Cytokine Network & NETs.

The process of forming and releasing neutrophil extracellular traps (NETs) can be regulated by exogenous and endogenous factors, including cytokines. The study aimed to assess the impact of proinflammatory cytokines, IL-15, IL-17, IL-18, and anti-inflammatory IL-10 on the formation of NETs, all in comparison to IL-8 and pathogenic factors: LPS, fMLP. Also, the expression of myeloperoxidase (MPO), one of the main elements of neutrophil traps, was evaluated. After isolating the neutrophils with Polymorphprep™, the cells were sorted using CD16 MACS® microbeads and incubated with selected factors. The formation of NETs was registered using a BD Pathway 855 microscope system and the expression of MPO was evaluated using flow cytometry. The amounts of circulating DNA in cell supernatants was fluorescently quantified. Microscopic photographs indicated that rhIL-15, rhIL-17, rhIL-18 and fMLP induce formation and release of NETs at a similar timespan, while in the presence of rhIL-10, the formation of the traps was delayed. The presence of the studied cytokines indicated two populations of neutrophils displaying differing MPO expression (MPOlow and MPOhigh). Moreover, stimulation of neutrophils with LPS and fMLP revealed two populations of these cells that differed not only in the expression of MPO, but also in size.

Erythropoietin accelerates tumor growth through increase of erythropoietin receptor (EpoR) as well as by the stimulation of angiogenesis in DLD-1 and Ht-29 xenografts.

Anemia is a relatively common symptom coexisting with colorectal carcinoma. Besides having a positive impact on hematological parameters, erythropoietin (Epo) has the serious adverse effect of promoting the neoplastic process. The role of Epo in colon cancer has not been clearly shown. The aim of this study was to assess the effects of Epo therapy on colorectal carcinoma cells both in in vitro and in animal models. Human colon adenocarcinoma cells DLD-1 and Ht-29 were cultured in medium with Epo beta in normoxia. Cell proliferation was measured with an automated cell counter. Expression of erythropoietin receptor (EpoR) mRNA, Akt mRNA, and their proteins were assessed by RT-PCR and confocal microscopy, respectively. Nude mice were inoculated with adenocarcinoma cells and treated with a therapeutic dose of Epo. Expression of EpoR, VEGF, Flt-1 and CD31 was evaluated in xenograft tumors. We identified that Epo through EpoR activates Akt, which promotes colon cancer cell growth and proliferation. Epo, and high levels of phosphorylated EpoR, directly accelerates tumor growth through its proliferative and proangiogenic effects. This study demonstrated that Epo had enhanced carcinogenesis through increase of EpoR and Flt-1 expression, and thereby contributed to tumor development. These results suggest that both EpoR-positive and EpoR-negative cancer cells could be regulated by exogenous Epo. However, an increased response to erythropoietin was observed in the EpoR-positive cells. Thus, erythropoietin increases the risk of tumor progression in colon cancer and should not be used to treat anemia in this type of cancer.

Cross-talk between integrin receptor and insulin-like growth factor receptor in regulation of collagen biosynthesis in cultured fibroblasts.

PURPOSE: Cellular processes are regulated by signals generated by adhesion receptors and growth factor receptors. IGFbinding protein 1 (IGFBP-1) is a molecule which may affect the both signaling pathways through inactivation of IGF-I (ligand for IGF-IR) and binding to RGD region of integrin receptors. Whether this phenomenon is important in communication between insulin-like growth factor receptor (IGF-IR) and ß1-integrin receptor in regulation of prolidase activity and collagen biosynthesis is the aim of this study. MATERIAL AND METHOD: We studied the effects of IGFBP-1, IGF-I, thrombin (integrin activator), echistatin (disintegrin), phosphatidylinositol 3-kinase inhibitor (LY-294002) and ERK 1/2 inhibitors (PD98059 and UO126) on prolidase activity, collagen biosynthesis and expression of proteins participating in pathways generated by these receptors. RESULTS: Stimulation of ß1-integrin and IGF-I receptors by standard ligands was proved to up-regulate collagen synthesis in cultured fibroblasts. IGFBP-1, similarly as echistatin and studied inhibitors, contributed to down-regulation of ERK1/2, Akt, mTOR expression and up-regulation of NFκB. It was accompanied by parallel decrease in prolidase activity and collagen biosynthesis. CONCLUSION: The data suggest that "cross talk" between IGF-I receptor and integrin receptor may play important role in regulation of prolidase activity and collagen biosynthesis.

Metronidazole affects breast cancer cell lines.

PURPOSE: The aim of our study was to evaluate the impact of metronidazole (MTZ) on cytotoxicity and DNA synthesis in MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative) breast cancer cell lines. MATERIAL/METHODS: Toxicity of MTZ was determined by MTT test. MCF-7 and MDA-MB-231 cells were incubated with metronidazole used in different concentrations for 24, 48 and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. RESULTS: We showed that MTZ in concentration 250 µg/ml significantly increases the growth of MCF-7 cell lines after 24 hours of incubation, but it reduces cell viability in concentrations 1 and 10 µg/ml 72 hours after the drug application. Significant increase of MDA-MB-231 cell viability was obtained in MTZ concentration of 250 µg/ml after 24 and 72 hours. The increase of [3H]-thymidine incorporation in MCF-7 cell line treated with MTZ in concentration 250 µg/ml was statistically significant after 24 hours. Great suppression of cell proliferation was obtained in MDA-MB-231 breast cell line after application of the following concentrations of MTZ: 0.1 µg/ml (after 24 hours) and 0.1, 10, 50, 250 µg/ml (after 72h). CONCLUSIONS: We found that metronidazole exerts different dose- and time- dependent effects on human breast cancer cell lines characterized by presence or absence of estrogen receptors. We suggest that these discrepancies may be influenced by the estrogen signaling.

The effect of prolactin and estrogen cross-talk on prolidase- dependent signaling in MCF-7 cells.

Estrogen and prolactin play important role in mammary carcinogenesis. The present study was undertaken to evaluate the effect of prolactin and estrogen cross-talk on HIF-1 α level and expression of some HIF-1 α- dependent signaling proteins. Since up-regulation of prolidase activity inhibits HIF-1 α degradation, the enzyme was considered as an interface of estrogen/prolactin signaling. The experiments were performed on MCF-7 cells cultured with prolactin in the presence or absence of estradiol. It was found that in the presence of estradiol, prolactin inhibits prolidase activity and its down-stream signaling proteins: HIF-1α, mTOR, AKT and MAPK p-38, while in the absence of estradiol, an opposite effect was observed. These results suggest that prolactin/estrogen cross-talk exert beneficial effect on prolidase-dependent down regulation of HIF-1α. It suggests that dual action of prolactin and estrogen may be considered as a strategy in therapy of breast cancer.

Estrogen-dependent regulation of PPAR-gamma signaling on collagen biosynthesis in adenocarcinoma endometrial cells.

The link between estrogen and metabolic developmental factors of endometrial carcinoma is well established. PPAR- gamma, (an important modulator of metabolism) and estrogen receptor belong to a family of nuclear hormone receptors that were shown to interact with each other. The interaction may affect transcriptional activity of these transcription factors. The anti-diabetic troglitazone (TGZ) is well known PPAR- gamma ligand. The effect of troglitazone-induced PPAR- gamma activation on estrogen-dependent stimulation of collagen biosynthesis was studied in the Ishikawa endometrial adenocarcinoma cell line. We have found that the presence of estrogen activity in growth medium (1nM) augmented collagen biosynthesis in the cells. An addition of PPAR- gamma agonists, as troglitazone or clofibrat to the growth medium induced inhibition of collagen biosynthesis. The inhibition was effective only when estrogen receptor was stimulated, since removal of estrogen receptor by ICI 182- 780-dependent degradation did not affect collagen biosynthesis. The mechanism of the inhibition was found at the level of NF-kB (known inhibitor of collagen gene expression) and MAPK signaling. PPAR- gamma ligands stimulated expression of NF-kB, while they inhibited expression of p-38 but not ERK1/ERK2. The data document for the first time that inhibitory effect of PPAR- gamma ligands on collagen biosynthesis in endometrial adenocarcinoma cells requires functional estrogen receptor.

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.

The effect of hyaluronic acid on interleukin-1-induced deregulation of collagen metabolism in cultured human skin fibroblasts.

Although hyaluronic acid (HA) has been used in the treatment of osteoarthritis for 30 years, the mechanism of its protective action on collagen metabolism disturbances in tissues during inflammation is not known. The present study was undertaken to evaluate the mechanism of Interleukin-1 (IL-1)-induced deregulation of collagen metabolism in cultured human skin fibroblast and the effect of HA on the process. In normal fibroblasts IL-1 strongly induced inhibition of collagen biosynthesis, while HA counteracted the process. The mechanism of this phenomenon was independent of prolidase activity, an enzyme that plays an important role in collagen biosynthesis at the post-translational level. Instead, IL-1 was found to inhibit the expression of insulin-like growth factor-I receptor (IGF-IR) and MAP kinases-ERK1 and ERK2, while HA was shown to counteract this process. Since insulin-like growth factor-I (IGF-I) is a most potent stimulator of collagen biosynthesis in fibroblasts the mechanism of IL-1-dependent inhibition of collagen biosynthesis may be related to inhibition of IGF-IR expression and signaling. The data suggest that hyaluronic acid protects collagen against IL-1-induced inhibition of biosynthesis of this protein in cultured human skin fibroblasts at the level of IGF-IR signaling.

Estrogenic and antiestrogenic effects of raloxifene on collagen metabolism in breast cancer MCF-7 cells.

We compared the effects of different concentrations of raloxifene (1, 4 and 10 microM) on collagen biosynthesis, gelatinolytic and prolidase activities and matrix metalloproteinase (MMP) expression (MMP-2 and MMP-9) in estradiol-stimulated (2 nM) breast cancer MCF-7 cells. Raloxifene inhibited in a dose-dependent manner the proliferation of MCF-7 cells, independently of the presence or absence of estradiol in the growth medium. Raloxifene at concentrations of 1 microM and 4 microM inhibited collagen biosynthesis by about 10-fold and prolidase activity by about 50%, while at a concentration of 10 microM it inhibited these processes by only about 25%. This phenomenon was accompanied by differences in gelatinolytic activity and MMP (MMP-2 and MMP-9) expression as demonstrated by zymography and Western immunoblot analysis, respectively. In estrogen-stimulated MCF-7 cells, cultured in the presence of 1 microM raloxifene, a dramatic increase in the activity of both collagenases was found. In contrast, addition of raloxifene at a concentration of 10 microM to the medium of the cells resulted in restoration of gelatinolytic activity to that found in control cells. Similarly, but at both doses (1 and 10 microM), raloxifene was able to reduce MMP-2 expression in the cells. However, when used alone (without estradiol) a concentration of 1 microM raloxifene strongly stimulated MMP-2 expression, while at a concentration of 10 microM the effect was not observed. In the case of MMP-9, only trace amounts of this gelatinase were detected, although in contrast to MMP-2, an increase in its expression was noticed at a concentration of 10 microM raloxifene. The data raise the possibility that in estrogen-stimulated MCF-7 cells, raloxifene at low concentrations (1 and 4 microM) evokes antiestrogenic effect on collagen biosynthesis and prolidase activity on the one hand, and an estrogenic effect on gelatinolytic activity on the other, while at higher concentrations (about 10 microM) it evokes an estrogenic effect on collagen biosynthesis and prolidase activity, and an antiestrogenic effect on gelatinolytic activity. Our data suggest that the effects of raloxifene on collagen synthesis, prolidase and metalloproteinase activities in breast cancer may explain its role in the prevention of breast cancer development.

Phosphorylation of prolidase increases the enzyme activity.

Prolidase [EC 3.4.13.9] is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline-containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. Although, the increase in the enzyme activity is correlated with increased rate of collagen turnover, the mechanism by which prolidase is regulated remain largely unknown. In the present study we found that phosphorylation of fibroblast's prolidase may be an underlying mechanism for up regulation of the enzyme activity. Supporting evidence comes from the following observations: (1) immunoprecipitated prolidase was detected as a phosphotyrosine protein as shown by western immunoblot analysis, (2) tyrosine kinase inhibitor-erbstatin induced (in a dose dependent manner) a decrease in prolidase activity in cultured human skin fibroblasts, (3) anti-phosphotyrosine antibody reduced and phosphotyrosine phosphatase 1B antibody (anti-PTP 1B) increased (in a dose dependent manner) the prolidase activity in extract of fibroblast's homogenate, (4) decrease in prolidase activity from collagenase treated or serum starved fibroblasts can be partially prevented by incubating fibroblast's homogenate extract with anti-PTP 1B antibody. These results provide evidence that prolidase is phosphotyrosine enzyme and suggest that the activity of prolidase may be up regulated by the enzyme phosphorylation.

Melanin potentiates daunorubicin-induced inhibition of collagen biosynthesis in human skin fibroblasts.

One of the recognized side effects of antineoplastic anthracyclines is poor wound healing, resulting from an impairment of collagen biosynthesis. The most affected tissue is skin. The mechanism underlying the tissue specificity of the side effects of anthracyclines has not been established. In view of the fact that a number of pharmacologic agents are known to form complexes with melanin and melanins are abundant constituents of the skin, we determined whether daunorubicin interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. Results indicated that daunorubicin forms complexes with melanin. Scatchard analysis showed that the binding of daunorubicin to melanin was heterogeneous, suggesting the presence of two classes of independent binding sites with K1 = 1.83 x 10(5) M(-1) and K2 = 5.52 x 10(3) M(-1). The number of strong binding sites was calculated as n1 = 0.158 micromol/mg of melanin and the number of weak binding sites as n2 = 0.255 micromol/mg of melanin. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracycline-induced inhibition of collagen synthesis. We found that daunorubicin induced inhibition of prolidase activity (IC50 = 10 microM), collagen biosynthesis (IC50 = 70 microM) and DNA biosynthesis (IC50= 10 microM) in human skin fibroblasts. Melanin (100 microg/ml) by itself produced about 25% inhibition of DNA synthesis and prolidase activity but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to daunorubicin-treated cells (at IC50 concentration) augmented the inhibitory action of daunorubicin on collagen and DNA biosynthesis without having any effect on prolidase activity. The same effect was achieved when the cells were treated with daunorubicin at one-fourth of the IC50 given at 0, 6, 12 and 18 h during a 24-h incubation. The data suggest that the melanin-induced augmentation of the inhibitory effects of daunorubicin on collagen and DNA biosynthesis may result from: (i) accumulation of the drug in the extracellular matrix, (ii) gradual dissociation of the complex, and (iii) constant action of the released drug on cell metabolism. The phenomenon may explain the potential mechanism for the organ specificity of daunorubicin-induced poor wound healing in patients administered this drug.

Defects of type I procollagen metabolism correlated with decrease of prolidase activity in a case of lethal osteogenesis imperfecta.

We have studied the structure and metabolism of type I procollagen in a case of perinatal lethal osteogenesis imperfecta (OI) type II. Cultured skin fibroblasts from the proband synthesized both normal and abnormal forms of type I procollagen. Some abnormal, overmodified molecules were secreted by OI cells, although less efficiently than normal molecules from control cells. The OI fibroblasts accumulated large amounts of abnormal proalpha1(I) and proalpha2(I) chains intracellularly. The extracellular collagenolytic activity was decreased compared to control cells. Furthermore, OI cells produced less type I procollagen and demonstrated lower capacity to synthesize DNA than control cells. We have found that in contrast to prolinase activity, the activity of prolidase (an enzyme essential for collagen synthesis and cell growth) is also significantly reduced in OI cells. No differences were found in the amount of the enzyme protein recovered from both the OI and control cells. However, we found that expressions of beta1 integrin and insulin-like growth factor-I receptor (receptors known to play an important role in up regulation of prolidase activity) were decreased in OI cells compared to control cells. The decrease in prolidase activity may provide an important mechanism of altered cell growth and collagen metabolism involved in producing the perinatal lethal form of the OI phenotype.

FAK-independent regulation of prolidase activity and collagen biosynthesis in MCF-7 cells.

Prolidase [E.C. 3.4.13.9] plays an important role in the recycling of proline for collagen synthesis and cell growth and this enzyme activity determines the rate of collagen turnover. It has been previously suggested that prolidase activity is regulated through signal mediated by the interaction of ECM proteins, with b1 integrin receptor and that this interaction is disturbed in MCF-7 cells. The potential candidates for mediating signal transduction are the nonreceptor tyrosine kinase p125FAK and two mitogen-activated protein (MAP) kinases, ERK-1 and ERK-2, which are activated upon attachment of cells to ECM. We found that serum starvation of MCF-7 cells for 24 hours contributed to a significant decrease (by about 30%) in prolidase activity and collagen biosynthesis. These phenomena were accompanied by suppression of MAP kinases expression without any effect on the expression of FAK. The data suggest that prolidase activity and collagen biosynthesis respond to signal mediated by MAP kinases, independently of FAK expression in MCF-7 cells.

Collagen metabolism disturbances are accompanied by an increase in prolidase activity in lung carcinoma planoepitheliale.

One of the consequences of neoplastic transformation is deregulation of tissue collagen metabolism. Although metalloproteinases initiate the breakdown of collagen in lung carcinoma, the final step of collagen degradation is mediated by prolidase (E.C.3.4.13.9). We investigated whether prolidase activity could reflect disturbances of collagen metabolism in human lung carcinoma planoepitheliale (Ca pl.). Ten human lung Ca pl. and 10 samples of normal lung parenchyma were compared with respect to prolidase activity and expression (western immunoblot), the content of collagen and collagen degradation products (free and bound hydroxyproline determination), beta1 integrin subunit expression (western immunoblot) and collagenolytic activity (zymography). An increase in collagen content (66%, P < 0.05), free proline pool (50%, P < 0.05) and collagenolytic activity was accompanied by a significant increase in the prolidase activity (106%, P < 0.05) and its expression in Ca pl. No differences were found between Ca pl. and the control lung tissue with respect to beta1 integrin expression. Prolidase activity may reflect disturbances in tissue collagen metabolism in lung Ca pl. and it may, therefore, serve as a sensitive marker of the disease.