Ilona Zareba 1 , Thi Yen Ly Huynh 1 , Adam Kazberuk 1 , Joanna Teul 2 , Agnieszka Klupczynska 1,3 , Jan Matysiak 3 , Arkadiusz Surazynski 1 , Jerzy Palka 4 Show Affiliations »
Abstract
BACKGROUND/AIMS: Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells. METHODS: The model of MCF-7 cells with prolidase overexpression (MCF-7PL) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. RESULTS: Prolidase overexpression in MCF-7PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7PL cells. The effects of studied compounds in MCF-7PL cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9. CONCLUSION: The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death. © Copyright by the Author(s). Published by Cell Physiol Biochem Press.
BACKGROUND/AIMS: Proline availability for proline dehydrogenase/proline oxidase (PRODH /POX) may represent switching mechanism between PRODH /POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells. METHODS: The model of MCF-7 cells with prolidase overexpression (MCF-7PL ) was obtained. In order to targeting proline for PRODH /POX-dependent pathways substrate for prolidase , glycyl-proline (GP ) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. RESULTS: Prolidase overexpression in MCF-7PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7PL cells. The effects of studied compounds in MCF-7PL cells were accompanied by increase in the expression of Atg7 , LC3A/B , Beclin-1 , HIF-1α and decrease in the expression of PRODH /POX, active caspases-3 and -9 . CONCLUSION: The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH /POX-dependent autophagic cell death . © Copyright by the Author(s). Published by Cell Physiol Biochem Press.
Entities: CellLine
Chemical
Disease
Gene
Keywords:
Prolidase; Proline; Autophagy; Collagen biosynthesis; Breast cancer cells MCF-7
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Substances： See more »
Year: 2020
PMID： 32918543 DOI： 10.33594/000000275
Source DB: PubMed Journal: Cell Physiol Biochem ISSN： 1015-8987