miR-23b targets proline oxidase, a novel tumor suppressor protein in renal cancer.

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.

Proline oxidase, a p53-induced gene, targets COX-2/PGE2 signaling to induce apoptosis and inhibit tumor growth in colorectal cancers.

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/beta-catenin pathway. First, POX markedly reduced COX-2 expression, suppressed the production of prostaglandin E2 (PGE(2)) 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/beta-catenin signaling was decreased by POX in that phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) was decreased on the one hand and phosphorylation of beta-catenin was increased on the other. There changes led to decreased accumulation of beta-catenin and decreased beta-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.

Prolidase-dependent regulation of collagen biosynthesis.

Prolidase [EC.3.4.13.9] is a cytosolic imidodipeptidase, which specifically splits imidodipeptides with C-terminal proline or hydroxyproline. The enzyme plays an important role in the recycling of proline from imidodipeptides (mostly derived from degradation products of collagen) for resynthesis of collagen and other proline-containing proteins. The enzyme activity is up-regulated by beta(1)-integrin receptor stimulation. The increase in the enzyme activity is due to its phosphorylation on serine/threonine residues. Collagen is not only structural component of extracellular matrix. It has been recognized as a ligand for integrin receptors, which play an important role in signaling that regulate ion transport, lipid metabolism, kinase activation and gene expression. Therefore, changes in the quantity, structure and distribution of collagens in tissues may affect cell signaling, metabolism and function. Several line of evidence suggests that prolidase activity may be a step-limiting factor in the regulation of collagen biosynthesis. It has been shown in different physiologic and pathologic conditions. It is of great importance during wound healing, inflammation, aging, tissue fibrosis and possibly skeletal abnormalities seen in Osteogenesis Imperfecta. The mechanism of prolidase-dependent regulation of collagen biosynthesis was found at both transcriptional and post-transcriptional levels. In this study, we provide evidence for prolidase-dependent transcriptional regulation of collagen biosynthesis. The mechanism was found at the level of NF-kB, known inhibitor of type I collagen gene expression. Modulation of integrin-dependent signaling by stimulatory (i.e. thrombin) or inhibitory (i.e. echistatin) beta(1)-integrin ligands or by nitric oxide donors (i.e. DETA/NO) affect prolidase at post-transcriptional level. All those factors may represent novel approach to pharmacotherapy of connective tissue disorders.

Proline oxidase activates both intrinsic and extrinsic pathways for apoptosis: the role of ROS/superoxides, NFAT and MEK/ERK signaling.

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.

Neural inhibition by c-Jun as a synergizing factor in bone morphogenetic protein 4 signaling.

The transcription factor, activator protein 1 (AP-1) complexes (c-Jun and c-Fos heterodimers) has been shown to interact with transforming growth factor beta signaling in mammalian cells and Drosophila embryo. Here we show that c-Jun alone is involved in the anti-neuralizing activity of bone morphogenetic protein 4, a transforming growth factor beta superfamily member, in Xenopus neurogenesis. Co-injection of mRNAs encoding c-jun and a dominant negative bone morphogenetic protein receptor completely inhibits dominant negative bone morphogenetic protein receptor-induced neuralization and reverses the epidermal fate in the animal cap. Surprisingly, a dominant negative c-Jun does not induce neural tissue in the animal cap, but it synergizes with dominant negative bone morphogenetic protein receptor for neural induction. Temporal analysis using a dexamethasone-inducible c-Jun shows that exogenous c-Jun activity must be turned on before or at stage 11 to fulfill the anti-neuralizing effect. Neural inhibition by c-Jun does not occur until stage 13 suggesting that c-Jun probably acts by suppressing neural maintenance rather than neural initiation. This is also supported by the fact that c-Jun does not inhibit expression of the neural-initializing gene Zic-r1 but the neural cofactor Sox2, and that ectopic expression of Sox2 attenuates the anti-neuralizing effect of c-Jun. Finally, we display that the c-Jun effect is enhanced by an auto-regulatory loop between c-Jun and bone morphogenetic protein. These studies suggest that c-Jun/AP-1 is a converging point in both the fibroblast growth factor and transforming growth factor beta signaling pathways. Based on our findings, we propose that c-Jun synergizes with bone morphogenetic protein 4 signaling to inhibit neural development in Xenopus ectoderm.

Diet and cancer prevention studies in p53-deficient mice.

Progress in mechanism-based cancer prevention research may be facilitated by the use of animal models displaying specific genetic susceptibilities for cancer such as mice deficient in the p53 tumor suppressor gene, the most frequently altered gene in human cancer. We observed in p53-knockout (p53-/-) mice that calorie restriction (CR; 60% of the control group's intake of carbohydrate energy) increased the latency of spontaneous tumor development (mostly lymphomas) approximately 75%, decreased serum insulin-like growth factor (IGF)-1 and leptin levels, significantly slowed thymocyte cell cycle traverse and induced apoptosis in immature thymocytes. In heterozygous p53-deficient (p53+/-) mice, CR and 1 d/wk of food deprivation each significantly delayed spontaneous tumor development (a mix of lymphomas, sarcomas and epithelial tumors) and decreased serum IGF-1 and leptin levels even when begun late in life. We have also developed a rapid and relevant p53+/- mouse mammary tumor model by crossing p53-deficient mice with MMTV-Wnt-1 transgenic mice, and found that CR and 1 d/wk food deprivation significantly increased mammary tumor latency (greater than twofold) and reduced the mean serum IGF-1 and leptin levels to <50% of that of control mice (P < 0.0001). In addition, fluasterone, fenretinide and soy each delayed tumor development but had little effect on IGF-1 or leptin levels. We have capitalized on the susceptibility of p53+/- mice to chronic, low dose, aromatic amine-induced bladder carcinogenesis to develop a useful model for evaluating bladder cancer prevention approaches such as cyclooxygenase-2 inhibition. As demonstrated by these examples, mice with specific (and human-like) genetic susceptibilities for cancer provide powerful new tools for testing and characterizing interventions that may inhibit the process of carcinogenesis in humans.

Testosterone and prostate specific antigen stimulate generation of reactive oxygen species in prostate cancer cells.

Prostate specific antigen, the clinical marker for prostate cancer, is a neutral serine protease whose function is to lyse seminal proteins. Recent work by our laboratory has suggested that prostate specific antigen stimulates the generation of reactive oxygen species in prostate cancer cells. Using 2',7'-dichlorofluorescin diacetate, a dye that fluoresces in the presence of hydrogen peroxide or hydroxyl radicals, we found that prostate specific antigen markedly stimulated reactive oxygen species generation in LNCaP cells. The effect was concentration dependent and its specificity was supported by the fact that anti-prostate specific antigen antibodies abolished the response. Since testosterone stimulates the production of prostate specific antigen, we considered that the reactive oxygen species response to testosterone may be linked to prostate specific antigen. We found that the testosterone effect on reactive oxygen species was blocked by flutamide and by anti-prostate specific antigen antibody. Additionally, though PC3 and DU145 could not respond to testosterone, they readily increased reactive oxygen species in response to prostate specific antigen. Focusing on the mechanism of the prostate specific antigen effect, we tested two other serine proteases, trypsin and chymotrypsin, but found no effect on reactive oxygen species in LNCaP cells. Nevertheless, serine protease inhibitors, alpha(1)-antichymotrypsin, alpha(2)-macroglobulin and Bowman-Birk inhibitor, blocked reactive oxygen species generation stimulated by prostate specific antigen. This apparent paradox was investigated with the use of a specific anti-'prostate specific antigen' antibody which recognizes an epitope away from the catalytic site and which does not inhibit protease activity. Despite the lack of inhibition of proteolytic activity, this antibody blocked the effect of prostate specific antigen on reactive oxygen species generation. These findings suggest that although the integrity of the prostate specific antigen molecule is necessary for stimulating reactive oxygen species generation, its proteolytic activity is not. The underlying mechanism is currently under investigation.

Proline oxidase, encoded by p53-induced gene-6, catalyzes the generation of proline-dependent reactive oxygen species.

The p53-dependent initiation of apoptosis is accompanied by the induction of proline oxidase (POX), a mitochondrial enzyme catalyzing the conversion of proline to pyrroline-5-carboxylate with the concomitant transfer of electrons to cytochrome c. However, the contribution of increased POX activity to apoptosis, if any, remains unknown. Using Adriamycin to initiate p53-dependent apoptosis, we showed that the expression of POX is up-regulated in a time- and dose-dependent manner in a human colon cancer cell line (LoVo). In cells expressing POX, the addition of proline increases reactive oxygen species (ROS) generation in a concentration-dependent manner; glutamate, a downstream product of proline oxidation, had no effect. Induction of POX was dependent on the p53 status of the cell. In the conditionally immortalized murine colonic epithelial cell line YAMC, where the p53 phenotype can be modulated by temperature, proline oxidase expression and ROS production could only be induced when the cells were phenotypically p53-positive. To confirm that the observed ROS production was not secondary to some other effect of p53, we also conditionally expressed POX in a p53-negative colon cancer line. Again, we found a proline-dependent ROS increase with POX expression. We hypothesize that proline oxidation supports the generation of ROS by donating reducing potential to an electron transport chain altered either by p53-dependent mechanisms or by overexpression of POX.

Differential formation of beta-catenin/lymphoid enhancer factor-1 DNA binding complex induced by nitric oxide in mouse colonic epithelial cells differing in adenomatous polyposis coli (Apc) genotype.

Increased cytoplasmic beta-catenin levels and the associated nuclear beta-catenin/T-cell factor (Tcf)-lymphoid enhancer factor (LEF) complex formation have been frequently found in colon cancer. In this context, overproduction of nitric oxide (NO) attributable to inflammatory stimuli in diseases such as ulcerative colitis and Crohn's disease may-contribute to colonic carcinogenesis. Therefore, we examined the modulation by NO of cytoplasmic beta-catenin levels and the formation of the nuclear beta-catenin/LEF-1 DNA binding complex in conditionally immortalized mouse colonic epithelial cells that differed in adenomatous polyposis coli (Apc) genotype, namely young adult mouse colon (YAMC; Apc+/+) and immortal mouse colon epithelium (IMCE; ApcMin/+). Unlike most colon cancer cell lines, this pair of cell lines has either nondetectable or low basal level of beta-catenin when they are cultured under nonpermissive and nonproliferative conditions. Using electrophoretic mobility shift assays, we found that NO-releasing agents (E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide and S-nitroso-N-acetylpenicillamine greatly enhanced the formation of beta-catenin/LEF-1 DNA binding complex in a concentration- and time-dependent fashion in YAMC and IMCE cells. Significantly, IMCE cells showed a markedly greater amount of nuclear beta-catenin/LEF-1 DNA binding complex in response to NO. Super shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Western blot analysis of the soluble cytoplasmic fractions demonstrated that these NO donors caused differential accumulation of cytoplasmic beta-catenin in YAMC and IMCE. In conclusion, this study indicates that the defective beta-catenin degradation machinery attributable to ApcMin/+ mutation in IMCE cells not only affects basal levels but also contributes to NO-induced dysregulation of cytoplasmic beta-catenin and nuclear beta-catenin/LEF-1 DNA binding complex formation.

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells.

Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc(+/+)). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Super-shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of beta-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex formation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formation of nuclear beta-catenin/LEF-1 DNA complex.

Differential expression of prostaglandin endoperoxide H synthase-2 and formation of activated beta-catenin-LEF-1 transcription complex in mouse colonic epithelial cells contrasting in Apc.

Mutations in Apc underlie the intestinal lesions in familial adenomatous polyposis and are found in >85% of sporadic colon cancers. They are frequently associated with overexpression of prostaglandin endoperoxide H synthase-2 (PGHS-2) in colonic adenomas. It has been suggested that Apc mutations are linked mechanistically to increased PGHS-2 expression by elevated nuclear accumulation of beta-catenin-Tcf-LEF transcription complex. In the present study, we show that PGHS-2 is differentially expressed in mouse colonic epithelial cells with distinct Apc status. Cells with a mutated Apc expressed markedly higher levels of PGHS-2 mRNA and protein and produced significantly more prostaglandin E2 than cells with normal Apc. Using electrophoretic mobility shift assays, we demonstrate that DNA-beta-catenin-LEF-1 complex formation is differentially induced in these two cell lines in an Apc-dependent manner. Our data indicate that the differential induction of beta-catenin-LEF-1 complex correlates closely with differential expression of PGHS-2. These findings support the hypothesis that the differential expression of PGHS-2 is mediated through the proposed beta-catenin/Tcf-LEF signaling pathway.

Mechanism-based cancer prevention approaches: targets, examples, and the use of transgenic mice.

Humans are exposed to a wide variety of carcinogenic insults, including endogenous and man-made chemicals, radiation, physical agents, and viruses. The ultimate goal of carcinogenesis research is to elucidate the processes involved in the induction of human cancer so that interventions may be developed to prevent the disease, either in the general population or in susceptible subpopulations. Progress to date in the carcinogenesis field, particularly regarding the mechanisms of chemically induced cancer, has revealed several points along the carcinogenesis pathway that may be amenable to mechanism-based prevention strategies. The purpose of this review is to examine the basic mechanisms and stages of chemical carcinogenesis, with an emphasis on ways in which preventive interventions can modify those processes. Possible ways of interfering with tumor initiation events include the following: i) modifying carcinogen activation by inhibiting enzymes responsible for that activation or by direct scavenging of DNA-reactive electrophiles and free radicals; ii) enhancing carcinogen detoxification processes by altering the activity of the detoxifying enzymes; and iii) modulating certain DNA repair processes. Possible ways of blocking the processes involved in the promotion and progression stages of carcinogenesis include the following: i) scavenging of reactive oxygen species; ii) altering the expression of genes involved in cell signaling, particularly those regulating cell proliferation, apoptosis, and differentiation; and iii) decreasing inflammation. In addition, the utility for mechanism-based cancer prevention research of new animal models that are based on the overexpression or inactivation of specific cancer-related genes is examined.

p53-independent inhibition of nitric oxide generation by cancer preventive interventions in ex vivo mouse peritoneal macrophages.

We have previously reported that dietary interventions with purported anti-inflammatory activity delay spontaneous tumorigenesis in p53-deficient (p53-/-) mice. In the present study, 4 weeks of calorie restriction or the dietary administration of chemopreventive steroids dehydroepiandrosterone and 16alpha-fluoro-5-androsten-17-one significantly reduced the inducible generation of nitric oxide (NO) in ex vivo peritoneal macrophages from male p53 wild-type and p53-/- mice relative to the respective ad libitum-fed controls; expression of inducible nitric oxide synthase II (NOS2) protein was also markedly decreased compared with respective controls. These findings suggest that the p53-independent suppression of inducible NO production observed in this study may contribute to the anti-cancer effects of these preventive interventions in p53-/- mice.

Calorie restriction reduces ulcerative dermatitis and infection-related mortality in p53-deficient and wild-type mice.

In rodents calorie restriction (CR) reduces cancer incidence, improves health by delaying age-related declines in physiologic measures, and extends both median and maximal life span. The mechanisms underlying the various beneficial effects of CR remain undefined. In this study, heterozygous p53-deficient (p53(+/-)) mice (in which the inactivation of one allele of the p53 tumor suppressor gene increases susceptibility to spontaneous and carcinogen-induced tumor development) and wild-type (WT) litter mates were subjected to a two-stage skin carcinogenesis protocol with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. Instead of skin carcinomas, however, the chemical treatment protocol caused ulcerous skin lesions, and 89% of mice fed ad libitum died from infection/septicemia. When WT mice were restricted to 60% of the average calorie intake of the respective ad libitum group, however, only 33% developed such lesions, and the CR mice survived twice as long on average as the ad libitum mice. CR also extended life span in p53(+/-) mice, but 50% of p53(+/-) mice subjected to CR still developed skin ulcers and mean life span was shorter than that seen in WT mice. Differences in response to CR between WT and p53(+/-) mice may be due to the reduction in p53 gene dosage, dissimilarity in the application of the CR treatment, or both. These results suggest that some of the beneficial effects of CR may need full expression of p53 for complete realization.

Differential expression and activity of phosphatases and protein kinases in adriamycin sensitive and resistant human breast cancer MCF-7 cells.

Multidrug resistance is one of the major obstacles in cancer chemotherapy. In tumor cells, overexpression of the transmembrane P-glycoprotein 170 (P-gp) is associated with the multidrug resistance phenotype and serves as a drug efflux pump. The activation of P-gp has been suggested to occur at the post-translational level. Protein kinase C mediated phosphorylation may be associated with the drug effux mechanism but the overall phosphorylation pathway has not been completely defined. we report the novel finding of an increase in phosphatase 1B (a tyrosine phosphatase) and a decrease in PP1 and PP2A (serine/threonine phosphatases) expression and activity in our series of early (R65) and late (R500) stage adriamycin resistant MCF-7 cells. In addition, we show a decrease in protein kinase A (PKA) activity and an increase in protein kinase C (PKC) in our drug resistant cells. Analyses of PKC isoforms alpha through epsilon revealed that PKCbeta was not expressed and that all other isoforms increased with increasing resistance, except PKCgamma which was detected only in R65 cells. Our findings suggest that in drug resistant cells, there is a pattern consistant with the maintenance of serine and threonine residues in a phosphorylated state.

Increased UDP-glucuronosyltransferase activity and decreased prostate specific antigen production by biochanin A in prostate cancer cells.

Our laboratory has characterized androgen metabolism in an androgen-responsive prostate cancer cell line (LNCaP) and showed that these cells accumulated intracellular testosterone primarily as glucuronidated metabolites. Using a cell-free assay with testosterone as substrate, we showed that LNCaP had UDP-glucuronosyltransferase (UDPGT) activity. Because dietary factors, such as flavonoids in soy products, may reduce the risk for hormone-dependent cancers, we studied the effects of flavonoids on testosterone-UDPGT activity. LNCaP cells were exposed to selected flavonoids for up to 6 days. The increase in UDPGT-specific activity was linear over this period. Of the compounds tested, biochanin A was the most potent, with increased activity at concentration range 0.5-50 microM. Activities were linear for time and protein and were unaffected by flavonoids added directly to the assay. Kinetics studies showed no change in Km for testosterone in the face of these large increases in specific activity. Cellular metabolism of testosterone reflected the increase in enzyme activity. Intact cells treated with biochanin A produced testosterone-glucuronide from testosterone at twice the rate of controls. The steroid form of the UDPGT transcript was expressed in LNCaP cells and was enhanced in biochanin A-treated LNCaP cells. Additionally, biochanin A markedly decreased prostate specific antigen (PSA) level against the effect of testosterone on PSA production. Biochanin A significantly decreased the testosterone-stimulated release of PSA, presumably because biochanin A increased UDPGT and increased the intracellular glucuronidation of testosterone. These studies suggest that the modulation of hormone metabolism by dietary factors may be important in the prevention and treatment of prostate cancer.

Prolidase in human breast cancer MCF-7 cells.

Prolidase (EC 3.4.13.9) is an ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of dipeptides containing C-terminal proline or hydroxyproline. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. We have shown previously that prolidase activity in normal human skin fibroblasts is regulated by the interaction of type I collagen with beta1 integrin receptor. In the present study, we investigate prolidase activity in MCF-7 cells and find it is only one-third of that in normal human skin fibroblasts. The relative difference in prolidase activity is corroborated by enzyme protein with Western immunoblot analysis. We propose that the decrease in prolidase activity is due to derangement of regulation by the collagen-beta1 integrin receptor axis. Supporting evidence comes from the following observations: (1) relative collagen content elaborated by MCF-7 cells as compared to fibroblasts is lower by 30% in sparse cells and by 80% at confluence; (2) collagenase treatment of both cell types results in decreased enzyme activity; (3) in contrast to fibroblasts, prolidase activity in MCF-7 cells is not stimulated by the addition of type I collagen or beta1 integrin antibodies (agonist for beta1 integrin receptor); and (4) in contrast to fibroblasts, MCF-7 cells express only trace amounts of beta1 integrin receptor as shown by Western immunoblot analysis. Thus, we conclude that depressed prolidase activity in MCF-7 cells may be a result of disturbances in signaling mediated by beta1 integrin-collagen interaction.

Prolidase activity in fibroblasts is regulated by interaction of extracellular matrix with cell surface integrin receptors.

Prolidase (EC 3.4.13.9) is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. An increase in enzyme activity is correlated with increased rates of collagen turnover indicative of extracellular matrix (ECM) remodeling, but the mechanism linking prolidase activity and ECM is poorly understood. Thus, the effect of ECM-cell interaction on intracellular prolidase activity is of special interest. In cultured human skin fibroblasts, the interaction with ECM and, more specifically, type I collagen mediated by the beta 1 integrin receptor regulates cellular prolidase activity. Supporting evidence comes from the following observations: 1) in sparse cells with a low amount of ECM collagen or in confluent cells in which ECM collagen was removed by collagenase (but not by trypsin or elastase) treatment, prolidase activity was decreased; 2) this effect was reversed by the addition of type I collagen or beta 1 integrin antibody (agonist for beta 1 integrin receptor); 3) sparse cells (with typically low prolidase activity) showed increased prolidase activity when grown on plates coated with type I collagen or on type IV collagen and laminin, constituents of basement membrane; 4) the relative differences in prolidase activity due to collagenase treatment and subsequent recovery of the activity by beta 1 integrin antibody or type I collagen treatment were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells, as shown by Western immunoblot analysis. Thus, we conclude that prolidase activity responded to ECM metabolism (tissue remodeling) through signals mediated by the integrin receptor.

Calorie restriction induces a p53-independent delay of spontaneous carcinogenesis in p53-deficient and wild-type mice.

We reported previously that calorie restriction (CR) delays spontaneous carcinogenesis in p53-deficient (p53-/-) mice, suggesting that CR modulates carcinogenesis by p53-independent mechanisms. To further evaluate the role of p53, we monitored tumor development in p53-/- and wild-type (p53+/+) mice fed ad libitum (AL) or a CR regimen (60% of AL calorie intake). CR delayed tumor mortality in p53-/- and p53+/+ mice (mean time to death, 169 and 648 days, respectively) relative to AL feeding (104 and 470 days). The estimated age-specific cancer death rate AL:CR ratios were 4.3 for p53-/- mice and 4.4 for p53+/+ mice. Thus, despite the accelerated onset of carcinogenesis in p53-/- mice, the tumor-delaying effect of CR was similar in the two genotypes.

Effects of dehydroepiandrosterone and calorie restriction on the Bcl-2/Bax-mediated apoptotic pathway in p53-deficient mice.

Modulation of apoptosis through altered expression of Bcl-2 and/or Bax may be a mechanism by which dehydroepiandrosterone (DHEA) administration and calorie restriction (CR) exert their chemopreventive effects in p53-deficient (p53-/-) mice. Using immunohistochemical detection we found that treatment with both DHEA and CR resulted in decreased expression of the PCNA proliferation marker in the thymus. In addition, treatment with DHEA also increased the rate of apoptosis in the thymus, resulting in marked thymic atrophy. Thus, both DHEA and CR appear to shift cell number homeostasis by favoring apoptosis. To further understand the molecular mechanisms by which DHEA and CR exert their effects, we examined two components of the apoptotic pathway, Bcl-2 and Bax. We found that p53-/- mice have much higher levels of Bcl-2 mRNA in the thymus than wild-type (p53+/+) mice. Treatment of p53-/- animals with DHEA resulted in decreased Bcl-2 but not Bax mRNA levels in the thymus. In contrast, CR did not change either Bcl-2 or Bax mRNA expression. The present study provides molecular evidence that DHEA and CR may modulate tumorigenesis through alterations in the apoptotic and/or proliferative pathways.