectively induced cell death in at least six lung cancer cell lines. It might provide possible target therapy for future clinical use.圖次………………………………………………………………………VⅢ次……………………………………………………………………… IX錄………………………………………………………………………X 要……………………………………………………………………… IVbstract……………………………………………………………………VI論………………………………………………………………………1料與方法………………………………………………………………12果………………………………………………………………………20論………………………………………………………………………29論與展望………………………………………………………………36…………………………………………………………………………37…………………………………………………………………………40錄………………………………………………………………………56考文獻…………………………………………………………………5