Proline oxidase (POX), localized on inner mitochondrial membranes, is encoded by a p53-induced gene and metabolically participates in p53-induced apoptosis. Previously, we showed that POX catalyzed the generation of reactive oxygen species (ROS). We and others have demonstrated that overexpression of POX, independent of p53, causes apoptotic cell death in a variety of cancer cells. But a necessary role for ROS remains uncertain. Therefore, we asked whether superoxide dismutases (SOD) and catalase (CAT), important antioxidant enzymes, might interfere with the POX-dependent induction of apoptosis. In this study, we used DLD-1 colorectal cancer cells stably transfected with the POX gene under the control of a tetracycline-inducible promoter. When doxycycline was removed from the culture medium and the expression of POX was induced, apoptotic cell death was initiated. To examine the importance of the ROS-dependent component of the pathway, we infected DLD-1 POX cells with recombinant adenoviruses containing MnSOD, CuZnSOD, CAT or varying combinations of these adenoviruses followed by induced expression of POX. The expression of MnSOD inhibited POX-induced apoptosis, but others did not. Mechanistically, mitochondria-localized MnSOD dramatically reduced the release of cytochrome c to cytosol by POX. Compared with control cells, MnSOD-expressing DLD-1 POX cells generated a higher concentration of H ₂ O ₂ owing to dismutation of superoxide radicals, which was elevated by POX. Thus, these data further suggest that the generation of superoxide radicals plays a crucial role in POX-induced apoptosis and the process is partially blocked by MnSOD.

Proline oxidase (POX), a flavoenzyme localized at the inner mitochondrial membrane, catalyzes the first step of proline degradation by converting proline to pyrroline-5-carboxylate (P5C). POX is markedly elevated during p53-induced apoptosis and generates proline-dependent reactive oxygen species (ROS), specifically superoxide radicals, to induce apoptosis through both mitochondrial and death receptor pathways. These previous studies also showed suppression of the mitogen-activated protein kinase pathway leading us to broaden our exploration of proliferative signaling. In our current report, we used DLD-1 colorectal cancer cells stably transfected with the POX gene under the control of a tetracycline-inducible promoter and found that three pathways which cross talk with each other were downregulated by POX: the cyclooxygenase-2 (COX-2) pathway, the epidermal growth factor receptor (EGFR) pathway and the Wnt/β-catenin pathway. First, POX markedly reduced COX-2 expression, suppressed the production of prostaglandin E2 (PGE₂) and importantly, the growth inhibition by POX was partially reversed by treatment with PGE_2. Phosphorylation of EGFR was decreased with POX expression and the addition of EGF partially reversed the POX-dependent downregulation of COX-2. Wnt/β-catenin signaling was decreased by POX in that phosphorylation of glycogen synthase kinase-3β (GSK-3β) was decreased on the one hand and phosphorylation of β-catenin was increased on the other. There changes led to decreased accumulation of β-catenin and decreased β-catenin/TCF/LEF-mediated transcription. Our newly described POX-mediated suppression of proliferative signaling together with the previously reported induction of apoptosis suggested that POX could function as a tumor suppressor. Indeed, in human colorectal tissue samples, immunohistochemically-monitored POX was dramatically decreased in tumors compared with normal counterparts. Thus, POX metabolism of substrate proline affects multiple signaling pathways, modulating both apoptosis and tumor growth, and could be an attractive target to metabolically control the cancer phenotypes.

Summary.: Previously we have found deregulation of collagen metabolism in human pancreatitis and pancreatic cancer tissues. Insulin‐like growth factor‐I (IGF‐I) is known to stimulate collagen biosynthesis through interaction with IGF‐I receptor. IGF‐I binding proteins (BPs) regulate the activity of IGF‐I. We investigated whether serum and tissue IGF‐I and IGF‐BPs as well as tissue IGF‐I receptor expression may reflect disturbances of collagen metabolism in patients with pancreatitis and pancreatic cancer. In pancreatitis tissue, a significant increase in IGF‐I and IGFBP‐3 content was accompanied by a distinct increase in IGF‐I receptor expression, compared to control pancreas tissue. In contrast, serum from patients with pancreatitis did not show significant increases in IGF‐I and IGFBP‐3 levels, however, significant increases in IGFBP‐1 level (2.5 fold). Moreover, a distinct decrease in radioactive IGF‐binding to the BPs, compared to control serum, was found. Pancreatic cancer tissue and serum of patients with pancreatic cancer showed significant increases in IGF‐I, IGFBP‐3 and IGFBP‐1 content, accompanied by dramatic increases in IGF‐I tissue receptor expression, compared to controls. In serum of patients with pancreatic cancer distinct increases in radioactive IGF‐binding to 46 kDa BP, compared to control serum, were observed. The data suggest that disturbances in tissue collagen metabolism during pancreatic diseases may result from deregulation of IGF‐I homeostasis and that elevated serum levels of IGF‐I, IGFBP‐3 and IGFBP‐1 may serve as markers of pancreatic cancer.