Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX–Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model

Abstract

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.

The mechanism for differential effect of nelfinavir and indinavir on collagen metabolism in human skin fibroblasts

“…In the presence of glutamine, NEL does not affect collagen biosynthesis in Gly‐Pro treated cells because proline comes from glutamine. Decrease of collagen biosynthesis by Gly‐Pro was also observed in POX knock‐down breast adenocarcinoma MCF‐7 cells . In such conditions, prolidase activity and proline concentration were increased.…”

“…Decrease of collagen biosynthesis by Gly-Pro was also observed in POX knock-down breast adenocarcinoma MCF-7 cells. [44] In such conditions, prolidase activity and proline concentration were increased.…”

A novel plausible mechanism of NSAIDs-induced apoptosis in cancer cells: the implication of proline oxidase and peroxisome proliferator-activated receptor

“…Activation of HIF-1α related pro-survival signaling pathways undergoes through inflammatory and pro-angiogenic genes (eg. COX-2, TNFα, IL-1, NFκB, VEGF) [106]. It suggests that prolidase activity plays important role in regulation of HIF-1α-dependent functions.…”

Extracellular Prolidase (PEPD) Induces Anabolic Processes through EGFR, β1-integrin, and IGF-1R Signaling Pathways in an Experimental Model of Wounded Fibroblasts

“…The total concentration of protein was measured by the method of Lowry et al [54]. The procedure of Western blot analysis was described previously [55]. Equal amounts (30 µg/lane) of protein were diluted in lysis buffer.…”