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The role of proline dehydrogenase/proline oxidase (PRODH/POX) in the mechanism of antineoplastic activity of metformin (MET) was studied in C32 melanoma cells. PRODH/POX is a mitochondrial enzyme-degrading proline that is implicated in the regulation of cancer cell survival/apoptosis. The enzyme is activated by AMP kinase (AMPK). It has been found that MET induced a significant decrease in cell viability and DNA biosynthesis accompanied by an increase in the expressions of AMPK and PRODH/POX in C32 cells. The mechanism for MET-dependent cytotoxicity on C32 cells was found at the level of PRODH/POX-induced ROS generation and activation of Caspase-3 and Caspase-9 expressions in these cells. The effects were not observed in MET-treated PRODH/POX knock-out C32 cells. Of interest is an MET-dependent increase in the concentration of proline, which is a substrate for PRODH/POX. This phenomenon is due to the MET-dependent inhibition of collagen biosynthesis, which is the main proline-utilizing process. It has been found that the underlying mechanism of anticancer activity of MET involves the activation of AMPK, PRODH/POX, increase in the cytoplasmic concentration of proline, inhibition of collagen biosynthesis, and stimulation of PRODH/POX-dependent ROS generation, which initiate the apoptosis of melanoma cells.

Simple Summary PRODH/POX (proline dehydrogenase/proline oxidase) is a mitochondrial enzyme that catalyzes proline degradation generating reactive oxygen species (ROS). Estrogens limit proline availability for PRODH/POX by stimulating collagen biosynthesis. It has been considered that estrogens determine efficiency of troglitazone (TGZ)-induced PRODH/POX-dependent apoptosis in breast cancer cells. The studies were performed in wild-type and PRODH/POX-silenced estrogen-dependent MCF-7 cells and estrogen-independent MDA-MB-231 cells. DNA and collagen biosynthesis were determined by radiometric method, ROS production was measured by fluorescence assay, protein expression was determined by Western blot and proline concentration by LC/MS analysis. We found that: i/TGZ-induced apoptosis in MDA-MB-231 occurs only in the absence of estradiol or ERβ, ii/the process is mediated by PRODH/POX, iii/and is facilitated by proline availability for PRODH/POX by TGZ-dependent inhibition of collagen biosynthesis (proline utilizing process). The data suggest that combined TGZ and anti-estrogen treatment could be considered in experimental therapy of ER negative breast cancers. Abstract The impact of estradiol on troglitazone (TGZ)-induced proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied in wild-type and PRODH/POX-silenced estrogen receptor (ER) dependent MCF-7 cells and ER-independent MDA-MB-231 cells. DNA and collagen biosynthesis were determined by radiometric method, prolidase activity evaluated by colorimetric method, ROS production was measured by fluorescence assay. Protein expression was determined by Western blot and proline concentration by LC/MS analysis. PRODH/POX degrades proline yielding reactive oxygen species (ROS). Estrogens stimulate collagen biosynthesis utilizing free proline and limiting its availability for PRODH/POX-dependent apoptosis. TGZ cytotoxicity was highly pronounced in wild-type MDA-MB-231 cells cultured in medium without estradiol or in the cells cultured in medium with estradiol but deprived of ERβ (by ICI-dependent degradation), while in PRODH/POX-silenced cells the process was not affected. The TGZ cytotoxicity was accompanied by increase in PRODH/POX expression, ROS production, expression of cleaved caspase-3, caspase-9 and PARP, inhibition of collagen biosynthesis, prolidase activity and decrease in intracellular proline concentration. The phenomena were not observed in PRODH/POX-silenced cells. The data suggest that TGZ-induced apoptosis in MDA-MB-231 cells cultured in medium without estradiol or deprived of ERβ is mediated by PRODH/POX and the process is facilitated by proline availability for PRODH/POX by TGZ-dependent inhibition of collagen biosynthesis. It suggests that combined TGZ and antiestrogen treatment could be considered in experimental therapy of estrogen receptor negative breast cancers.

Although the role of platelet-rich plasma (PRP) in tissue regeneration has been confirmed in many studies, the mechanism of this process is still not fully understood. Human keratinocytes (HaCaT) cells were used as an experimental model for studies on the effects of PRP on cell proliferation, migration, collagen biosynthesis, prolidase activity, and its expression and anabolic signaling. The activation of epidermal growth factor receptor (EGFR), &beta, 1-integrin, and insulin-like growth factor-1 receptor (IGF-1R) by PRP were investigated by western blot and immunocytochemistry. It has been found that PRP induced keratinocytes migration and proliferation through activation of cell cycle progression and EGFR downstream signaling. Similar biological effects were achieved by an addition to the culture medium of prolidase (PEPD), a ligand of EGFR (PRP is a rich source of PEPD&ndash, 2 ng/mL). PRP-dependent stimulation of collagen biosynthesis was accompanied by an increase in the expression of NF-&kappa, &beta, IGF-1R-downstream signaling proteins, and PEPD activity. The data suggest that PRP activates a complex of growth factors and adhesion receptors that stimulate cell proliferation, migration, and collagen biosynthesis. PRP induces PEPD-dependent human keratinocyte proliferation through activation of the EGFR receptor. Our study provides a novel mechanism of PRP-dependent wound healing.

The role of prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR) was studied in an experimental model of wound healing in cultured fibroblasts. The cells were treated with PEPD (1&ndash, 100 nM) and analysis of cell viability, proliferation, migration, collagen biosynthesis, PEPD activity, and the expressions of EGFR, insulin-like growth factor 1 (IGF-1), and &beta, 1-integrin receptor including downstream signaling proteins were performed. It has been found that PEPD stimulated proliferation and migration of fibroblasts via activation of the EGFR-downstream PI3K/Akt/mTOR signaling pathway. Simultaneously, PEPD stimulated the expression of &beta, 1-integrin and IGF-1 receptors and proteins downstream to these receptors such as FAK, Grb2, and ERK1/2. Collagen biosynthesis was increased in control and &ldquo, wounded&rdquo, fibroblasts under PEPD treatment. The data suggest that PEPD-induced EGFR signaling may serve as a new attempt to therapy wound healing.

Proline oxidase (POX) is mitochondrial proline-degrading enzyme of dual apoptosis/survival function. POX expression and proline availability are considered an underlying mechanism for differential POX functions. The mechanism for POX-dependent regulation of cell death/survival was studied in wild-type (MCF-7WT) and shRNA POX-silenced breast cancer cells (MCF-7iPOX). Proline concentration and proteomic analyses were determined by LC/MS/QTOF and LC/MS/ORBITRA, respectively. Inhibition of collagen biosynthesis (proline utilizing process) by 2-methoxyestradiol (2ME) contributed to induction of apoptosis in MCF-7WT cells, as detected by increase in the expression of active caspase-3, -9 and p53. The process was not shown in MCF-7iPOX. In MCF-7iPOX cells prolidase activity and expression as well as proline concentration were drastically increased, compared to MCF-7WT cells. Down-regulation of p53 in MCF-7iPOX cells was corroborated by proteomic analysis showing decrease in the expression of p53-related proteins. The mechanism for down-regulation of p53 expression in MCF-7iPOX cells was found at the level of p53–PEPD complex formation that was counteracted by hydrogen peroxide treatment. In this study, we found that silencing POX modulate pro-survival phenotype of MCF-7 cells and suggest that the mechanism of this process undergoes through down-regulation of p53-dependent signaling.

Recent reports have indicated prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR). Since this receptor is involved in the promotion of cell proliferation, growth, and migration, we aimed to investigate whether prolidase may participate in wound healing in vitro. All experiments were performed in prolidase-treated human keratinocytes assessing cell vitality, proliferation, and migration. The expression of downstream signaling proteins induced by EGFR, insulin-like growth factor 1 (IGF-1), transforming growth factor &beta, 1 (TGF-&beta, 1), and &beta, 1-integrin receptors were evaluated by Western immunoblotting and immunocytochemical staining. To determine collagen biosynthesis and prolidase activity radiometric and colorimetric methods were used, respectively. Proline content was determined by applying the liquid chromatography coupled with mass spectrometry. We found that prolidase promoted the proliferation and migration of keratinocytes through stimulation of EGFR-downstream signaling pathways in which the PI3K/Akt/mTOR axis was involved. Moreover, PEPD upregulated the expression of &beta, 1-integrin and IGF-1 receptors and their downstream proteins. Proline concentration and collagen biosynthesis were increased in HaCaT cells under prolidase treatment. Since extracellular prolidase as a ligand of EGFR induced cell growth, migration, and collagen biosynthesis in keratinocytes, it may represent a potential therapeutic approach for the treatment of skin wounds.

In stress conditions, as neoplastic transformation, amino acids serve not only as nutrients to maintain the cell survival but also as mediators of several regulatory pathways which are involved in apoptosis and autophagy. Especially, under glucose deprivation, in order to maintain the cell survival, proline and glutamine together with other glutamine-derived products such as glutamate, alpha-ketoglutarate, and ornithine serve as alternative sources of energy. They are substrates for production of pyrroline-5-carboxylate which is the product of conversion of proline by proline dehydrogenase/ proline oxidase (PRODH/POX) to produce ATP for protective autophagy or reactive oxygen species for apoptosis. Interconversion of proline, ornithine, and glutamate may therefore regulate PRODH/POX-dependent apoptosis/autophagy. The key amino acid is proline, circulating between mitochondria and cytoplasm in the proline cycle. This shuttle is known as proline cycle. It is coupled to pentose phosphate pathway producing nucleotides for DNA biosynthesis. PRODH/POX is also linked to p53 and AMP-activated protein kinase (AMPK)-dependent pathways. Proline availability for PRODH/POX-dependent apoptosis/autophagy is regulated at the level of collagen biosynthesis (proline utilizing process) and prolidase activity (proline supporting process). In this review, we suggest that amino acid metabolism linking TCA and Urea cycles affect PRODH/POX-dependent apoptosis/autophagy and the knowledge might be useful to targeted cancer therapy.