Kyo-Cheol Mun

Impaired Biological Activity of Erythropoietin by Cyanate Carbamylation

Aims: During advanced renal failure, particularly in patients with end-stage renal disease (ESRD), proteins are carbamylated as a result of a reaction with cyanate. Some or all of the cyanate is derived from urea. If the carbamylation of proteins adversely alters their biologic activities, then urea must be viewed as an uremic toxin, rather than a surrogate. Therefore, we studied the effect of cyanate carbamylation on the erythropoietic activity of erythropoietin (EPO) in a rodent model. Methods: EPO was carbamylated by incubation with cyanate at 37°C. The extent of carbamylation was monitored using trinitrobenzenesulfonic acid. In Sprague-Dawley rats the erythrocyte count, hemoglobin concentration, and hematocrit were measured after the twice-weekly subcutaneous injection of either EPO or carbamylated EPO for 3 weeks. Two additional control groups received physiologic saline or 0.2 ml of 1 M cyanate. Results: The level of carbamylated EPO was increased as the time of exposure to cyanate increased from 1 to 6 h, and as the cyanate concentration increased from 8 to 2,000 mM. EPO injections caused significantly large increases in all erythropoietic measures. Physiologic saline or 1 M cyanate-injected controls and the carbamylated EPO-injected animals demonstrated no change from baseline in erythropoietic parameters. Conclusion: These results support that EPO exposed to high levels of cyanate in vitro demonstrates diminished biologic activity in healthy Sprague-Dawley rats. This effect may be manifested by the carbamylation of EPO by the cyanate. Should this occur in ESRD patients, it may contribute to the suboptimal erythropoietic response to EPO therapy associated with high urea levels, especially related to inadequate dialysis. Targeting dialysis doses specifically to urea concentrations may be more important than previously considered.

(-)-Epigallocatechin gallate attenuates glutamate-induced cytotoxicity via intracellular Ca2+ modulation in PC12 cells

1. The effects of (–)‐epigallocatechin gallate (EGCG), a green tea polyphenol, on glutamate‐induced increases in intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity in PC12 cells were investigated.

Inhibition of erythropoietin activity by cyanate

Objective: Increased urea concentration is a measure of advanced renal failure and the adequacy of renal replacement therapy in end‐stage renal disease (ESRD). Altered biologic activity due to changes in protein structure occurs when cyanate, formed spontaneously from urea, reacts with proteins. Carbamylation results in impaired erythropoietin (EPO) activity when high concentrations of cyanate react with EPO. In this study, the activity of carbamylated EPO (C‐EPO), formed at a cyanate concentration which may occur in vivo, was studied in Sprague–Dawley rats. Material and Methods: The extent of carbamylation, causing loss of free amino groups, was monitored using trinitrobenzenesulfonic acid. Erythrocyte, hemoglobin, hematocrit and leukocyte levels were measured after either EPO, incubated EPO, C‐EPO, physiologic saline or cyanate (1.5 μM; 0.2 ml) were injected subcutaneous twice weekly for 3 weeks in rats. Results: In vitro carbamylation of EPO was time‐ and concentration‐dependent. C‐EPO concentration increased as the duration of exposure to cyanate increased from 6 to 72 h, or as cyanate concentration increased from 15 nM to 1.5 μM. Injections of EPO caused significant increases in vivo in all erythropoietic measures. In contrast, injections of C‐EPO, physiologic saline or 1.5 μM cyanate caused no change from baseline. Conclusions: These results demonstrated diminished biologic activity in healthy rats by C‐EPO formed in vitro at cyanate concentrations that may be found in vivo. C‐EPO and high urea‐derived cyanate levels may contribute to suboptimal erythropoietic responses to EPO therapy for chronic renal failure and ESRD, and may provide another measurement indicating inadequate dialysis.

Ciglitazone enhances ovarian cancer cell death via inhibition of glucose transporter-1

Ciglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist and improves insulin sensitivity. Apart from antidiabetic activity, ciglitazone elicits inhibitory effects on cancer cell growth. Recent studies indicate that glucose metabolism plays a key role in malignant diseases. Significant increase in glucose consumption is found under malignant conditions. The role of ciglitazone in cancer cell death in relation to glucose metabolism is unclear. Thus we designed this study to determine the effect of ciglitazone on glucose metabolism. First, we found ciglitazone inhibited glucose uptake in ovarian cancer cells but did not affect hexokinase activity. Ciglitazone decreased expression levels of glucose transporter-1 (GLUT-1). We also found that ciglitazone and siGLUT-1 treatments induced cell death in ovarian cancer cells. We identified that ciglitazone decreased expressions of specific protein 1 (Sp-1) and β-catenin while increased phosphorylation levels of AMP-activated protein kinase. In vivo study using NOD-scid IL2Rgamma(null) mice confirmed that ciglitazone significantly decreased ovarian cancer mass transplanted onto the back of the mice. Finally, we determined GLUT-1 expressions in patients with serous type ovarian cancer and found that GLUT-1 expression was markedly increased in cancer patients and expression level was proportional to the degree of cancer stages. These results suggest that ciglitazone induces apoptosis in ovarian cancer cells by the inhibition of GLUT-1 and provides a possible therapeutic effect of ciglitazone as an adjuvant drug in the treatment of ovarian cancer.

Mutations of the BAK gene are infrequent in advanced gastric adenocarcinomas in Koreans

The bcl-2 homologue antagonist/killer (BAK) is a potently apoptosis-inducing gene and plays an important role in modulating apoptosis in epithelial cells. We have analyzed the mutation of the entire coding region of BAK gene in 107 Korean advanced gastric adenocarcinomas by polymerase chain reaction-single strand conformation polymorphism and sequencing. Homozygous deletions were not found in these samples. Only three cases of 107 gastric adenocarcinomas (2.8%) exhibited the BAK mutations. Two of them exhibited missense mutations and the remaining one had a silent mutation. All of these mutations were exclusively detected in exon 2. Mutations in the BAK gene were observed only in advanced gastric adenocarcinomas with extensive metastases of regional lymph nodes. The data presented here suggest that the mutations of BAK gene rarely occurred in advanced gastric adenocarcinomas.

CYANATE AS A HEMOLYTIC FACTOR

During advanced renal failure, and particularly in patients with end-stage renal disease, proteins are carbamylated as a result of a reaction with cyanate. If the carbamylation of proteins adversely alters their biologic activities and structures, then urea must be viewed as an uremic toxin, rather than a surrogate. Therefore, we studied in this paper the role of cyanate as a hemolytic factor of erythrocytes to explain anemia observed in patients with high blood urea levels due to inadequate dialysis. Cyanate was added to make the final concentration 150, 300 and 600 nmol to each test tube containing the final concentration of 140×106 with human erythrocytes per mL of phosphate buffered saline solution. And they were incubated at 37°C for 24, 48 and 72 hours. The extent of hemolysis and carbamylation was monitored. The levels of hemolysis and carbamylated erythrocytes increase as the time of exposure to cyanate increased from 24 hours to 72 hours. Furthermore, those increased as cyanate concentration in the incubation media rose from 150 nmol to 600 nmol. Cyanate can induce hemolysis by carbamylation of erythrocytes. Urea, through cyanate, may contribute to hemolysis. If one extrapolates these results to patients with end-stage renal disease, it may help explain one of the reasons for the anemia in patients with high levels of BUN due to inadequate dialysis.