- 學位論文
光動力療法所引起的細胞死亡與Procaspase-3的後轉譯修飾作用
Cell death and post-translational modification of procaspase-3 induced by Photofrin-mediated Photodynamic treatment in mammalian cells
摘要
Photofrin® is the first generation photosensitizer for photodynamic treatment (PDT) of cancer, and Photofrin-PDT-triggered cell death represents its major therapeutic effect. Although diverse cell death phenotypes induced by Photofrin-PDT have been observed for some time, the mechanism(s) underlying this diversity remain elusive. Using human epidermoid carcinoma A431 cells as a model, we previously showed that distinct cell death types could be triggered which depend on Photofrin location and PDT dosage [Hsieh et al., (2003) J. Cell. Physiol. 194, 363-375]. In this thesis, the effects of Photofrin-PDT on A431 cells with intracellular organelle-localized Photofrin were further investigated. My study showed that PDT of A431 cells with intracellular organelle-localized Photofrin caused dilation of endoplasmic reticulum (ER), which results in the perinuclear vacuole formation. These dilated vacuoles are just proximal to the intracellular DCFDA-sensitive reactive oxygen species (ROS) elicited post Photofrin-PDT. The PDT-induced alteration of ER structure is similar to that triggered by thapsigargin, a ER Ca2+-ATPase inhibitor that perturb Ca2+ homeostasis, suggesting a role of Ca2+ in the formation of PVs. These results unravel a novel cell response to ER stress elicited by PDT when intracellular organelles are selectively loaded with Photofrin. To explore the control mechanism(s) of multiple cell death phenotypes caused by PDT treatment, the possible modification/regulation of caspase-3, a critical executioner of apoptosis, by Photofrin-PDT was further investigated. The results revealed for the first time that Photofrin-PDT can modify and inactivate procaspase-3, some of which turned into novel species with extraordinary high molecular weight (HMW) (~90 kDa) observed in SDS-PAGE. Photofrin-PDT generated ROS and blocked the activation of procaspase-3 by the upstream protease, caspase-8. Mass spectrometry-based analysis of post-translational modification of procaspase-3 using FT-MS and 16O/18O- and 14N/15N-labeling quantitative methods showed that Photofrin-PDT caused Met oxidation of procaspase-3 in this process mainly on Met-27, 39 and 44 in a Photofrin dose-dependent manner, but the active site Cys163 remained largely unmodified. Site direct mutagenesis (Met to Leu) experiments further denoted Met-44 as a critical residue for regulating procaspase-3 activation. In addition, the caspase-3-containing HMW products could be induced in various human cell lines in an oxidative stress-dependent manner, as well as in a mouse xenograft tumor model. Experiments using ectopically expressed procaspase-3 tagged with different tags indicated that the HMW products were resulted from cross-linking of procaspase-3 itself. In conclusion, this thesis have characterized different types of cell death elicited by Photofrin-PDT, in which Met oxidation and HMW product formation of procaspase-3 may play important roles in regulating caspase-3 activity and hence the cell fate determination.
並列摘要
Photofrin 是光動力療法中用來治療癌症的第一代光敏劑，主要的治療效果可導致癌細胞的死亡。許多研究發現PDT可以導致各種不同型態的細胞死亡，但是其中的機制還不是非常清楚。我們利用A431細胞進行PDT的研究，發現Photofrin不同處理時間，會分布在細胞不同的區域，照光處理後可以造成細胞不同的死亡型態 [Hsieh et al., (2003) J. Cell. Physiol. 194, 363-375]。在本研究中發現，當Photofrin分布在細胞內胞器中時，處理光照後2-8小時會導致細胞核周圍的空泡形成，我們證明空泡時由ER形成的，並且和ROS的形成是相關的；利用一個ER膜上的Ca2+-ATPase (SERCA) 的抑制劑處理，也可以看到相同的空泡的形成。本研究發現了一個新的細胞反應，PDT處理後會造成ER的壓力並導致空泡的形成。此外，caspase-3在細包凋亡中扮演一個執行者的角色，我們證實了PDT處理後會導致procaspase-3被修飾並且失去活性，一部分的procaspase-3還會形成三倍的高分子量產物。高劑量的PDT處理可以阻止內生性的以及重組的procaspase-3被上游的蛋白質(caspase-3)活化。利用FT-MS分析蛋白質量差異，或用16O/18O以及 15N穩定同位素標定的定量實驗中，我們發現氧化是PDT所造成的主要修飾作用。定量結果顯示，Met-27, -39 及 -44是氧化最明顯的胺基酸，而活性中心Cys163沒有明顯的變化。在procaspase-3-D3A中點突變的實驗結果顯示，M44L的突變株活性相較於其他突變株低 (M27L, M39L and M222L)；在野生型中點突變的實驗結果顯示，M44L的突變株被上游caspase-8活化的情況較差；綜合以上的結果顯示高劑量PDT處理後造成Methionine氧化以致procaspase-3不易被活化，這或許可以解釋PDT造成的複雜的細胞死亡；更進一步的，我們認為高分子產物的行程也可能在細胞死亡中扮演一個角色。我們發現高分子量產物會在各種細胞形成，並且與氧化壓力相關，而高分子量的形成也可以在動物實驗中看到。高分子量產物是經由修飾已生成的蛋白質所形成，並造成原來的procaspase-3的減少，在細胞中表現myc- 以及EGFP-procaspase-3 經PDT處理後更證明了高分子量產物是procaspase-3之間彼此共價鍵結形成。但高分子量產物的鍵結不是非常穩定，在純化的過程中會再度斷裂變回單體結構，目前我們正在尋找純化的方法以及鑑定共價鍵結的位置。綜合上述結論，在PDT處理後，我們鑑定不同的細胞死亡型態；另一方面，PDT處理後造成caspase-3氧化，形成高分子量產物，並失去活性，這些caspase-3的改變可能調控了細胞死亡的路徑。