engleski

ADAP Caused Apoptotic Cell Death without Stimulation of Topoisomeraze II-mediated DNA Cleavage

4-Methyl-2, 7-diamino-5, 10-diphenyl-4, 9-diazapyrenium hydrogensulfate (ADAP), newly synthesized DNA and RNA intercalator of a high fluorescence and binding specificity. Fluorescence microscopy of MIAPaCa-2 cells was used to analyse enter and distribution of ADAP into the cells and nuclei. Cytotoxic effects of ADAP on human cell lines MIAPaCa-2, Hep-2, HeLa, HT-29, Caco-2, SW620, MCF-7, HBL and WI38 were tested using the tetrazolium dye (MTT) assay. For detection of apoptotic characteristics of treated cells the annexin-V-fluorescein assay was used. Cell-free assay was performed for screening of topoisomerase II-targeted effects of ADAP. Fluorescence microscopy showed that ADAP dyed in red nucleus and/or cytoplasm dependent on concentration and treatment time, and was seen intracellular 5 minutes after administration even at sub μ M concentrations. ADAP, in concentrations 10 μ M and 1 μ M, caused strong cytotoxic effects on all treated tumour cells, but it did not inhibit the WI38 cell's growth. Exposure of phosphatidylserine, one of the hallmarks of apoptosis, was detected on Caco-2 cells. ADAP in the presence of human topoisomerase IIalpha did not stimulate generation of linear plasmid form. Based on growth inhibition, morphological changes and apoptotic features of treated human tumour cells we can conclude that ADAP in 1 μ M concentration caused apoptotic cell death of the treated tumour cells. Although ADAP was binding as intercalator to isolated DNA and RNA, it did not cause topoisomerase II-mediated DNA cleavage, suggesting mode of action in tumour cells different than DNA intercalation.

ADAP; DNA-intercalators; topoizomeraze II

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