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Proline oxidase silencing induces proline-dependent pro-survival pathways in MCF-7 cells

Proline degradation by proline dehydrogenase/proline oxidase (PRODH/POX) contributes to apoptosis or autophagy. The identification of specific pathway of apoptosis/survival regulation is the aim of this study. We generated knocked-down PRODH/POX MCF-7 breast cancer cells (MCF-7shPRODH/POX). PRODH/POX silencing did not affect cell viability. However, it contributed to decrease in DNA and collagen biosynthesis, increase in prolidase activity and intracellular proline concentration as well as increase in the expression of iNOS, NF-κB, mTOR, HIF-1α, COX-2, AMPK, Atg7 and Beclin-1 in MCF-7shPRODH/POX cells. In these cells, glycyl-proline (GlyPro, substrate for prolidase) further inhibited DNA and collagen biosynthesis, maintained high prolidase activity, intracellular concentration of proline and up-regulated HIF-1α, AMPK, Atg7 and Beclin-1, compared to GlyPro-treated MCF-7 cells. In MCF-7 cells, GlyPro increased collagen biosynthesis, concentration of proline and expression of caspase-3, cleaved caspases -3 and -9, iNOS, NF-κB, COX-2 and AMPKβ. PRODH/POX knock-down contributed to pro-survival autophagy pathways in MCF-7 cells and GlyPro-derived proline augmented this process. However, GlyPro induced apoptosis in PRODH/POX-expressing MCF-7 cells as detected by up-regulation of active caspases -3 and -9. The data suggest that PRODH/POX silencing induces autophagy in MCF-7 cells and GlyPro-derived proline supports this process.


Cell viability, DNA and collagen biosynthesis, prolidase activity and intracellular proline concentration in MCF-7 PRODH/POX knockdown cells (MCF-7 shPRODH/POX ) versus MCF-7 cells
The studies were performed on 3 clones of PRODH/ POX silenced MCF-7 cells and MCF-7 control cells treated with GlyPro (glicyl-proline) as a substrate for prolidase in order to increase cytoplasmic level of proline (Supplementary Figure 2). There was no significant difference in cell viability between studied clones of MCF-7 cells, the cells transfected with empty vector and MCF-7 control cells either treated or untreated with GlyPro (Supplementary Figure 2A). However, in clone 2 and clone 3 of MCF-7 cells, DNA biosynthesis was significantly decreased compared to MCF-7 control cells ( Figure 2B). Although, GlyPro further inhibited the DNA biosynthesis in cells of clone 1 and clone 2, there was not significant effect of GlyPro on the process in clone 3 MCF-7 cells.
Collagen biosynthesis was decreased in clone 2 MCF-7 cells (but not in clone 1) and GlyPro inhibited the process in clone 1 and clone 2 (Supplementary Figure 2C). However, collagen biosynthesis was not affected in clone 3 MCF-7 cells in both GlyPro treated and untreated cells.
Prolidase activity and intracellular proline concentration were significantly increased in clone 2 MCF-7 cells in both GlyPro treated and untreated cells (Supplementary Figure 2D, Supplementary Figure 2E).
The data suggest that clone 2 MCF-7 cells represent the most characteristic phenotype to study the role of deregulation of proline generation/utilization processes on apoptosis/autophagy. Therefore, we used clone 2 MCF-7 cells for further studies.

Cell viability assay
The cell viability was determined using Nucleo Counter NC-3000 (ChemoMetec, Copenhagen, Denmark). Prior the experiment MCF-7 and MCF-7 shPRODH/POX cells were cultured in six-well plates at 1 × 10 5 cells/well with 2 ml of growth medium. After 24 h incubation of the cells in glutamine-free DMEM with or without GlyPro, medium was discarded and the cells were rinsed three times with phosphate buffered saline (PBS). Then the cells were harvested, washed and stained with VitaBright-48 (VB-48) (ChemoMetec), acridine orange (AO) (ChemoMetec), propidium iodide (PI) (ChemoMetec) and analyzed using NC-3000 cell counter.