A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model
Tekijät: Zareba I, Surazynski A, Chrusciel M, Miltyk W, Doroszko M, Rahman N, Palka J
Kustantaja: KARGER
Julkaisuvuosi: 2017
Journal: Cellular Physiology and Biochemistry
Tietokannassa oleva lehden nimi: CELLULAR PHYSIOLOGY AND BIOCHEMISTRY
Lehden akronyymi: CELL PHYSIOL BIOCHEM
Vuosikerta: 43
Numero: 2
Aloitussivu: 670
Lopetussivu: 684
Sivujen määrä: 15
ISSN: 1015-8987
eISSN: 1421-9778
DOI: https://doi.org/10.1159/000480653
Rinnakkaistallenteen osoite: https://research.utu.fi/converis/portal/detail/Publication/28259514
Background/Aims: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. Methods: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7(shPRODH/POX)) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7(shPRODH/POX) cells. All studied compounds decreased cell viability in MCF-7 and MCF-7(shPRODH/POX) cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7(shPRODH/POX) and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7(shPRODH/POX) cells and contributed to the induction of pro-survival mode only in MCF-7(shPRODH/POX) cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. Conclusion: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7(shPRODH/POX) cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. (C) 2017 The Author(s) Published by S. Karger AG, Basel
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