以5,10-biaminophenyl-15,20-bipyridinyl porphyrin為光感藥物作光動力治療誘導子宮頸癌細胞死亡之模式探討

Abstract

目的: 以5,10-biaminophenyl-15,20-bipyridinyl porphyrin (BABPP)對子宮頸癌細胞光敏化後, 探討以光動力治療造成之細胞死亡, 其壞死與凋亡的特徵. 材料與方法:我們用3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 分析與乳酸脫氫酶(LDH)滲漏分析決定光動力治療後的細胞毒性. 流式細胞儀分析用於測定細胞週期及細胞壞死與凋亡的分布比例.結果: MTT分析顯示BABPP在黑暗中具有極低的細胞毒性.此外, 細胞存活率隨藥物濃度與照光劑量之提高而逐漸降低. 細胞存活率在照光後隨時間推移而漸次下降, 與LDH滲漏分析的結果有良好的對應關係.藉由流式細胞儀的分析, 我們發現在光動力治療後出現的壞死細胞在某個時間點之後會漸漸減少, 最後凋亡會逐漸變成細胞死亡的主要方式. 以碘化丙啶(PI)對細胞染色後作流式細胞儀分析則顯示亞G1峰在光動力治療後3小時就出現, 且隨時間推移而更趨顯著; 同時G2/M期的比例則逐步縮小. 結論: 在照光後的時間過程裡, 由凋亡或壞死細胞特徵之表現與LDH滲漏分析的結果可以辨識出兩種細胞破壞的機制. 以BABPP為光感藥物之光動力治療, 其產生之細胞死亡係透過凋亡與壞死兩種方式進行. 最後, 細胞凋亡的程度隨照光後時間流逝而漸次上揚, 且依照光劑量與藥物濃度而定. BABPP是一種值得進一步探索之有潛力的新型光感藥物.

Purpose: To investigate the necrotic and apoptotic features of cell death in response to 5,10-biaminophenyl-15,20-bipyridinyl porphyrin (BABPP) photosensitization of HeLa cells. Material and Methods: We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) leakage assay to determine cytotoxicity in HeLa cells after photodynamic therapy (PDT). Flow cytometric analysis was used to evaluate the cell cycle and the distributive ratio of apoptotic versus necrotic cells. Results: The MTT assay revealed low cytotoxicity in the dark; further, cell viability declined in a dose-dependent manner for drugs and illumination. The survival rate gradually decreased during the postirradiation period, and it correlated well with the results of the LDH leakage assay. By flow cytometric analysis, we observed that the necrotic cells gradually disappeared at some point after PDT, and eventually, apoptosis became the main pattern of cell death. The flow cytometric analysis of propidium iodide (PI)-stained HeLa cells revealed a subG1 peak 3 hr after PDT that progressively became more prominent during the postexposure time; simultaneously, the proportion of the G2/M phase slowly diminished. Conclusion: There are 2 mechanisms of cellular destruction identified from the time course of LDH leakage and from the manifestation of markers of apoptosis or necrosis. BABPP-based PDT-mediated cell death can advance through both necrotic as well as apoptotic pathways. Finally, the extent of apoptosis was time-related and displayed drug and light dose-dependent characteristics. Thus, BABPP is a potential photosensitizer worthy of further investigation.