Kleban Ján, Szilárdiová Beáta, Mikes Jaromír, Horváth Viktor, Sacková Veronika, Brezáni Peter, Hofmanová Jirina, Kozubík Alois, Fedorocko Peter
Institute of Biology and Ecology, Faculty of Sciences, P.J. Safárik University, Moyzesova 11, Kosice 041 65, Slovakia.
J Photochem Photobiol B. 2006 Aug 1;84(2):79-88. doi: 10.1016/j.jphotobiol.2006.02.003. Epub 2006 Mar 20.
It may be hypothesized that the lipoxygenase (LOX) metabolic pathway plays an important role in photodynamic therapy (PDT) of malignant tumours, and modification of this pathway may result in administration of lower doses of photodynamic active agents accompanied by reduced side effects. In this study, we examine in more detail the cytokinetic parameters of human colon adenocarcinoma HT-29 cells pre-treated for 48 or 24h with LOX inhibitor MK-886, followed by PDT induced by hypericin. Based on MTT assay the concentrations of both agents (MK-886 and hypericin) with relatively slight (non-significant) cytotoxic effects were selected. These concentrations were used for combined treatment, where MTT response, total cell number, floating cells quantification, viability, cell cycle progression and DNA synthesis were detected. Hoechst/PI staining, PARP fragmentation and mitochondrial membrane potential (MMP) were evaluated to determine the extent of apoptosis. While MK-886 alone caused mainly necrosis, 48h pre-treatment of cells with MK-886 followed by PDT with hypericin clearly shifted the type of cell death to apoptosis. PDT with hypericin alone caused apoptosis in 19% of the cell population. Some combined modalities significantly potentiated the apoptotic effect (31% of apoptotic cells; 2.5microM MK-886/0.1microM hypericin), i.e., by 60% more than after single treatment with hypericin. Increased apoptosis was confirmed by PARP (116kDa) cleavage to characteristic 89kDa fragments and changes in MMP. Increasing concentration of MK-886 was accompanied by massive changes in the cell cycle progression. Combined treatment with lower concentrations of MK-886 and hypericin increased accumulation of cells in the S phase, accompanied by inhibition of DNA synthesis. Increasing concentration of MK-886 in this combination caused the opposite effect, manifesting significant accumulation of cells in the G0/G1 phase. More pronounced effects were observed after the 48h pre-treatment schedule. This anti-proliferative effect was confirmed by BrdU incorporation. These results indicate that combined treatment involving PDT and LOX inhibitor MK-886 may improve the therapeutic effectiveness of PDT.
可以推测，脂氧合酶（LOX）代谢途径在恶性肿瘤的光动力疗法（PDT）中起重要作用，对该途径的修饰可能导致光动力活性剂剂量降低，同时副作用减少。在本研究中，我们更详细地研究了用LOX抑制剂MK-886预处理48或24小时后的人结肠腺癌HT-29细胞的细胞动力学参数，随后用金丝桃素诱导PDT。基于MTT分析，选择了具有相对轻微（无统计学意义）细胞毒性作用的两种药物（MK-886和金丝桃素）的浓度。这些浓度用于联合治疗，检测MTT反应、总细胞数、漂浮细胞定量、活力、细胞周期进程和DNA合成。通过Hoechst/PI染色、PARP裂解和线粒体膜电位（MMP）评估来确定凋亡程度。虽然单独使用MK-886主要导致坏死，但先用MK-886预处理细胞48小时，然后用金丝桃素进行PDT，明显将细胞死亡类型转变为凋亡。单独用金丝桃素进行PDT导致19%的细胞群体发生凋亡。一些联合治疗方式显著增强了凋亡作用（31%的凋亡细胞；2.5μM MK-886/0.1μM金丝桃素），即比单独用金丝桃素治疗后增加了60%。PARP（116kDa）裂解为特征性的89kDa片段以及MMP的变化证实了凋亡增加。MK-886浓度增加伴随着细胞周期进程的大量变化。较低浓度的MK-886和金丝桃素联合治疗增加了S期细胞的积累，同时抑制了DNA合成。在这种联合治疗中增加MK-886的浓度会产生相反的效果，表现为G0/G1期细胞的显著积累。在48小时预处理方案后观察到更明显的效果。这种抗增殖作用通过BrdU掺入得到证实。这些结果表明，涉及PDT和LOX抑制剂MK-886的联合治疗可能提高PDT的治疗效果。
