Mucin 1 (MUC1) is a highly glycosylated type I transmembrane glycoprotein, which is overexpressed in various cancer cells especially in breast cancer cells. The aim of the study was to investigate the proapoptotic properties Pt₂ (4-ethylpyridine)₄(berenil)₂ (Pt12) with anti-MUC1 using flow cytometry assessment of annexin V binding, analysis of mitochondrial membrane potential and defragmentation of DNA by TUNEL assay in MCF-7 breast cancer cell line. The cell death was measured by flow cytometric analysis after annexin V-FITC and propidium iodide staining. The incubation of MCF-7 breast cancer cells with Pt12, cisplatin, Pt12 with anti-MUC1 and cisplatin with anti-MUC1 induced the visible phosphatidylserine exposure after 24 and 48 hours of treatment. The apoptotic effect of Pt12 was found to be stronger than that caused by cisplatin. The ratio of early and late apoptotic cells was increased after 24h of combined treatment with Pt12 with anti-MUC1 (10 μM + 10 μg/mL) in MCF-7 from 3.0 to 19.0%. After combined treatment with cisplatin with anti-MUC1 used in the same doses as Pt12 with anti-MUC1 (10 μM + 10 μg/mL) in MCF-7 for 24 h the ratio of early and late apoptotic cells was increased from 3.0% to 12.0% . The ratio of necrotic cells was, however, also increased after treatment with the Pt12 with anti-MUC1 for 24 h (from 2.3% to 12.2%) compared to treatment with cisplatin with anti-MUC1 (from 2.3% to 6.7%). The dissipation effect of Pt12 with anti-MUC1 on mitochondrial membrane potential (ΔΨm)  is particularly conspicuous, the red-to-green fluorescence ratio at 10 μM reaches to 16% of the control value in MCF-7 cells, suggesting that the mitochondrial membrane has been severely damaged. These results are in accord with those obtained in the Annexin V/PI assay and indicate that the apoptosis induced by Pt12 with anti-MUC1 may go through the mitochondrial pathway.    To detect the yield of DNA strand breaks, which are intimately associated with an apoptotic response, the TUNEL assay was performed after treating MCF-7 cells with compounds for 24h and 48h. A highest increase in the percent of TUNEL positive cells was observed after incubation of Pt12 with anti-MUC1 in comparison to cisplatin with anti-MUC1 after 24 and 48h. Control cells cultured in normal growth media showed minimal DNA fragmentation.We have found that the apoptotic effect of Pt12 with anti-MUC1 was stronger than evoked by cisplatin, anti-MUC1, Pt12 and cisplatin used with anti-MUC1.

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