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.

Background Even with considerable improvement in treatment of epithelial ovarian cancer achieved in recent years, an increasing chemotherapy resistance and disease 5-year relapse is recorded for a majority part of patients that encourages the search for better therapeutic options. Gold nanoparticles (Au NPs) due to plethora of unique physiochemical features are thoroughly tested as drug delivery, radiosensitizers, as well as photothermal and photodynamic therapy agents. Importantly, due to highly controlled synthesis, it is possible to obtain nanomaterials with directed size and shape. Methods In this work, we developed novel elongated-type gold nanoparticles in the shape of nanopeanuts (AuP NPs) and investigated their cytotoxic potential against ovarian cancer cells SKOV-3 using colorimetric and fluorimetric methods, Western blot, flow cytometry, and fluorescence microscopy. Results Peanut-shaped gold nanoparticles showed high anti-cancer activity in vitro against SKOV-3 cells at doses of 1–5 ng/mL upon 72 hours treatment. We demonstrate that AuP NPs decrease the viability and proliferation capability of ovarian cancer cells by triggering cell apoptosis and autophagy, as evidenced by flow cytometry and Western blot analyses. The overproduction of reactive oxygen species (ROS) was noted to be a critical mediator of AuP NPs-mediated cell death. Conclusion These data indicate that gold nanopeanuts might be developed as nanotherapeutics against ovarian cancer.

Recent studies on the regulatory role of amino acids in cell metabolism have focused on the functional significance of proline degradation. The process is catalysed by proline dehydrogenase/proline oxidase (PRODH/POX), a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate (P5C). During this process, electrons are transferred to electron transport chain producing ATP for survival or they directly reduce oxygen, producing reactive oxygen species (ROS) inducing apoptosis/autophagy. However, the mechanism for switching survival/apoptosis mode is unknown. Although PRODH/POX activity and energetic metabolism were suggested as an underlying mechanism for the survival/apoptosis switch, proline availability for this enzyme is also important. Proline availability is regulated by prolidase (proline supporting enzyme), collagen biosynthesis (proline utilizing process) and proline synthesis from glutamine, glutamate, α-ketoglutarate (α-KG) and ornithine. Proline availability is dependent on the rate of glycolysis, TCA and urea cycles, proline metabolism, collagen biosynthesis and its degradation. It is well established that proline synthesis enzymes, P5C synthetase and P5C reductase as well as collagen prolyl hydroxylases are up-regulated in most of cancer types and control rates of collagen biosynthesis. Up-regulation of collagen prolyl hydroxylase and its exhaustion of ascorbate and α-KG may compete with DNA and histone demethylases (that require the same cofactors) to influence metabolic epigenetics. This knowledge led us to hypothesize that up-regulation of prolidase and PRODH/POX with inhibition of collagen biosynthesis may represent potential pharmacotherapeutic approach to induce apoptosis or autophagic death in cancer cells. These aspects of proline metabolism are discussed in the review as an approach to understand complex regulatory mechanisms driving PRODH/POX-dependent apoptosis/survival.

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.

A growing interest in metabolomics studies of cultured cells requires development not only untargeted methods capable of fingerprinting the complete metabolite profile but also targeted methods enabling the precise and accurate determination of a selected group of metabolites. Proline metabolism affects many crucial processes at the cellular level, including collagen biosynthesis, redox balance, energetic processes as well as intracellular signaling. The study aimed to develop a robust and easy-to-use targeted metabolomics method for the determination of the intracellular level of proline and the other two amino acids closely related to proline metabolism: glutamic acid and arginine. The method employs hydrophilic interaction liquid chromatography followed by high-resolution, accurate-mass mass spectrometry for reliable detection and quantification of the target metabolites in cell lysates. The sample preparation consisted of quenching by the addition of ice-cold methanol and subsequent cell scraping into a quenching solution. The method validation showed acceptable linearity (r > 0.995), precision (%RSD < 15%), and accuracy (88.5–108.5%). Pilot research using HaCaT spontaneously immortalized human keratinocytes in a model for wound healing was performed, indicating the usefulness of the method in studies of disturbances in proline metabolism. The developed method addresses the need to determine the intracellular concentration of three key amino acids and can be used routinely in targeted mammalian cell culture metabolomics research.

Celecoxib (Cx), an inhibitor of cyclooxygenase 2, induces apoptosis of cancer cells. However, the mechanism of the chemopreventive effect remains not fully understood. We aimed to investigate the role of PRODH/POX that is involved in the regulation of apoptosis induced by celecoxib. MCF-7 breast cancer cell line and the corresponding MCF-7 cell line with silenced PRODH/POX (MCF-7shPRODH/POX) were used. The effects of Cx on cell viability, proliferation, and cell cycle were evaluated. The expressions of protein markers for apoptosis (Bax, caspase 9, and PARP) and autophagy (Atg5, Beclin 1, and LC3A/B) were investigated by Western immunoblotting. To analyze the proline metabolism, collagen biosynthesis, prolidase activity, proline concentration, and the expression of proline-related proteins were evaluated. The generation of ATP, ROS, and the ratio of NAD+/NADH and NADP+/NADPH were determined to test the effect of Cx on energetic metabolism in breast cancer cells. It has been found that Cx attenuated MCF-7 cell proliferation via arresting the cell cycle. Cx induced apoptosis in MCF-7 breast cancer cells, while in MCF-7shPRODH/POX, autophagy occurred more predominantly. In MCF-7 breast cancer cells, Cx affected proline metabolism through upregulation of proline biosynthesis, PRODH/POX and PYCRs expressions, PEPD activity, and downregulation of collagen biosynthesis. In MCF-7shPRODH/POX clones, these processes, as well as energetic metabolism, were remarkably suppressed. The data for the first time suggest that celecoxib induces apoptosis through upregulation of PRODH/POX in MCF-7 breast cancer cells.

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.