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 β1 (TGF-β1), and β1-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 β1-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.

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), β1-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–2 ng/mL). PRP-dependent stimulation of collagen biosynthesis was accompanied by an increase in the expression of NF-κβ, 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.

It has been considered that proline dehydrogenase/proline oxidase (PRODH/POX) is involved in antineoplastic activity of metformin (MET). The aim of this study is identification of key metabolites of glycolysis, pentose phosphate pathway (PPP), tricarboxylic acids (TCA), urea cycles (UC) and some amino acids in MET-treated MCF-7 cells and PRODH/POX-knocked out MCF-7 (MCF-7crPOX) cells. MCF-7crPOX cells were generated by using CRISPR-Cas9. Targeted metabolomics was performed by LC-MS/MS/QqQ. Expression of pro-apoptotic proteins was evaluated by Western blot. In the absence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to similar inhibition of glycolysis (drastic increase in intracellular glucose and pyruvate) and increase in the utilization of phospho-enol-pyruvic acid, glucose-6-phosphate and some metabolites of TCA and UC, contributing to apoptosis. However, in the presence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to utilization of some studied metabolites (except glucose), facilitating pro-survival phenotype of MCF-7 cells in these conditions. It suggests that MET treatment or PRODH/POX-knock out induce similar metabolic effects (glucose starvation) and glycolysis is tightly linked to glutamine metabolism in MCF-7 breast cancer cells. The data provide insight into mechanism of anticancer activity of MET as an approach to further studies on experimental breast cancer therapy.

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.