Mechanism of anticancer action of novel berenil complex of platinum(II) combined with anti-MUC1 in MCF-7 breast cancer cells.

Mucin 1 (MUC1) is a high molecular weight transmembrane glycoprotein, that is overexpressed in >90% of breast cancers. It serves a crucial role in anti-apoptosis and tumor progression. MUC1 interacts with proteins in the extracellular matrix, at the cell membrane, in the cytoplasm and in the nucleus. The aim of the present study was to investigate the mechanism of anticancer action induced by novel berenil complex of platinum(II) (Pt12) together with a monoclonal antibody against MUC1 in breast cancer MCF-7 cells. The effect of combined treatment on the concentration of selected markers of apoptosis including proapoptotic B-cell lymphoma 2 associated X protein (Bax), caspase-8, cytochrome c and caspase-9, as well as selected proteins involved in intracellular signal transduction pathways including p53, phosphoinositide 3-kinase and phosphorylated protein kinase B (p-Akt) were analyzed. The results of the present study demonstrated that combined treatment may be a promising strategy in anticancer treatment and represents an alternative to monotherapy. All compounds used alone (Pt12, cisplatin and the anti-MUC1 antibody) increased the concentration of proapoptotic Bax, cytochrome c and caspase-9 in comparison with control, thus suggesting that they activated the mitochondrial apoptotic pathway. Pt12 alone significantly increased the concentration of caspase-8, which is responsible for the initiation of the extrinsic apoptotic pathway. However, the strongest effect was observed following Pt12 (20 µM) treatment combined with the anti-MUC1 antibody (10 µg/ml). These two compounds together strongly induced apoptosis in MCF-7 breast cancer cells via the external and internal apoptotic pathways. It was also demonstrated that combined treatment based on Pt12 and the anti-MUC1 antibody significantly reduced p-Akt concentration.

Determining normal values of urinary phosphorus excretion in 3913 healthy children aged 2-18 to aid early diagnosis and treatment for urolithiasis.

AIM: This study determined the specific reference values for urinary phosphorus excretion in healthy children and adolescents aged 2-18 years and evaluated whether they changed with age during growth and were gender dependent. METHODS: We enrolled 3913 healthy children and adolescents aged 2-18 years to this study. The study population was divided into age groups, and the analysis was performed in one-year periods, separately for boys and girls. Urinary phosphorus excretion was analysed using four categories: P1 in mmol/24 hour units, P2 in mmol/kg/24 hours, P3 in mmol/1.73 m2 /24 hours and P4 in mmol/mmol creatinine. RESULTS: Clear differences in urinary exertion for girls and boys were observed as well as systematic changes with age. The boys presented with significantly higher daily urinary phosphorus excretion independent of its manner of expression (p < 0.001). The median urinary phosphorus (P1) rose with age (p < 0.001). Percentile tables of phosphorous exertion are presented. CONCLUSION: This was the largest study of urinary phosphate excretion based on a randomly selected sample of girls and boys aged 2-18 years. It highlights the importance of determining phosphorus reference values for children of different ages to provide early diagnosis and treatment for urolithiasis.

Changes of glycosylation of IgG in rheumatoid arthritis patients treated with methotrexate.

PURPOSE: In patients with active rheumatoid arthritis (RA) decrease of galactosylation is correlated with disease activity. The aim of our study was to evaluate an effect of methotrexate therapy on glycosylation disturbances of IgG in RA patients. MATERIALS/METHODS: IgG glycosylation in 40 patients with active RA treated with methotrexate for 12 months prior to and after treatment were compared. The control group consisted of 20 healthy volunteers. IgG glycosylation was assessed using biotinylated lectins and immunosorbent ELISA assay. For galactose specificity Datura stramonium lectin (DSA), for sialic acid Sambucus nigra (SNA) and Maackia amurensis (MAA) and for fucose residue Areulia auranta (AAA) lectins were used. RESULTS: In RA-cases N-glycan galactosylation and sialylation of IgG before treatment were significantly lower than in healthy subjects (for DSA, MAA lectins p<0.001 and SNA p<0.05). Significant increase of IgG galactosylation and sialylation in RA patients after therapy (for DSA, MAA and SNA lectin p<0.05) was detected. Moreover the glycosylation disturbances of N-glycan IgG were strongly associated with changes of disease activity based on disease activity score. For fucose residues significantly higher absorbency of AAA lectin in RA patients before treatment was observed compared to control subjects (p<0.05) and slightly, not significantly decreased after MTX therapy. CONCLUSIONS: Defect of galactosylation of IgG in RA patients is a useful marker of disease activity that may be used for the assessment of therapy effectiveness. The role of IgG fucosylation and sialylation in RA pathogenesis has still to be determined.

The combined treatment with novel platinum(II) complex and anti-MUC1 increases apoptotic response in MDA-MB-231 breast cancer cells.

New strategy of cancer's targeting treatment is combining monoclonal antibodies with chemotherapeutic agents. An important goal of targeted therapy appears to be a transmembrane glycoprotein type I-mucin 1 (MUC1), which is overexpressed in tumors of epithelial origin, especially in breast cancer. The goal of the study was to check the effect of monoclonal antibody against MUC1 with novel platinum(II) complex (Pt12) on selected aspects of apoptosis in human MDA-MB-231 breast cancer cells. The number of apoptotic and necrotic cells was measured using annexin V binding assay. The decrease of mitochondrial membrane potential (MMP) and DNA fragmentation was analyzed. Finally, the influence of novel platinum(II) complex (Pt12) used with anti-MUC1 on the concentration of selected markers of apoptosis such as Bax, caspase-8, -9, and caspase-3 was performed using ELISA. The results from combined treatment were compared with those obtained using monotherapy. In our study, we proved that anti-MUC1 used in combination with Pt12 strongly induced apoptosis in MDA-MB-231 breast cancer cell line. The effect was stronger than treatment with Pt12, cisplatin, anti-MUC1, and anti-MUC1 used with cisplatin. We also observed the highest decrease of MMP and the strongest DNA fragmentation after such a combined treatment. The results obtained from ELISA showed increased concentration of Bax, caspases-8, -9, -3 compared to monotherapy. Our study proved that Pt12 together with anti-MUC1 strongly induced apoptosis in estrogen-negative breast cancer cell line (MDA-MB-231). The apoptosis may go through extrinsic pathway associated with caspase-8 as well as intrinsic pathway connected with caspase-9.

Does obesity or hyperuricemia influence lithogenic risk profile in children with urolithiasis?

BACKGROUND: There are indications that obesity and hyperuricemia may influence the formation and composition of urinary stones. The aim of our study was to determine the effect of obesity and hyperuricemia on the urinary lithogenic risk profile in a large cohort of pediatric patients. METHODS: The study population comprised 478 children with urolithiasis and 517 healthy children (reference group). We studied the effects of obesity on the lithogenic profile by dividing the patients with urolithiasis into two groups based on body mass index Z-score (patients who were overweight/obese vs. those with normal weight for age) and comparing the two groups. To study the effect of hyperuricemia on the lithogenic profile, we divided the patients with urolithiasis into two groups based on the presence or not of hyperuricemia (110 patients with urolithiasis accompanied by hyperuricemia vs. 368 patients with urolithiasis and normal serum uric acid levels) and compared the groups. RESULTS: Among the children and adolescents with urolithiasis and hyperuricemia, there was a significantly lower excretion of crystallization inhibitors (citrates, magnesium). We also found significantly negative correlations between serum uric acid levels and the urine citrate/creatinine ratio (citrate/cr.; r = -0.30, p < 0.01), as well as the magnesium/cr. ratio (Mg/cr.; r = -0.33, p < 0.01). There was no statistically significant differences in the urinary excretion of oxalates, citrates, calcium, phosphorus, magnesium and uric acid between children with urolithiasis who were either overweight or obese and children with urolithiasis who had a normal body weight. CONCLUSIONS: In our pediatric patient cohort, hyperuricemia was associated with a decrease in the excretion of crystallization inhibitors in the urine, but the clinical relevance of this observation needs to be confirmed in future studies. Obesity and overweight had no direct influence on the lithogenic risk profile in the urinary stone formers in our study, but there was an indication that higher serum uric acid may be associated with impairment in renal function, which in turn could influence the excretion of lithogenic parameters.

Cytotoxic efficacy of a novel dinuclear platinum(II) complex used with anti-MUC1 in human breast cancer cells.

Mucin 1 (MUC1) is overexpressed in various cancer cells especially in breast cancer cells. There are known research works on the use of anti-MUC1 antibody with docetaxel in ovarian cancer, but there are no data about combined therapy platinum compounds with anti-MUC1 in breast cancer. The aim of the study was to evaluate the antiproliferative properties of a new dinuclear platinum(II) complex (Pt12) used with anti-MUC1 in human breast cancer cells. The dinuclear platinum(II) complex (Pt12) has been synthesized, and its cytotoxicity with anti-MUC1 has been tested in both MCF-7 and MDA-MB-231 breast cancer cells. In this study, the effects of Pt12 with anti-MUC1 on collagen and DNA biosynthesis in human breast cancer cells were compared to those evoked by cisplatin and cisplatin with anti-MUC1. The mechanism of action of Pt12 with anti-MUC1 was studied employing flow cytometry assessment of annexin V binding assay. It was found that Pt12 with anti-MUC1 was more active inhibitor of DNA and collagen synthesis as well more cytotoxic agent than Pt12 alone and cisplatin with anti-MUC1. Cytotoxicity of Pt12 with anti-MUC1 against breast cancer cells is due to apoptotic cell death as well as necrotic cell death. These results indicate that the use of Pt12 with anti-MUC1 may constitute a novel strategy in the chemotherapy of breast cancer tumors.

Mucin levels in saliva of adolescents with dental caries.

BACKGROUND: Human saliva, a complex secretion that contains a mixture of inorganic and organic molecules, plays an essential role in the maintenance of oral health. Mucins are the major macromolecular component of the secretion and are considered the first line of defense for epithelial tissues. The aim of this study was to compare levels of mucins (MUC5B, MUC7, and MUC1) in saliva of young subjects with dental caries. MATERIAL AND METHODS: All patients had DMF (decay/missing/filled) higher than value 0. Eight subjects with DMF=3 (control group) and 27 adolescents with DMF >11 (research group) were recruited for this study. Clinical evaluation procedures were oral examination, including tooth, periodontal, oral mucosal status, and collection of saliva samples. Saliva was collected for mucin assay. Enzyme-linked immunosorbent assay was used to quantitate MUC5B, MUC7, and MUC1. RESULTS: Our results indicate that adolescents with very high intensity of dental caries disease had increased levels of MUC1 and MUC5B. The membrane mucin MUC1 protein levels in the group with DMF>11 (research group) were higher compared to the group with DMF=3 (control group), and the increase was statistically significant (p=0.011). Similarly, secreted mucin MUC5B protein levels were higher (p=0.06) in the group with DMF>11 (research group). Although MUC7 protein levels were slightly reduced in symptomatic subjects, the decrease was statistically insignificant (p=0.918). CONCLUSIONS: Our data suggest links between the production of mucins, especially MUC1 and MUC5B in saliva, and dental caries disease.

The levels of sMUC-1 in patients with multiple myeloma.

Mucins have been shown to be aberrantly overexpressed in various diseases including cystic fibrosis, asthma, and cancer. Recent studies have uncovered the roles of these mucins in the pathogenesis of cancer. The presence of MUC-1 has also been detected on the cell surface of multiple myeloma (MM) cells in peripheral blood and showed direct correlation with tumor mass. In this study, we evaluated the levels of soluble MUC-1 (sMUC-1) in 50 new MM patients and correlated this with the levels of sMUC-1 after treatment. High levels of sMUC-1 were found in 20/50 (40%) MM patients, and in 2/50 (4%) healthy individuals (p = 0.001). According to the ISS, we found significant differences of mean sMUC-1 levels between the first stage of the disease (0.63 ± ± 0.26) and the third (0.93 ± 0.24; p = 0.03), but not with the second stage (0.80 ± 0.22; p = 0.08). Our study confirmed the correlation between elevated sMUC-1 and high elevated lactate dehydrogenase (p = 0.03) and the level of IgG in groups of patients with MM IgG at every stage of disease (p = 0.001). We showed for the first time that levels of sMUC-1 after treatment, in a group of patients with initially elevated levels of MUC-1, were statistically lower than in a group of patients with initially lower levels of sMUC-1 (21% vs. 42,6%; p = 0.05). At 37 months median of follow-up, we found a statistically significant difference between patients with normal versus elevated sMUC-1 in terms of progression-free survival (median 12 months vs. 8.1 months; p = 0.03).

Expression of MUC1 mucin in human umbilical vein endothelial cells (HUVEC).

Mucin 1 (MUC1) is a membrane-bound glycoprotein that is expressed by various epithelial cell types. MUC1 functions include modulation of cell adhesion, signal transduction, lubrication and hydration of epithelial surfaces, and their protection from infection. In this study we demonstrated that MUC1 is expressed in human umbilical vein endothelial cells (HUVECs) and could be released/shed from cellular membrane. MUC1 presence in these cells was verified using three methods: Western blotting, flow cytometry and metabolic labeling. We also showed that mucin expression is stimulated by proinflammatory cytokines: about a 2-fold increase was observed after TNF-α treatment and lower after IFN-γ alone and in combination with TNF-α treatment. It can be assumed that the presence of MUC1 in endothelial cells may have an important role in the interactions with different cell types in physiological and pathological processes.

Glutathione reductase activity correlates with concentration of extracellular matrix degradation products in synovial fluid from patients with joint diseases.

The mechanisms underlying cartilage matrix degradation in joint diseases is not fully understood but reactive oxygen species are implicated as main causative factors. Comparative studies of glutathione reductase (GR) activity in synovial fluid from patients with rheumatoid arthritis (RA), reactive arthritis (ReA) and osteoarthritis (OA) as well as correlations between GR activity and concentration of the major cartilage components in synovial fluid are presented in this study. We found significantly higher activity of GR in RA (about three-fold) and ReA (about two-fold) than in OA. In RA and ReA patients, GR activity in synovial fluid correlates negatively with the concentrations of collagen and degradation products of sulfated glycosaminoglycans. In OA patients the activity of GR was significantly lower than in RA and ReA, which positively correlated with the concentration of collagen and showed a tendency for positive correlation with the degradation products of sulfated glycosaminoglycans. Our results suggest that in RA and ReA patients increased activity of GR does not prevent the increased degradation of collagen and proteoglycans by ROS.

Reference values of plasma oxalate in children and adolescents.

Oxalate homeostasis is a derivative of absorption and transportation in the digestive system and renal/intestinal excretion of oxalate. The objective of this cross-sectional study was to determine normative values of plasma oxalate in relation to age, gender, and body size. A group of 1,260 healthy Caucasian children and adolescents aged 3 months to 18 years [mean +/- standard deviation (SD) 10.5 +/- 4.3] was studied. Each 1-year group comprised 70 subjects. Oxalate levels were assessed in blood plasma samples obtained from fasted individuals using the precipitation-enzymatic method with oxalate oxidase. Median oxalate levels in healthy infants was 3.20 micromol/L (5th-95th percentiles: 1.56-5.58) and was higher compared with older children [2.50 micromol/L (5th-95th percentiles: 0.95-5.74); p < 0.01]. No differences were found in plasma oxalate levels between boys and girls. There were no associations between plasma oxalate levels and anthropometric traits. In the healthy population aged 1-18 years, plasma oxalate concentration is independent of age, gender, and body size. Infants demonstrate higher plasma oxalate levels compared with older children, which suggests possible immature mechanisms of renal excretion. This study appears to be the first extensive report providing normative data for plasma oxalate in children and adolescents.

[The concentration of plasma anion oxalate in children treatment antibiotics beta lactame]. / Stezenie anionu szczawianowego w osoczu dzieci leczonych antybiotykami beta-laktamowymi.

UNLABELLED: The aim of work was the assessment of plasma anion oxalate (Ox) concentration in children during antibacterial treatment depending on way and time of antibiotic administration. MATERIAL AND METHODS: The examinations were carried out in 80 children, without nephrolithiasis, aged 10.1 +/- 4.3 years with bronchopneumonia, treated with beta-lactame antibiotics. The children were divided in two groups: I--children treated with oral amoxicillin + clavulanic acid or cefuroxime axetil (n=40), II--children treated with the same antibiotics intravenously (n=40). The Ox concentration in plasma and urine was measured using an enzymatic method with oxalate oxidase, four times. (0)--before treatment, (a)--in third day and (b)--in last day of administration (10 to 14 day), (c)--3 weeks after finishing treatment with antibiotics. RESULTS: The result showed that in children before treatment (0) mean plasma Ox concentration was 2.439 +/- 0.645 micromol/l. In 3rd day (a) the Ox concentration increased to 7.848 +/- 0.999 micromol/l (p < 0.01), in last day of treatment (b) decreased to 5.681 +/- 0.871 micromol/l, and after 3 weeks (c) came back to initial values (p > 0.05). Intravenous antibiotics administration did not influence plasma Ox concentration. CONCLUSIONS: Plasma oxalate concentration increases during oral administration of beta-lactame antibiotics caused by increased intestinal absorption, as a result of saprophytic microflora deterioration. However intravewous administration of the same antibiotics does not change the concentration of plasma oxalate.

The influence of N- and O-glycosylation inhibitors on the glycosylation profile of cellular membrane proteins and adhesive properties of carcinoma cell lines.

The effects of N- and O-glycosylation inhibitors on the expression of membrane proteins (MUC1 and some integrins) were evaluated in human endometrial (Ishikawa) and breast (MCF-7) cancer cells. Subconfluent cells were treated with 1-3 mg% concentration of tunicamycin and 2-10 mM of benzyl-N-acetyl-alpha-galactosaminide for 1-2 days, and used for flow cytometry, immunohistochemical staining, adhesion test and Western blotting. Benzyl-N-acetyl-alpha-galactosaminide inhibits MUC1 expression on the surface of breast more than endometrial cancer cells. Tunicamycin reduces MUC1 concentration on the cellular surface more than benzylglycoside, and greatly reduces glycosylation of glycoproteins, causing an increase in cell adhesion in both types of cancer cells. The expression of alpha2beta1 integrins on the surface of these cells was weak and decreased after treatment with inhibitors. Two different glycoforms of MUC1 proteins in endometrial cells and three in breast cancer cells were expressed and their molecular weights were reduced after treatment with glycosylation inhibitors. It was confirmed with lectin detection of carbohydrate epitopes (Tn and T) in MUC1 proteins. These observations show that glycosylation inhibitors altered the N- and O-glycan patterns in a sufficient manner, and positively modified the biological features of cancer cells.

Effect of estradiol and raloxifene on MUC1 expression and adhesive properties of Ishikawa cells.

The MUC1 is a transmembrane protein with a large mucin-like extracellular domain protruding high above the cell surface. Steroid regulation of MUC1 gene expression is essential, since overexpression of MUC1 may influence the metastatic potential of cancer cells. Our earlier results demonstrated that tamoxifen, alone and combined with estradiol, inhibits MUC1 biosynthesis in endometrial adenocarcinoma cells, in contrast to estradiol. In the present study, we examine the effect of administering raloxifene or estradiol at concentrations of 1 x 10(-8)-5 x 10(-7) M, and both drugs together, on the expression of MUC1 protein, its incorporation into the cell membrane, shedding to culture medium and adhesive properties of cancer cells to the extracellular matrix (ECM). The obtained results demonstrate that raloxifene, to a lesser degree than estradiol, stimulates [3)H]Thr incorporation to the cellular, as well as the extracellular MUC1 protein. Raloxifene-treated cells show a higher cell adhesion to collagen than estradiol-treated cells, especially at lower concentrations of these drugs, probably the result of smaller amounts of sialic acid residues in the terminal glycan chains with T and Tn antigens. Sole administration of raloxifene has a lesser effect on the expression of alpha(2)beta(1) integrin than estradiol, which is in contrast to the combined action of estradiol and raloxifene. Therefore, raloxifene, which stimulates MUC1 expression in cancer cells and inhibits their adhesion to collagen to a lesser degree than estradiol, may be a clinically safe treatment for the endometrium.

Inhibition of the O-glycan elongation limits MUC1 incorporation to cell membrane of human endometrial carcinoma cells.

We have studied how benzyl-N-acetyl-alpha-D-galactosaminide, O-glycosylation inhibitor, affects the polymorphism and shedding of membrane-bound MUC1 mucin, and change in adhesive properties of cancer cells. In endometrial adenocarcinoma cells (Ishikawa line), high molecular weight MUC1 mucin was shed from cellular membrane and could be detected in culture medium 24 h after [14C]threonine labelling. Short-time (2 days) exposure of these cells to benzyl-N-acetyl-alpha-D-galactosaminide was associated with a reduction in sialic acid level and increase in T antigen content in cellular MUC1 mucin. These changes could be inverted after removal of the inhibitor. A longer, 6-day action of the inhibitor induced a decrease in sialic acid and T antigen levels in cellular MUC1 mucin. Benzyl-N-acetyl-alpha-D-galactosaminide treatment caused the occurrence of a few incompletely glycosylated glycoforms of MUC1 in cells, but not in culture medium. Adhesion of endometrial cells to ECM compounds (type I collagen) was increased by benzyl-N-acetyl-alpha-D-galactosaminide treatment, indicating that glycosylation of extracellular domain of MUC1 can modulate adhesive properties of cells.