Mechanisms involved in the cell cycle and apoptosis of HT-29 cells pre-treated with MK-886 prior to photodynamic therapy with hypericin

Autoři: Kleban, J., Mikes, J., Horvath, V., Sackova, V., Hofmanova, J., Kozubik, A., Fedorocko, P.
Rok: 2008


in our previous study we have proved that colon cancer cells HT-29 pre-treated with specific 5-lipoxygenase inhibitor MK-886 became more susceptible to photodynamic therapy (PDT) with hypericin and we also found that this mutual combination induced cell cycle arrest and stimulated onset of apoptosis (Kleban et al., 2007.J. Photochem. Photobiol. B 84,2). To further explain events associated with MK-886 mediated sensitization of tumor cells toward PDT with hypericin, more detailed study of signaling pathways leading to increase in apoptosis as well as cell cycle perturbations was performed and is presented herein. Intensive accumulation of HT-29 cells in G(0)/G(1) phase of cell cycle led to expression analyses of several G(0)/G(1) checkpoint molecules (cyclin A, cyclin E, cdk-2, pRb). Similarly, accumulation of apoptotic cells invoked analyses of key molecules involved in apoptotic signaling (caspase-3, -8, -9; PARP; Lamin B; Mcl-1; Bax) by Western blotting and caspase activity assay. Long term survival of cells was examined by clonogenicity test. As the effect of PDT is mediated by ROS production, levels of hydrogen peroxides and superoxide anion were monitored by flow cytometric analyses. in addition, an impact of MK-886 on LTB4 production and expression of 5-LOX was monitored. Massive G(0)/G(1) arrest in the cell cycle accompanied by increase in cyclin E level and decrease/absention of cyclin A, cdk-2 and pRb expression indicated incapability for G(1)/S transition. Minimal changes in cleavage of procaspases observed in cells treated with non-toxic concentrations of either agent alone or their mutual combination were not quite in line with their activity (caspase-3, -8, -9) which was significantly increased mainly in combinations. Treatment with non-toxic concentration of MK-886 had minimal influence over ROS production compared to control cells. In contrast, hypericin alone markedly increased the level of ROS, but no additional effect of MK-886 pre-treatment was detected. Further analyses of particular ROS groups unveiled an impact of increasing MK-886 concentration on superoxide accumulation accompanied with depletion of hydrogen peroxide level within the cells. The clonogenicity test revealed disruption of colony formation after mutual combination of both agents as compared to MK-886 or PDT alone. In conclusion, we presume that stimulation of apoptosis in our experimental model was accomplished preferentially through the mitochondrial pathway, although caspase-8 activation was also noticed. Interestingly, pre-treatment with MK-886 modulated distribution of ROS production in mutual combination with PDT. (c) 2008 Elsevier B.V. All rights reserved.