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James M. Phang - One of the best experts on this subject based on the ideXlab platform.
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Abstract 4613: Effects of co-regulation of Proline Oxidase and succinate dehydrogenase on oxidative respiration.
Molecular and Cellular Biology, 2013Co-Authors: Chad N. Hancock, James M. PhangAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Proline Oxidase (POX) is an inner mitochondrial membrane protein that oxidizes Proline to generate superoxide as well as ATP. Work in our laboratory has documented that POX activity is highly regulated during genotoxic and metabolic stress. Inducers of POX include p53, and Proline-dependent ROS play a role in p53- and mitochondrial-mediated apoptosis. Succinate dehydrogenase, also known as Complex II of the electron transport chain (ETC), oxidizes the TCA cycle metabolite succinate to fumarate as it reduces ubiquinone to ubiquinol, generating ATP through oxidative respiration. Complex II and the p53-DNA damage repair pathway are intimately linked. Mutations in Complex II have been shown to increase risk of breast and thyroid cancer through destabilization of p53, and tocopherol-succinate has been demonstrated to reduce radiation-induced apoptosis. Thus, the ETC and activities of p53 appear to be linked through Complex II. Because POX can generate ATP from Proline, we explored the relationship between POX and the ETC. We found that POX binds directly to ubiquinone, and that ubiquinone is a very effective acceptor of Proline-derived electrons. Increased POX catalytic activity due to ubiquinone required Complex III and Complex IV of the ETC to be functional, indicating that ATP generated by POX is through oxidative respiration. In addition, experiments using the Seahorse XF24 analyzer confirmed that while POX can utilize Proline as a short term source of electrons during nutrient stress conditions, long term expression of POX results in reactive oxygen species (ROS)-dependent suppression of respiration, consistent with its role in mitochondrial-mediated apoptosis. We found that the POX and Complex II were linked functionally, and that the presence of succinate dramatically affects POX activity. Lineweaver-Burk analysis showed that succinate inhibits POX catalytic activity using anti-competitive inhibition, a rare mechanism of enzymatic inhibition. Two highly selective inhibitors of the Complex II holoenzyme, TTFA and carboxin, inhibit the ubiquinone-mediated activities of POX; in addition, POX co-immunoprecipitates with Complex II, indicating a physical interaction between these two enzymes. Interestingly, Proline appears to stimulate oxidation of succinate by Complex II in vitro, and acute co-administration of Proline and succinate to POX-expressing cells results in a burst of oxidative respiration well above that seen with either substrate alone. Finally, addition of succinate to cell culture inhibits ROS generation by POX and inhibits the long-term effects of POX on respiration. Together, these data support a model in which POX is an integral contributor in oxidative respiration during nutrient stress, and in which respiration is regulated by level and duration of POX expression, as well as levels of Proline relative to the TCA cycle metabolite, and Complex II substrate, succinate. Citation Format: Chad N. Hancock, James M. Phang. Effects of co-regulation of Proline Oxidase and succinate dehydrogenase on oxidative respiration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4613. doi:10.1158/1538-7445.AM2013-4613
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Proline Oxidase promotes tumor cell survival in hypoxic tumor microenvironments
Cancer research, 2012Co-Authors: Wei Liu, Kristine Glunde, Zaver M. Bhujwalla, Venu Raman, Anit Sharma, James M. PhangAbstract:Proline is a readily released stress substrate that can be metabolized by Proline Oxidase (POX) to generate either reactive oxygen species (ROS) to induce apoptosis or autophagy or ATP during times of nutrient stress. However, the contribution of Proline metabolism to tumorigenesis in hypoxic microenvironments has not been explored. In this study, we investigated the different functions of POX under hypoxia and glucose depletion. We found that hypoxia induced POX expression in cancer cells in vitro and that POX upregulation colocalized with hypoxic tissues in vivo. In addition, the combination of hypoxia and low glucose showed additive effects on POX expression. Similar to conditions of low glucose, hypoxia-mediated POX induction was dependent on AMP-activated protein kinase activation but was independent of HIF-1α and HIF-2α. Under low-glucose and combined low-glucose and hypoxic conditions, Proline catabolized by POX was used preferentially for ATP production, whereas under hypoxia, POX mediated autophagic signaling for survival by generating ROS. Although the specific mechanism was different for hypoxia and glucose deprivation, POX consistently contributed to tumor cell survival under these conditions. Together, our findings offer new insights into the metabolic reprogramming of tumor cells present within a hostile microenvironment and suggest that Proline metabolism is a potential target for cancer therapeutics. Cancer Res; 72(14); 3677–86. ©2012 AACR.
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Abstract 1119: The oxidation of Proline by Proline Oxidase provides a regulated source of ROS for mitochondria derived cellular signaling
Molecular and Cellular Biology, 2012Co-Authors: Chad N. Hancock, James M. PhangAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL A role for reactive oxygen species (ROS) in intracellular signaling has been generally accepted, but the organellar localization, the enzymatic source and the regulation of ROS generation are not well understood. Recent work by others has focused on mitochondria as a source of ROS and specifically on the reduction of oxygen by electrons from complex III to form superoxide. The accessibility of complex III to the mitochondrial intermembrane space and cytosol provides an attractive hypothesis for mitochondrial ROS as regulators of cellular redox signaling. We now report that the regulated metabolism of Proline makes it a special substrate for this process. Proline Oxidase (POX) is an inner mitochondrial membrane protein that oxidizes Proline to generate superoxide as well as ATP. Work in our laboratory has documented that POX activity is highly regulated during genotoxic and metabolic stress. Inducers of POX include p53, and Proline-dependent ROS play a role in mitochondrial apoptosis. PPAR-γ and its ligands also induce POX but depending on the metabolic context, POX initiates autophagy as well as apoptosis. Mechanistic models for ROS generation by POX have come from microorganisms. PutA, the prokaryotic homolog of POX can generate ROS by electrons reducing oxygen directly from its catalytic center. On the other hand, in S. cerevisiae, the POX homolog, Put1, has been shown to directly interact with ubiquinone, thereby contributing electrons derived from Proline oxidation into the electron transport chain. In this work, we now report that human POX interacts directly with ubiquinone and that POX activity is markedly increased by the presence of ubiquinone. In addition, by using selective inhibitors of electron transport, we demonstrate that electrons derived from Proline can be converted to superoxide by complex III of the electron transport chain. These radicals are directed into both the mitochondrial matrix as well as into the intermembrane space and the cytosol. Thus, POX is not only specifically regulated but also specially located and coupled to the mitochondrial electron transport chain to mediate ROS signaling. These POX- mediated mechanisms provide important insights into its novel apoptotic and metabolic tumor suppressive functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1119. doi:1538-7445.AM2012-1119
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miR-23b targets Proline Oxidase, a novel tumor suppressor protein in renal cancer.
Oncogene, 2010Co-Authors: Wenbin Liu, Zabirnyk O, Hongshan Wang, Yih Horng Shiao, Michael L. Nickerson, Khalil S, Lucy M. Anderson, Alan O. Perantoni, James M. PhangAbstract:Proline Oxidase (POX) is a novel mitochondrial tumor suppressor that can suppress proliferation and induce apoptosis through the generation of reactive oxygen species (ROS) and decreasing hypoxia-inducible factor (HIF) signaling. Recent studies have shown the absence of expression of POX in human cancer tissues, including renal cancer. However, the mechanism for the loss of POX remains obscure. No genetic or epigenetic variation of POX gene was found. In this study, we identified the upregulated miR-23b in renal cancer as an important regulator of POX. Ectopic overexpression of miR-23b in normal renal cells resulted in striking downregulation of POX, whereas POX expression increased markedly when endogenous miR-23b was knocked down by its antagomirs in renal cancer cells. Consistent with the POX-mediated tumor suppression pathway, these antagomirs induced ROS, inhibited HIF signaling and increased apoptosis. Furthermore, we confirmed the regulation of miR-23b on POX and its function in the DLD1 Tet-off POX cell system. Using a luciferase reporter system, we verified the direct binding of miR-23b to the POX mRNA 3'-untranslated region. In addition, pairs of human renal carcinoma and normal tissues showed a negative correlation between miR-23b and POX protein expression, providing its clinical corroboration. Taken together, our results suggested that miR-23b, by targeting POX, could function as an oncogene; decreasing miR-23b expression may prove to be an effective way of inhibiting kidney tumor growth.
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Oxidized low-density lipoproteins upregulate Proline Oxidase to initiate ROS-dependent autophagy.
Carcinogenesis, 2009Co-Authors: Olga Zabirnyk, Wei Liu, Anit Sharma, Shadi Khalil, James M. PhangAbstract:Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated Proline Oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible for the latter. To elucidate the role of POX in oxLDL-mediated cytotoxicity, we knocked down POX via small interfering RNA and found that this (i) further reduced viability of cancer cells treated with oxLDL; (ii) decreased oxLDL-associated reactive oxygen species generation; (iii) decreased autophagy measured via beclin-1 protein level and light-chain 3 protein (LC3)-I into LC3-II conversion. Using POX-expressing cell model, we established that single POX overexpression was sufficient to activate autophagy. Thus, it led to autophagosomes accumulation and increased conversion of LC3-I into LC3-II. Moreover, beclin-1 gene expression was directly dependent on POX catalytic activity, namely the generation of POX-dependent superoxide. We conclude that POX is critical in the cellular response to the noxious effects of oxLDL by activating protective autophagy.
Yongmin Liu - One of the best experts on this subject based on the ideXlab platform.
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Proline Oxidase functions as a mitochondrial tumor suppressor in human cancers.
Cancer research, 2009Co-Authors: Yongmin Liu, Steven P. Donald, Gregory L. Borchert, Bhalchandra A. Diwan, Miriam R. Anver, James M. PhangAbstract:Tumor metabolism and bioenergetics have become important topics for cancer research and are promising targets for anticancer therapy. Although glucose serves as the main source of energy, Proline, an alternative substrate, is important, especially during nutrient stress. Proline Oxidase (POX), catalyzing the first step in Proline catabolism, is induced by p53 and can regulate cell survival as well as mediate programmed cell death. In a mouse xenograft tumor model, we found that POX greatly reduced tumor formation by causing G2 cell cycle arrest. Furthermore, immunohistochemical staining showed decreased POX expression in tumor tissues. Importantly, HIF-1α signaling was impaired with POX expression due to the increased production of α-ketoglutarate, a critical substrate for prolyl hydroxylation and degradation of HIF-1α. Combined with previous in vitro findings and reported clinical genetic associations, these new findings lead us to propose POX as a mitochondrial tumor suppressor and a potential target for cancer therapy. [Cancer Res 2009;69(16):6414–22]
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Proline Oxidase (dehydrogenase) generates switchable bioenergetics for cancer cell survival or programmed cell death
Cancer Research, 2008Co-Authors: James M. Phang, Jui Pandhare, Yongmin LiuAbstract:4934 Cancer cells survive by surmounting temporal and spatial gauntlets of stress. Identifying and targeting these stress responders may lead to novel regimens for cancer prevention and treatment. With Proline mobilized in the tumor microenvironment from matrix metalloprotein-ases-dependent collagen degradation, cancer cells can activate Proline Oxidase (dehydrogenase) as a stress responder for either bioenergetics or to produce mediators of programmed cell death. Bound to mitochondrial inner membranes, Proline Oxidase (POX) is encoded by a p53-induced gene, and it generates mitochondrial superoxide. Overexpression of POX in DLD-tet-off-POX colorectal cancer cells initiated apoptosis by both intrinsic (mitochondrial) and extrinsic (death receptor) pathways. Co-expression of superoxide dismutase 2 (SOD2) in mitiochondria blocked these effects. POX is also induced by PPARγ and its pharmacologic ligands, the thiazolidinediones. The PPARγ-dependent ROS in cancer cells is markedly decreased by knockdown of POX with antisense or siRNAs in colorectal cancer cells and non-small-cell lung cancer cells. The linkage to genotoxic and inflammatory stress suggested that POX may also be important during nutrient stress as invading cancer cells become isolated from their blood supply. Indeed, this was the case as glucose depletion downregulated mTOR-mediated phosphorylation of S6 kinase but upregulated POX more than 5-fold. The mediation by mTOR was established by showing that Rapamycin or AICAR upregulated POX.more than 10-fold. Under these conditions, Rapamycin modulated bioenergetics to maintain ATP levels in the cell, and POX was involved since the Rapamycin effect was decreased by inhibition of POX catalytic activity with dehydroProline. These findings show that POX is a critical responder to genotoxic, inflammatory and nutrient stress. Recent structural biology characterization by others suggested that POX contains a “switching” mechanism for production of ATP for bioenergetics as well as ROS for programmed cell death. Depending on the specific stress, POX can use Proline-derived electrons to reduce oxygen generating superoxide for programmed cell death or donate electrons to the electron transport chain yielding ATP.
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PPARγ and Proline Oxidase in Cancer
PPAR research, 2008Co-Authors: James M. Phang, Jui Pandhare, Olga Zabirnyk, Yongmin LiuAbstract:Proline is metabolized by its own specialized enzymes with their own tissue and subcellular localizations and mechanisms of regulation. The central enzyme in this metabolic system is Proline Oxidase, a flavin adenine dinucleotide-containing enzyme which is tightly bound to mitochondrial inner membranes. The electrons from Proline can be used to generate ATP or can directly reduce oxygen to form superoxide. Although Proline may be derived from the diet and biosynthesized endogenously, an important source in the microenvironment is from degradation of extracellular matrix by matrix metalloproteinases. Previous studies showed that Proline Oxidase is a p53-induced gene and its overexpression can initiate Proline-dependent apoptosis by both intrinsic and extrinsic pathways. Another important factor regulating Proline Oxidase is peroxisome proliferator activated receptor gamma (PPAR). Importantly, in several cancer cells, Proline Oxidase may be an important mediator of the PPAR-stimulated generation of ROS and induction of apoptosis. Knockdown of Proline Oxidase expression by antisense RNA markedly decreased these PPAR-stimulated effects. These findings suggest an important role in the proposed antitumor effects of PPAR. Moreover, it is possible that Proline Oxidase may contribute to the other metabolic effects of PPAR.
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Proline Oxidase activates both intrinsic and extrinsic pathways for apoptosis the role of ros superoxides nfat and mek erk signaling
Oncogene, 2006Co-Authors: Yongmin Liu, G L Borchert, A Surazynski, James M. PhangAbstract:Proline Oxidase (POX), often considered a 'housekeeping enzyme' might play an important role in apoptosis. We have shown that POX generated Proline-dependent reactive oxygen species (ROS), specifically superoxide radicals, and induced apoptosis through the mitochondrial (intrinsic) pathway. 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 POX-stimulated expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), DR5 and cleavage of caspase-8. Importantly, apoptosis measured by flow cytometry was partially inhibited by Z-IETD-FMK, a specific inhibitor of caspase-8. These findings suggest that the extrinsic (death receptor) pathway also is activated by POX. Furthermore, the mechanism of this effect on the extrinsic pathway, specifically, the induction of TRAIL by POX, may be mediated by NFAT transcription factors. Additionally, POX expression also dramatically decreased phosphorylation of MEK and ERK, and the decrease was partially reversed by expression of manganese superoxide dismutase (MnSOD). Overexpression of constitutively active form of MEK, acMEK, partially blocked POX-induced apoptosis. These findings suggest the involvement of MEK/ERK signaling and further confirm the role of ROS/superoxides in POX-induced apoptosis. Combined with previously published data, we conclude that POX may induce apoptosis through both intrinsic and extrinsic pathways and is involved in nuclear factor of activated T cells (NFAT) signaling and regulation of the MEK/ERK pathway. It is suggested that, as a nutrition factor, POX may modulate apoptosis signals induced by p53 or other anti-cancer agents and enhance apoptosis in stress situations.
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Proline Oxidase activates both intrinsic and extrinsic pathways for apoptosis: the role of ROS/superoxides, NFAT and MEK/ERK signaling
Oncogene, 2006Co-Authors: Yongmin Liu, G L Borchert, Surazynski A, James M. PhangAbstract:Proline Oxidase (POX), often considered a 'housekeeping enzyme' might play an important role in apoptosis. We have shown that POX generated Proline-dependent reactive oxygen species (ROS), specifically superoxide radicals, and induced apoptosis through the mitochondrial (intrinsic) pathway. 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 POX-stimulated expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), DR5 and cleavage of caspase-8. Importantly, apoptosis measured by flow cytometry was partially inhibited by Z-IETD-FMK, a specific inhibitor of caspase-8. These findings suggest that the extrinsic (death receptor) pathway also is activated by POX. Furthermore, the mechanism of this effect on the extrinsic pathway, specifically, the induction of TRAIL by POX, may be mediated by NFAT transcription factors. Additionally, POX expression also dramatically decreased phosphorylation of MEK and ERK, and the decrease was partially reversed by expression of manganese superoxide dismutase (MnSOD). Overexpression of constitutively active form of MEK, acMEK, partially blocked POX-induced apoptosis. These findings suggest the involvement of MEK/ERK signaling and further confirm the role of ROS/superoxides in POX-induced apoptosis. Combined with previously published data, we conclude that POX may induce apoptosis through both intrinsic and extrinsic pathways and is involved in nuclear factor of activated T cells (NFAT) signaling and regulation of the MEK/ERK pathway. It is suggested that, as a nutrition factor, POX may modulate apoptosis signals induced by p53 or other anti-cancer agents and enhance apoptosis in stress situations.
Hyae Gyeong Cheon - One of the best experts on this subject based on the ideXlab platform.
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apoptotic action of peroxisome proliferator activated receptor γ activation in human non small cell lung cancer is mediated via Proline Oxidase induced reactive oxygen species formation
Molecular Pharmacology, 2007Co-Authors: Kiyoung Kim, Jin Hee Ahn, Hyae Gyeong CheonAbstract:Peroxisome proliferator-activated receptor (PPAR)-γ ligands have been shown to inhibit human lung cancers by inducing apoptosis and differentiation. In the present study, we elucidated the apoptotic mechanism of PPARγ activation in human lung cancers by using a novel PPARγ agonist, 1-(trans-methylimino- N -oxy)-6-(2-morpholinoethoxy)-3-phenyl-(1 H -indene-2-carboxylic acid ethyl ester (KR-62980), and rosiglitazone. PPARγ activation selectively inhibited cell viability of non–small-cell lung cancer with little effect on small-cell lung cancer and normal lung cells. The cell death induced by PPARγ activation presented apoptotic features of oligonucleosomal DNA fragmentation in A549 human non–small-cell lung cancer cell line. Reactive oxygen species (ROS) production was accompanied by increased expression of Proline Oxidase (POX), a redox enzyme expressed in mitochondria, upon incubation with the agonists. POX RNA interference treatment blocked PPARγ-induced ROS formation and cytotoxicity, suggesting that POX plays a functional role in apoptosis through ROS formation. The apoptotic effects by the agonists were antagonized by bisphenol A diglycidyl ether, a PPARγ antagonist, and by knockdown of PPARγ expression, indicating the involvement of PPARγ in these actions. The results of the present study suggest that PPARγ activation induces apoptotic cell death in non–small-cell lung carcinoma mainly through ROS formation via POX induction.
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Apoptotic Action of Peroxisome Proliferator-Activated Receptor-γ Activation in Human Non–Small-Cell Lung Cancer Is Mediated via Proline Oxidase-Induced Reactive Oxygen Species Formation
Molecular pharmacology, 2007Co-Authors: Kiyoung Kim, Jin Hee Ahn, Hyae Gyeong CheonAbstract:Peroxisome proliferator-activated receptor (PPAR)-γ ligands have been shown to inhibit human lung cancers by inducing apoptosis and differentiation. In the present study, we elucidated the apoptotic mechanism of PPARγ activation in human lung cancers by using a novel PPARγ agonist, 1-(trans-methylimino- N -oxy)-6-(2-morpholinoethoxy)-3-phenyl-(1 H -indene-2-carboxylic acid ethyl ester (KR-62980), and rosiglitazone. PPARγ activation selectively inhibited cell viability of non–small-cell lung cancer with little effect on small-cell lung cancer and normal lung cells. The cell death induced by PPARγ activation presented apoptotic features of oligonucleosomal DNA fragmentation in A549 human non–small-cell lung cancer cell line. Reactive oxygen species (ROS) production was accompanied by increased expression of Proline Oxidase (POX), a redox enzyme expressed in mitochondria, upon incubation with the agonists. POX RNA interference treatment blocked PPARγ-induced ROS formation and cytotoxicity, suggesting that POX plays a functional role in apoptosis through ROS formation. The apoptotic effects by the agonists were antagonized by bisphenol A diglycidyl ether, a PPARγ antagonist, and by knockdown of PPARγ expression, indicating the involvement of PPARγ in these actions. The results of the present study suggest that PPARγ activation induces apoptotic cell death in non–small-cell lung carcinoma mainly through ROS formation via POX induction.
Steven P. Donald - One of the best experts on this subject based on the ideXlab platform.
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Proline Oxidase functions as a mitochondrial tumor suppressor in human cancers.
Cancer research, 2009Co-Authors: Yongmin Liu, Steven P. Donald, Gregory L. Borchert, Bhalchandra A. Diwan, Miriam R. Anver, James M. PhangAbstract:Tumor metabolism and bioenergetics have become important topics for cancer research and are promising targets for anticancer therapy. Although glucose serves as the main source of energy, Proline, an alternative substrate, is important, especially during nutrient stress. Proline Oxidase (POX), catalyzing the first step in Proline catabolism, is induced by p53 and can regulate cell survival as well as mediate programmed cell death. In a mouse xenograft tumor model, we found that POX greatly reduced tumor formation by causing G2 cell cycle arrest. Furthermore, immunohistochemical staining showed decreased POX expression in tumor tissues. Importantly, HIF-1α signaling was impaired with POX expression due to the increased production of α-ketoglutarate, a critical substrate for prolyl hydroxylation and degradation of HIF-1α. Combined with previous in vitro findings and reported clinical genetic associations, these new findings lead us to propose POX as a mitochondrial tumor suppressor and a potential target for cancer therapy. [Cancer Res 2009;69(16):6414–22]
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Regulation and Function of Proline Oxidase Under Nutrient Stress
Journal of cellular biochemistry, 2009Co-Authors: Jui Pandhare, Steven P. Donald, Sandra K. Cooper, James M. PhangAbstract:Under conditions of nutrient stress, cells switch to a survival mode catabolizing cellular and tissue constituents for energy. Proline metabolism is especially important in nutrient stress because Proline is readily available from the breakdown of extracellular matrix (ECM), and the degradation of Proline through the Proline cycle initiated by Proline Oxidase (POX), a mitochondrial inner membrane enzyme, can generate ATP. This degradative pathway generates glutamate and α-ketoglutarate, products that can play an anaplerotic role for the TCA cycle. In addition the Proline cycle is in a metabolic interlock with the pentose phosphate pathway providing another bioenergetic mechanism. Herein we have investigated the role of Proline metabolism in conditions of nutrient stress in the RKO colorectal cancer cell line. The induction of stress either by glucose withdrawal or by treatment with rapamycin, stimulated degradation of Proline and increased POX catalytic activity. Under these conditions POX was responsible, at least in part, for maintenance of ATP levels. Activation of AMP-activated protein kinase (AMPK), the cellular energy sensor, by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), also markedly upregulated POX and increased POX-dependent ATP levels, further supporting its role during stress. Glucose deprivation increased intracellular Proline levels, and expression of POX activated the pentose phosphate pathway. Together, these results suggest that the induction of Proline cycle under conditions of nutrient stress may be a mechanism by which cells switch to a catabolic mode for maintaining cellular energy levels.
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Overexpression of Proline Oxidase induces Proline-dependent and mitochondria-mediated apoptosis
Molecular and Cellular Biochemistry, 2006Co-Authors: Steven P. Donald, David Valle, Wei Wen Lin, Zhihe Liu, Gary Steel, Cassandra Obie, James M. PhangAbstract:Proline Oxidase (POX), a mitochondrial inner-membrane protein, catalyzes the rate-limiting oxidation of Proline to pyrroline- 5-carboxylate (P5C). Previously we showed that overexpression of POX is associated with generation of reactive oxygen species (ROS) and apoptosis in POX-inducible colorectal cancer cells, DLD-1.POX. We also showed expression of mitochondrial MnSOD partially blunts POX-induced ROS generation and apoptosis. To further investigate the molecular basis of POX-induced apoptosis, we utilized the DLD-1.POX cells to show that cells overproducing POX exhibit an L-Proline-dependent apoptotic response. The apoptotic effect is specific for L-Proline, detectable at 0.2 mM, maximal at 1 mM, and occurs during 48–72 h following the addition of L-Proline to cells with maximally induced POX. The apoptotic response is mitochondria-mediated with release of cytochrome c, activation of caspase-9, chromatin condensation/DNA fragmentation, and cell shrinkage. We conclude that in the presence of Proline, high POX activity is sufficient to induce mitochondria-mediated apoptosis.
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MnSOD inhibits Proline Oxidase-induced apoptosis in colorectal cancer cells
Carcinogenesis, 2005Co-Authors: Yongmin Liu, Steven P. Donald, Gregory L. Borchert, Arkadiusz Surazynski, Christine J. Weydert, Larry W. Oberley, James M. PhangAbstract:Proc Amer Assoc Cancer Res, Volume 45, 2004 763 Proline Oxidase (POX), localized at mitochondrial inner-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) which is freqeuently associated with mitochondria-dependent apoptosis. As an important antioxidant enzyme, superoxide dismutases (SOD) may 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 doxcycline 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-POX cells with recombinant adenoviruses containing MnSOD, CuZnSOD, catalase, or varying combinations of these adenoviruses followed by induced expression of POX. The expression of MnSOD dramatically inhibited POX-induced apoptosis. Compared with control cells, MnSOD-expressing DLD-POX cells generated higher concentration of H2O2 due to dismutation of superoxide radicals. These cells also expressed higher level of phospho-p38 MAPK concomitant to generation of H2O2. Thus, these data further confirm that POX-induced apoptosis is due to the generation of ROS (probably superoxide radicals), and the process is blocked by MnSOD. In addition, activation of p38 MAPK may play a role in the blockade of POX-induced apoptosis by MnSOD.
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Upregulation of Proline Oxidase transcriptional activity by peroxisome proliferator-activated receptor-gamma (PPAR-γ)
Cancer Research, 2004Co-Authors: Jui Pandhare, Yongmin Liu, Steven P. Donald, Sandra K. Cooper, James M. PhangAbstract:4350 Proline Oxidase (POX), also known as Proline dehydrogenase, is a mitochondrial membrane-bound enzyme catalyzing the conversion of Proline to pyrroline-5-carboxylate coupled to the transfer of electrons to cytochrome c . We and others have shown that POX is a p53-induced gene and hyperexpression of POX in colon cancer cells is sufficient for initiating the apoptotic cascade. Despite the demonstrated role of POX in p53-mediated growth inhibition and apoptosis, the molecular mechanisms regulating its expression are not defined. To gain an understanding of these mechanisms, we cloned the human POX promoter region and produced a POX promoter/luciferase reporter construct. By transient transfection studies using expression constructs of several transcription factors in human colon cancer (LoVo) cells and human embryonic kidney (HEK293) cells, we character-ized mechanisms involved in POX expression. Analysis of the promoter nucleotide sequence revealed a number of putative transcription factor-binding sites including C/EBP, AP-1, Sp1, RAR, PPAR, RXR and others which became the basis for our initial studies. In LoVo cells, several transcription factors (PEA3, p300, c-Jun, c-Fos, Fra 1, Jun-D) were modestly active in POX expression, but the most active was PPAR-γ. We found a similar response in HEK 293 cells in which expression of PPAR-γ resulted in a seven- to ten-fold activation of the POX promoter. This effect of PPAR-γ was further enhanced by PPAR-γ ligands, troglitazone and ciglitizone, in a concentration-dependent manner. Peroxisome proliferator-activated receptors (PPARs) have received recent attention because these nuclear hormone receptors, which function as ligand-activated transcription factors regulating lipid metabolism and homeostasis, are involved in genetic control of many cellular processes. Recent reports by others suggest that PPAR-γ ligands induce apoptotic cell death in colon cancer cells and suppress colon carcinogenesis in mice. Our current results establish a significant role for PPAR-γ in regulation of POX expression and suggest that POX is a target gene of PPAR-γ mediating the apoptotic process.
Jerzy Palka - One of the best experts on this subject based on the ideXlab platform.
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A novel plausible mechanism of NSAIDs-induced apoptosis in cancer cells: the implication of Proline Oxidase and peroxisome proliferator-activated receptor
Pharmacological Reports, 2020Co-Authors: Adam Kazberuk, Ilona Zareba, Jerzy Palka, Arkadiusz SurazynskiAbstract:Although pharmaco-epidemiological studies provided evidence for the anticancer potential of non-steroidal anti-inflammatory drugs (NSAIDs), the mechanism of their anti-cancer activity is not known. Several lines of evidence suggest that Proline dehydrogenase/Proline Oxidase (PRODH/POX) may represent a target for NSAIDs-dependent anti-cancer activity. PRODH/POX catalyzes conversion of Proline into Δ1-pyrroline-5-carboxylate releasing ATP or reactive oxygen species for autophagy/apoptosis. Since NSAIDs are ligands of peroxisome proliferator-activated receptor (PPARs) and PPARs are implicated in PRODH/POX-dependent apoptosis we provided a hypothesis on the mechanism of NSAIDs-induced apoptosis in cancer cells.
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Understanding the role of key amino acids in regulation of Proline dehydrogenase/Proline Oxidase (prodh/pox)-dependent apoptosis/autophagy as an approach to targeted cancer therapy
Molecular and Cellular Biochemistry, 2020Co-Authors: Thi Yen Ly Huynh, Ilona Zareba, Weronika Baszanowska, Sylwia Lewoniewska, Jerzy PalkaAbstract: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.
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understanding the role of key amino acids in regulation of Proline dehydrogenase Proline Oxidase prodh pox dependent apoptosis autophagy as an approach to targeted cancer therapy
Molecular and Cellular Biochemistry, 2020Co-Authors: Thi Yen Ly Huynh, Ilona Zareba, Weronika Baszanowska, Sylwia Lewoniewska, Jerzy PalkaAbstract: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.
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prolidase Proline dehydrogenase Proline Oxidase collagen biosynthesis axis as a potential interface of apoptosis autophagy
Biofactors, 2016Co-Authors: Ilona Zareba, Jerzy PalkaAbstract:Prolidase is a cytosolic imidodipeptidase that specifically splits imidodipeptides with C-terminal Proline or hydroxyProline. The enzyme plays an important role in the recycling of Proline from imidodipeptides for resynthesis of collagen and other Proline-containing proteins. The mechanism of prolidase-dependent regulation of collagen biosynthesis was found at both transcriptional and post-transcriptional level. The increase in the enzyme activity is due to its phosphorylation on serine/threonine residues. Prolidase-dependent transcriptional regulation of collagen biosynthesis was found at the level of NF-κB, known inhibitor of type I collagen gene expression. Proline dehydrogenase/Proline Oxidase (PRODH/POX) is flavin-dependent enzyme associated with the inner mitochondrial membrane. The enzyme catalyzes conversion of Proline into Δ(1) -pyrroline-5-carboxylate (P5C), during which reactive oxygen species (ROS) are produced, inducing intrinsic and extrinsic apoptotic pathways. Alternatively, under low glucose stress, PRODH/POX activation produces ATP for energy supply and survival. Of special interest is that PRODH/POX gene is induced by P53 and peroxisome proliferator-activated gamma receptor (PPARγ). Among down-regulators of PRODH/POX is an oncogenic transcription factor c-MYC and miR-23b*. On the other hand, PRODH/POX suppresses HIF-1α transcriptional activity, the MAPK pathway, cyclooxygenase-2, epidermal growth factor receptor and Wnt/b-catenin signaling. PRODH/POX expression is often down-regulated in various tumors, limiting mitochondrial Proline utilization to P5C. It is accompanied by increased cytoplasmic level of Proline. Proline availability for PRODH/POX-dependent ATP or ROS generation depends on activity of prolidase and utilization of Proline in process of collagen biosynthesis. Therefore, Prolidase-PRODH/POX-Collagen Biosynthesis axis may represent potential interface that regulate apoptosis and survival. © 2016 BioFactors, 42(4):341-348, 2016.
