Pre-treatment of HT-29 cells with 5-LOX inhibitor (MK-886) induces changes in cell cycle and increases apoptosis after photodynamic therapy with hypericin

Abstrakt

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 24 h 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, 48 It 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; 2.5 mu M MK-886/0.1 mu M hypericin), i.e., by 60% more than after single treatment with hypericin. Increased apoptosis was confirmed by PARP (116 kDa) cleavage to characteristic 89 kDa 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 48 h 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. (c) 2006 Elsevier B.V. All rights reserved.