In previously published studies, we have shown that apoptosis can be induced by overexpression of POX by stable transfection ( Liu Y, et al., Carcinogenesis, 26:1335, 2005; Liu Y, et al., Oncogene, 25:5640, 2006) or by increased expression with pharmacologic agents (Pandhare J, et al., J. Biol. Chem., 281:2044, 2006 ). Furthermore, we showed that POX activates both the intrinsic (mitochondrial) and extrinsic (death receptor) pathways (Liu Y, et al., Oncogene, 25:5640, 2006) for apoptosis. These effects are based on the generation of proline-dependent superoxide; co-expression of MnSOD which is localized in mitochondria, abolished the POX-dependent apoptotic effects. Not only is the apoptotic cascade induced by overexpression of POX, but also the cell cycle is disrupted. Varying the concentration of doxycycline in the medium yields a graduated expression of POX in DLD-tet-off-POX cells. At 0.2 ng/ml and 0.02 mg/ml of doxycycline, the level of POX on Westerns is about 35% and 75%, respectively, of that in cells without doxycycline. Importantly, graduated POX expression altered progression through the cell cycle. With increasing POX expression, an increase of cells in G2 and a decrease in S phase were observed. There were marked changes in regulatory proteins governing the G2/M checkpoint. Additional insights were gained from microarray analysis of POX-related gene expression which showed marked changes in the growth arrest and DNA damge inducible (GADD) gene family. To corroborate this finding, we performed studies overexpressing POX and monitored GADD expression using RT-PCR and Western blots. Using RT-PCR we found that isoforms of GADD45 were increased. On Western blots, GADD45alpha which was undetectable with POX suppressed, showed a robust signal with POX expression. These findings clearly showed that POX overexpression not only induces apoptosis, but also blocks the G2/M transition, perhaps by inducing GADD. To test whether this POX-dependent apoptotic mechanism can be translated to animal models, we performed studies using DLD-tet-off-POX xenograft tumors in athymic mice differentially administered doxycycline (100 micrograms/ml) in their drinking water. First, we injected DLD-tet-off-vector cells into the lumbar region of athymic mice and found that tumors rapidly grew unaffected by doxycycline. By the end of week 3, all the animals were euthanized because of the size of the tumors. When DLD-tet-off-POX cells were injected, tumor formation was directly related to intake of doxycycline. In animals on doxycycline (POX suppressed), all animals (n=22) rapidly formed tumors in a fashion no different from those injected with DLD-tet-off-vector cells. In contrast, animals without doxycycline (POX induced), had marked retardation of tumor growth. In fact, by the end of week 3, tumors were palpable in only 14% (3/22) of the animals. Even by the end of week 5, palpable tumors were found in only 32% (7/22). These studies showed that POX expression and the resultant proline-dependent apoptosis markedly decreased the formation of tumors in athymic mice. Data expressed as Kaplan-Meier survival curves showed that mice with POX expression suppressed by doxycycline were all dead by day 24 (euthanized for ethical reasons). In striking contrast, all mice with induced POX (doxycycline withheld) survived to the end of the experiment (day 30). To extend our findings into POX-dependent suppression of specific tumors, we will develop POX transgenic mice and cross them with mice with genetic tumor susceptibilities to determine whether POX expression affects expression of specific tumor phenotypes. PPARgamma and its ligands (the thiazolidinediones) induce apoptosis in cultured cancer cells. More importantly, the population of type 2 diabetes mellitus treated with thiazolidinediones have decreased risk for certain cancers (lung, but not colorectal or prostate). Our studies showed that the apoptotic effects of PPARgamma on RKO cells (colon cancer cells) was dependent on POX activity (Pandhare J, et al., J. Biol. Chem., 281:2044, 2006). This finding was recently corroborated in non-small cell lung cancer cells (Kim KY, et al., Mol. Pharm. 72:674, 2007). Since ex-smokers as well as smokers have increased risk for lung cancer, new prevention approaches are intensely sought. Thus, these new findings may be important for public health. We are collaborating with Dr. Eva Szabo of the Division of Cancer Prevention to pursue these studies. Although we are focused on translating the basic discoveries into cancer-relevant clinical applications, we have also made additional novel discoveries. The aforementioned studies have emphasized proline, and an alternative substrate, hydroxyproline, the hydroxylated derivative of proline plays an important role in metabolsim. In collagen, the most abundant protein in the body, hydroxyproline and proline are present in approximately equal amounts, and together they comprise 25% of collagen residues. Free proline and hydroxyproline are degraded by parallel pathways. However, their respective first degradative steps are catalyzed by POX and hydroxyproline oxidase (HyPOX), separate enzymes encoded by distinct genes, PRODH and PRODH2, located on separate chromosomes and with little substrate crossover. We now have shown that the degradation of hydroxyproline serves a function similar to that of proline in apoptosis. Overexpression of p53 by transient transfection or induction of p53 expression with cytotoxic agents markedly upregulated HyPOX activity resulting in increased levels of reactive oxygen species (ROS) and induction of apoptosis. Thus, hydroxyproline degradation back up the metabolic functions of proline degradation. This finding further supports the metabolic importance of ECM degradation as a source of both proline and hydroxyproline, and provides a plausible explanation for the rather mild consequences of isolated mutations in either PRODH or PRODH2 (Coopoer SK, Pandhare J, Donald SP, Phang SM. J. Biol. Chem. 283:10485-10492, 2008. During the year 2007-2008, advances have been made in two important areas of POX-mediated programmed cell death. First, a robust link between POX and human cancer has been established. Using DLD-tet-off POX cells, we showed that POX expression can downregulate three important pathways involved in carcinogenesis. Cyclooxygenase 2 (COX-2), an enzyme known to be highly expressed in tumors, is markedly down-regulated with POX expression. This is accompanied by a marked decrease in the production of prostaglandin E2 (PGE2). The phosphorylation of the epidermal growth factor receptor (EGFR) was also downregulated. Finally, POX downregulated the signaling of the Wnt/beta-catenin pathway by decreasing the phosphorylation of glycogen synthase kinase-3beta and increasing the phosphorylation of beta-catenin. Signaling by POX thereby provided a mechanism for suppressing carcinogenesis, and this was shown by immunohistochemical studies of human tumors which showed a marked decrease in POX as compared to paired adjacent normal tissues (Liu Y, Borchert GL, Surazynski A, Phang JM. Oncogene 2008 Sep 15 [Epub ahead of print] PMID 18794709). The second interesting area is the demonstration that POX-mediated apoptosis is involved [summary truncated at 7800 characters]
| Phang, James M (2008) Introduction to second proline symposium. Amino Acids 35:653-4 |
| Hu, Chien-an A; Donald, Steven P; Yu, Jian et al. (2007) Overexpression of proline oxidase induces proline-dependent and mitochondria-mediated apoptosis. Mol Cell Biochem 295:85-92 |
| Liu, Y; Borchert, G L; Surazynski, A et al. (2006) Proline oxidase activates both intrinsic and extrinsic pathways for apoptosis: the role of ROS/superoxides, NFAT and MEK/ERK signaling. Oncogene 25:5640-7 |
