In Vitro Anti-tumor Effect of the Unsymmetrical Distyryl Boron Dipyrromethene BODIPY-5 in Hepatocellular Carcinoma

光動力療法(PDT)是透過特定波長的光、光敏劑(photosensitizer)和氧氣之間的化學反應製造活性氧自由基(reactive oxygen species, ROS)，以達到抗腫瘤的作用。在本次探討中，一種不對稱二苯乙烯基氟化硼二吡咯BODIPY-5的光敏劑被用於治療體外的肝癌細胞 HepG2。這種光敏劑以細胞質膜為其主要定位目標。BODIPY-5能夠有效地殺死HepG2細胞，而其半抑制細胞增殖的濃度(IC50)約為30納摩爾每升(nM)。BODIPY-5亦被用於治療具多藥耐藥性 (包括DNA靶向抗癌藥物阿黴素,doxorubicin) 的肝癌細胞R-HepG2，並表現了有效的殺癌能力。另外，膜聯蛋白-V/碘化丙啶雙染色法顯示當BODIPY-5的濃度增加，更多的HepG2細胞會進行凋亡。以BODIPY-5為主導的光動力反應除了導致細胞質膜磷脂絲胺酸的外化，亦觸發細胞凋亡的其他事件，包括線粒體外膜去極化、caspase-3 的激活及脱氧核醣核酸 (DNA) 的破壞。在雙氯螢光黃乙酸乙酯測試中，BODIPY-5能在光的激活下產生大量一次ROS，亦能誘發HepG2細胞製造二次ROS。兩種ROS抑制劑 (分別是D-甘露醇和L-抗壞血酸) 均能降低BODIPY-5對HepG2細胞的毒性和其產生ROS的能力，這證明了BODIPY-5能夠同步進行第一型和第二型的光反應，製造不同的ROS，包括羥基自由基及單態氧。光動力療法抗腫瘤的成效受多個因素影響，其中包括光敏劑的劑量及處理時間。另外，光本身亦擔當著重要的角色。較長的照明時間能令BODIPY-5促使更多HepG2細胞凋亡。但是當BODIPY-5本身受光照的時間越久，其吸光和發射螢光的能力亦相應地降低。不過有趣的是，預先受到光照的BODIPY-5，當它再次被用作光敏劑時，其殺死HepG2細胞和製造ROS的效力均有所提高。透過高效液相色譜法的分析，新的光化產物在BODIPY-5受長時間的照明中產生，而它們的產生很有可能解釋上述BODIPY-5被光激活的實驗現象。為提高整體的抗腫瘤療效，進一步研究了BODIPY-5和其他藥物合併使用時的效果。BODIPY-5分別和另一光敏劑脫鎂葉綠酸鹽-a(pheophorbidea)及阿黴素同時及順序地使用於治療HepG2細胞，均表現了協同效應;藥物的處理程序與整體的合併效果沒有重大關聯。BODIPY-5也和阿黴素同時合併使用於治療R-HepG2細胞，兩者表現了加性效應，結果與HepG2細胞的有所不同。Photodynamic therapy (PDT) utilizes light of specific wavelength, photosensitizer and oxygen to generate reactive oxygen species (ROS) and kill tumor cells. In the present study, the anti-tumor effect of the unsymmetrical distyryl boron-dipyrromethene BODIPY-5, a photosensitizer found to localize mainly in cellular plasma membrane, was studied using the hepatocellular carcinoma HepG2 cells.BODIPY-5 killed HepG2 cells in a dose-dependent manner, with 50% inhibitory concentration determined as 30 nM. BODIPY-5 also demonstrated a moderate killing effect in the drug-resistant R-HepG2 cells which showed resistance to the DNA-targeted anti-cancer drug doxorubicin, indicating that BODIPY-5-PDT could overcome the drug resistance problem. More cells were subjected to apoptosis when they were treated with higher BODIPY-5 concentrations, as shown by annexin V and propidium iodide staining assay. Mitochondrial membrane depolarization, caspase 3 activation and DNA fragmentation, the hallmark events for apoptosis, were triggered by BODIPY-5-PDT and their extent was also concentration-dependent. By 2’,7’-dichlorodihydrofluorescin diacetate assay, both primary and secondary ROS were found to be generated during and after BODIPY-5-PDT respectively. BODIPY-5 was also found to trigger both Types I and II PDT reactions, which respectively generate hydroxyl radicals and singlet oxygen, as both ROS scavengers D-mannitol and L-ascorbic acid could inhibit its ROS generation as well as the killing effect.Apart from the photosensitizer’s concentration and incubation time, light is another important factor determining the potency of PDT. Longer light irradiation periods for BODIPY-5-PDT led to more cells undergoing apoptosis. On the other hand, light can also influence the stability of photosensitizer. Prolonged light irradiation on BODIPY-5 alone reduced its absorbance and emission fluorescence at the peaks. Nonetheless, the pre-irradiated photosensitizer demonstrated a higher potency in killing HepG2 cells, together with more ROS production. High performance liquid chromatography experiments indicated that new compounds were formed from BODIPY-5 after prolonged light irradiation.To improve the anti-tumor potency of BODIPY-5, combination studies together with other drugs were conducted. Synergy was demonstrated in both simultaneous and sequential administrations of BODIPY-5 and pheophorbide a, another photosensitizer. The overall killing effect was independent of the treatment schedule. BODIPY-5 was also combined in use with doxorubicin for treating HepG2 cells. A synergistic effect for BODIPY-5 and doxorubicin was observed and the effect was not significantly influenced by the treatment order. In contrast to HepG2 cells, combination of BODIPY-5 and doxorubicin demonstrated only an additive effect in R-HepG2 cells.Hung, Wai.Thesis M.Phil. Chinese University of Hong Kong 2016.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …).Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only