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.

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.

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.

Captopril-dependent inhibition of collagen biosynthesis in cultured fibroblasts.

The mechanism underlying the dermatological manifestations that accompany captopril therapy is not known. The facts that prolidase plays an important role in collagen biosynthesis and that captopril directly inhibits prolidase activity led us to evaluate its effect on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with milimolar concentrations (0.2-1 mM) of captopril (CAP) for 48 h. It was found that CAP-dependent decrease in prolidase activity was accompanied by parallel decrease in collagen biosynthesis. Since insulin-like growth factor receptor (IGF-IR) is the most potent regulator of both collagen biosynthesis and prolidase activity, and prolidase is regulated by beta1 integrin signaling, the effect of CAP on IGF-IR and beta1 integrin receptor expressions was evaluated. It was found that exposure of the cells to 0.3 mM CAP contributed to a decrease in IGF-IR, alpha2beta1 integrin receptor and MAPK/ERK1/2 expressions. The data suggest that CAP-dependent decrease of collagen biosynthesis in cultured human skin fibroblasts results from inhibition of prolidase activity that may occur through inhibition of alpha2beta1 integrin and IGF-IR signaling.

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.

Mechanism of betulinic acid inhibition of collagen biosynthesis in human endometrial adenocarcinoma cells.

Collagen as a ligand for integrin receptors plays important role in the integrin - dependent regulation of cellular metabolism. Since betulinic acid (BA) evokes anticancer activity, its effect on collagen biosynthesis was studied in cultured endometrial adenocarcinoma cells. Confluent cells were treated with different concentrations of BA for 24 hours. It was found that BA inhibit collagen biosynthesis ([3H] proline incorporation assay). The mechanism of this phenomenon was found at the level of insulin-like growth factor-I receptor (IGF-IR) and alpha2 integrin signalling (Western immunoblot analysis). The expressions of IGF-I receptor and alpha2 integrin subunit as well as integrin activated focal adhesion kinase (FAK) were decreased in the cells treated with BA. It was accompanied by a parallel decrease in the expression of Sos protein and phosphorylated MAP-kinases (ERK1, ERK2) and up - regulation of NF-kappaB. The data suggest that BA-dependent inhibition of collagen biosynthesis in cultured human endometrial adenocarcinoma cells undergoes through alpha2 integrin and IGF-IR signaling that activate NF-kappaB, potent inhibitor of collagen gene expression.

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.

Hyaluronic acid counteracts interleukin-1-induced inhibition of collagen biosynthesis in cultured human chondrocytes.

Although, hyaluronic acid (HA) is 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. Therefore, the present study was undertaken to evaluate the mechanism of IL-1beta action (inductor of experimental inflammation) on deregulation of collagen biosynthesis in cultured human chondrocytes and the effect of HA on the process. It has been found that IL-1beta strongly induced inhibition of collagen biosynthesis, while HA counteracted the process. The mechanism of this phenomenon was found at both transcriptional and post-transcriptional level. IL-1 was found to down regulate the expression of mRNA for type II collagen and to inhibit prolidase activity, an enzyme that plays an important role in collagen biosynthesis at post-translational level. HA was shown to counteract the IL-1beta-dependent inhibition of both processes. During experimental inflammation of chondrocytes cultured in 0.1% FBS there was no differences in the expression of beta(1)-integrin independently of cell number and the presence of HA in growth medium. In chondrocytes cultured in 5% FBS, IL-1beta up-regulated the expression of beta(1)-integrin receptor while HA abolished the effect. The data suggest that HA-dependent up-regulation of collagen biosynthesis in IL-1beta-treated chondrocytes may involve stimulation of prolidase activity in serum "starved" cells and may also originate at the transcriptional level in the cells cultured in standard conditions.

[Combined approach to the treatment of severe aortic stenosis and to three coronary vessel diseases in patients with brain meningioma and severe stenosis of internal carotid artery]. / Kombinovaný prístup pri resení tezké aortální stenózy a onemocnení tri koronárních tepen u nemocného s mozkovým meningeomem a významnou stenózou pravé vnitrní krkavice.

The authors present a case report of a 76-year-old man with a simultaneous finding of severe aortic stenosis, three coronary vessel diseases, severe stenosis of internal carotid artery and brain meningioma. The patient was scheduled for carotid endarterectomy 2 months prior the aortic valve replacement and coronary artery bypass grafting. Brain meningioma was removed 3 months after the cardiac procedure. The authors present a successful interdisciplinary co-operation in the treatment strategy.

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

Ototoxicity is one of the well known side effects of kanamycin. The mechanism underlying the organ specificity of the side effect is not understood. Since many pharmacologic agents are known to form complexes with melanin and melanin is an abundant constituent of the inner ear, we investigated whether kanamycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. We found that kanamycin forms complexes with melanin. The amount of kanamycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the kanamycin-melanin complex formation: strong binding sites with the association constant K1 - 3 x 10(5) M(-1), and the weak binding sites with K2 - 4 x 10(3) M(-1). The number of total binding sites (n1 + n2) was calculated as about 0.64 micromol kanamycin per 1 mg melanin. We found that kanamycin induced inhibition of collagen and DNA biosynthesis (IC50 - 5 microM). Melanin at 100 microg/ml produced about 25% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to kanamycin-treated cells (5 microM) augmented the inhibitory action of kanamycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in cell growth and collagen metabolism, may be one of the targets for kanamycin-induced inhibition of these processes. As shown by Western immunoblot analysis melanin augmented kanamycin-induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2. The obtained results demonstrate that melanin potentiates the inhibitory effect of kanamycin on IGF-I receptor-dependent signaling pathway in cultured fibroblasts. The data suggest a potential mechanism for the organ specificity of kanamycin-induced hearing loss in patients which may result from melanin-induced augmentation of the inhibitory effects of kanamycin on collagen and DNA biosynthesis.

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.

Doxycycline-induced inhibition of prolidase activity in human skin fibroblasts and its involvement in impaired collagen biosynthesis.

Several lines of evidence suggest that doxycycline, a semi-synthetic derivative of tetracycline, may be a useful agent in the treatment of osteoarthritis. It inhibits collagen synthesis and collagenase activity in hypertrophic chondrocytes, slowing the process of collagen turnover. However, the mechanism of doxycycline-induced inhibition of these processes has not been established. We considered prolidase, an enzyme involved in collagen metabolism, as a possible target for the doxycycline-induced inhibition of collagen synthesis. Cultured human skin fibroblasts, specialized for collagen synthesis, were used as model cells. Prolidase [E.C. 3.4.13.9] is a manganese-dependent cytosolic exopeptidase that cleaves imidodipeptides containing C-terminal proline, thus providing large amounts of proline for collagen resynthesis. Enzyme activity is regulated through the beta1 integrin receptor. Therefore, we compared the effect of doxycycline on prolidase activity and expression, collagen biosynthesis, gelatinolytic activity and beta1 integrin expression in 24-h treated cultured human skin fibroblasts. We found that doxycycline induced coordinately inhibition of prolidase activity and collagen biosynthesis (IC50 at about 150 microg/ml) and gelatinolytic activity in cultured human skin fibroblasts. The inhibitory effect of doxycycline on the processes was not due to the cytotoxicity of this drug, as shown in the cell viability tetrazoline test. However, an inhibitory effect of the drug on DNA synthesis was observed (IC50 at about 100 microg/ml). The decrease in prolidase activity in fibroblasts treated with doxycycline was not accompanied by any differences in the amount of prolidase or beta1 integrin recovered from these cells, as shown by Western immunoblot analysis. This suggests that the doxycycline-induced down-regulation of prolidase is a post-translational event. The data presented here raise the possibility that the doxycycline-induced decrease in collagen biosynthesis is mostly due to the inhibition of prolidase activity.

Cytotoxicity and effect on collagen biosynthesis of proline analogue of melphalan as a prolidase-convertible prodrug in cultured human skin fibroblasts.

Proline analogue of melphalan (MEL-PRO) was synthesised as a prodrug susceptible to the action of ubiquitously distributed, cytosolic imidodipeptidase--prolidase [E.C.3.4.13.9]. Conjugation of melphalan (MEL) with proline (PRO) through an imido-bond resulted in formation of a good substrate for prolidase. The susceptibility of MEL-PRO to the action of prolidase was found to be similar, compared to glycyl-proline--the most abundant, endogenous substrate for prolidase and about 6-fold higher compared to its substrate--glycyl-hydroxyproline. We have compared the transport of MEL and its prodrug through cell membrane, their antimitotic activity, cytotoxicity and effect on collagen biosynthesis in cultured, normal human skin fibroblasts. The prodrug was found to be more effectively transported into the cells than the free drug. Moreover, a lower cytotoxicity, antimitotic activity and inhibitory effect on collagen biosynthesis of the prodrug, compared to the free drug were observed after 24 h of incubation. MEL and MEL-PRO at concentrations of 12 microM led to the decrease in cell viability in confluent human skin fibroblasts by about 40 and 20%, respectively, during 24 h of incubation. IC50 of MEL for DNA synthesis (measured by thymidine incorporation assay) was found at about 7 microM, while MEL-PRO used at this concentration produced about 35% reduction in thymidine incorporation. Similarly, MEL and MEL-PRO used at 7 microM concentrations inhibited collagen biosynthesis in fibroblasts cultured for 24 h to about 30 and 80% of control values, respectively. However, when the cells were cultured with the drugs for 72 h, similar effects of both drugs on DNA and collagen biosynthesis were observed. The data suggest that MEL-PRO may serve as a prolidase-convertible prodrug that evokes lower cytotoxicity, antimitotic activity, and lower inhibitory effect on collagen biosynthesis in fibroblast cultures, compared to the free drug.

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.

The potential mechanism for glutamine-induced collagen biosynthesis in cultured human skin fibroblasts.

Although glutamine (Gln) is known as an important stimulator of collagen biosynthesis in collagen-producing cells, the mechanism and endpoints by which it regulate the process remain largely unknown. Intermediates of Gln interconversion: glutamate (Glu) and pyrroline-5-carboxylate (P5C) stimulate collagen biosynthesis in cultured cells but evoke different maxima of collagen biosynthesis stimulating activity at different times of incubation. P5C was found to be the most potent stimulator of collagen biosynthesis after 6 h of incubation (approx. three-fold increase); after 12 h, it induced increase in collagen biosynthesis to 260%, while at 24 h, the process was decreased to approximately 80% of control values. Glu induced increase in collagen biosynthesis to approximately 180%, 400% and 120% of control values, after 6, 12 and 24 h, respectively, suggesting that after 12 h of incubation, Glu was the most potent stimulator of collagen biosynthesis. Glu was also the most potent stimulator of type I procollagen expression at this time. After 6, 12 and 24 h incubation, Gln induced collagen biosynthesis to approximately 112, 115 and 230% of control values, respectively. Since prolidase is known to be involved in collagen metabolism, the enzyme activity assay was performed in fibroblasts cultured in the presence of Gln, Glu and P5C. While Gln and Glu required 24 h for maximal stimulation of prolidase activity, P5C induced it after 6-12 h. The data suggest that P5C induced collagen biosynthesis and prolidase activity in a shorter time than Gln and Glu. We considered that P5C directly stimulates the processes, while Gln acts through its intermediate-P5C. Reduction of P5C to proline is coupled to the conversion of glucose-6-phosphate (G6P) to 6-phospho-gluconate, catalyzed by G6P dehydrogenase. We have found that dehydroepiandrosterone (DHEA), a potent inhibitor of G6P dehydrogenase, inhibited a stimulatory effect of P5C on collagen synthesis, expression of type I collagen and prolidase activity. Our results postulate a potential mechanism of glutamine-induced collagen biosynthesis through its intermediate - P5C. P5C-dependent activation of nucleotide biosynthesis, prolidase activity and P5C conversion into proline may contribute to the stimulation of collagen biosynthesis.

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.

Potential role of beta 1 integrin and collagen biosynthesis in estrogen-dependent reduction of apoptosis in tamoxifen-treated breast cancer cells.

It was found that 10 microM tamoxifen induced apoptosis and a significant (approximately 50%) depletion of beta 1 integrin levels in human breast cancer cells. Estradiol-treated MCF-7 cells exhibited exceptional viability and adherence, high levels of beta 1 integrin and increased (by 100%) collagen biosynthesis. Pretreatment of MCF-7 cells with 1 nM estradiol prevented tamoxifen-induced cell death, loss of cell adherence and decrease in beta 1 integrin level. Tamoxifen and estradiol had an opposite effect on the beta 1 integrin level and adherence in breast cancer cells, suggesting that the decrease in the beta 1 integrin level may be an early event during tamoxifen-induced apoptosis in breast cancer cells.

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.

Decreased cytotoxicity and increased antimitotic activity of a proline analogue of chlorambucil as a prodrug susceptible to the action of fibroblast's prolidase.

We synthesized an proline analogue of chlorambucil (CH-pro) as a prodrug susceptible to the action of ubiquitously distributed, cytosolic imidopeptidase--prolidase [E.C.3.4.13.9]. A conjugation of chlorambucil (CH) with proline through an imido-bond resulted in the formation of a good substrate for prolidase. We have compared several aspects of biological actions of CH and its prodrug in cultured normal human skin fibroblasts. The prodrug was found to be more effectively transported into the cells than the free drug. Moreover, in opposition to CH, CH-pro had no inhibitory effect on fibroblast's prolidase activity against the endogenous substrate, glycyl-L-proline. Lower cytotoxicity and a higher antimitotic activity of the prodrug, compared to the free drug, was observed. CH and CH-pro at concentrations of 25 microM led to a 30% and 10%, decrease in cell viability in confluent human skin fibroblasts. IC50 values of CH and CH-pro for DNA synthesis was found to be 30 microM and 7 microM, suggesting higher antimitotic potency of the pro-drug compared to the free drug. CH-pro also evoked lower ability to inhibit collagen biosynthesis in cultured fibroblasts than the free drug. IC50 values of CH and CH-pro for collagen biosynthesis were found at about 15 microM and 30 microM, respectively. Targeting of prolidase as a prodrug-converting enzyme may serve as a novel strategy in pharmacotherapy of various diseases, leading to the increase in therapeutic efficacy and reduction in untoward side effects of antineoplastic agents.

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.