Taketoshi Kishimoto

Antitumor effects of Scutellariae radix and its components baicalein, baicalin, and wogonin on bladder cancer cell lines

OBJECTIVES To investigate the antitumor effects of Scutellariae radix and its components baicalein, baicalin, and wogonin on human bladder cancer cell lines (KU-1 and EJ-1) and a murine bladder cancer cell line (MBT-2). METHODS Bladder cancer cells were incubated with various concentrations of the agents. Antiproliferative activity against the bladder cancer cell lines was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diplenyl tetrazolium bromide assay. In an in vivo experiment, the mice were subcutaneously injected with MBT-2 cells, and Scutellariae radix was orally administered at a dose of 2 or 10 mg per mouse one time daily for 10 days from day 11 to day 20. RESULTS All the drugs inhibited cell proliferation in a dose-dependent manner, but baicalin exhibited the greatest antiproliferative activity. The concentration of baicalin necessary to obtain 50% inhibition was 3.4 microg/mL for KU-1, 4.4 microg/mL for EJ-1, and 0.93 microg/mL for MBT-2. For KU-1 and MBT-2, the percentage of cell survival significantly decreased (P <0.05) at a baicalin concentration of 1 microg/mL. In an in vivo experiment, antitumor effects of Scutellariae radix on C3H/HeN mice implanted with MBT-2 were investigated. All the control mice showed a progressive increase in tumor volume, reaching 2.81 +/- 0.18 cm(3) on day 20 and 5.36 +/- 0.44 cm(3) on day 25. However, when Scutellariae radix was orally administered at a dose of 10 mg per mouse one time daily for 10 days from day 11 to day 20, the tumor volume was 1.99 +/- 0.19 cm(3) on day 20 and 3.86 +/- 0.26 cm(3) on day 25, a significant inhibition of tumor growth (P <0.05). Conclusions. These results suggest that Chinese herbal medicines may become an attractive and promising treatment for bladder cancer.

Side Effects of Extracorporeal Shock-Wave Exposure in Patients Treated by Extracorporeal Shock-Wave Lithotripsy for Upper Urinary Tract Stone

The side effects of shock-wave exposure on patients treated by extracorporeal shock-wave lithotripsy were analyzed by measuring chemical substances including cell-escaped enzymes in the blood and urine. Free hemoglobin, glutamic oxaloacetic transaminase (GOT) and lactate dehydrogenase (LDH) increased as a function of shock-wave count. Total bilirubin, GOT and LDH reached a peak on the 1st postoperative day and decreased to pretreatment values by the 4th day. Creatine phosphokinase and myoglobin also significantly increased on the 1st postoperative day but returned to pretreatment levels by the 4th day. The urinary ratio of N-acetyl-beta-d-glucosaminase to creatinine significantly increased after treatment and was still high on the 4th day. These results suggest that significant hemolysis and myolysis developed owing to shock-wave exposure, and that the kidney was damaged to some extent. Therefore, these side effects must be taken into account in determining the dose and frequency of treatment.

Detection of Peptidoglycan and Endotoxin in Dialysate, Using Silkworm Larvae Plasma and Limulus Amebocyte Lysate Methods

Silkworm larvae plasma (SLP) reagent is activated by peptidoglycan (PG), a fragment of both the gram-positive and gram-negative bacterial cell wall, as well as beta-glucan (BG), a component of fungi. It is possible to measure contamination of gram-positive bacteria quantitatively by combining the conventional limulus amebocyte lysate (LAL) and PG measurement methods. Therefore, a more highly accurate analysis of dialysate can be made using both SLP and LAL methods to detect endotoxin (ET) and/or PG contamination. We studied the effects of contaminated dialysate on human peripheral blood mononuclear cells (PBMC) by producing various cytokines in vitro. Muramyl dipeptide (MDP) was used as the biologically active minimum constituent of PG. A total of 54 dialysate samples were obtained under sterile conditions from 4 sites: (1) reverse osmosis water unit; (2) proportioning unit; (3) multiple dialysate preparation console, and (4) personal dialysate preparation console, at 9 dialysis facilities. To detect bacterial contamination, the samples were measured with LAL(C), LAL(G) and SLP methods. PBMC were collected from 10 healthy controls and from 10 hemodialysis patients and cultured for 24 h with ET, MDP, ET + MDP and contaminated dialysate. IL-1 receptor antagonist (IL-1Ra), IL-1 beta and TNF-alpha in the culture medium supernatants were measured using the ELISA method. PG was not detected in dialysate from sites 1 or 2. However, dialysate from the inlet of the dialyzer at the bedside monitor of the central supply and personal console showed 4.1 +/- 6.1 ng/ml for site 3 (in 7 of 18 samples) and 3.3 +/- 4.6 ng/ml for site 4 (in 3 of 18 samples). Contamination by PG alone and complex contamination by PG and ET were also detected. Furthermore, IL-1Ra, IL-1 beta and TNF-alpha production by PBMC increased in accordance with the concentrations of MDP. Cytokine production was enhanced 5-10 times more where MDP and ET coexisted than where either MDP or ET existed alone, showing the synergic effects of MDP and ET. Based on these results, there is a high possibility that PG may also be a pyrogen in the dialysate prior to this study. ET had been considered the only pyrogen in dialysate. Therefore, it is essential to recognize the existence of both ET and PG in investigating dialysate contamination.

Magnesium supplementation prevents experimental chronic cyclosporine a nephrotoxicity via renin-angiotensin system independent mechanism.

Background. We have previously shown that correction of hypomagnesemia by magnesium (Mg) supplementation ameliorates chronic cyclosporine A (CsA) nephropathy via inhibiting gene expression of fibrogenic molecules. Experiments were conducted to further elucidate upstream mechanism of the beneficial effects upon CsA nephrotoxicity. Methods. CsA (15 mg/kg/day, subcutaneous [SC]) was administered daily to rats maintained on low sodium diet for 7, 14, and 28 days. Because blockade of renin-angiotensin system improves chronic CsA nephropathy, the effects of Mg supplementation and those of angiotensin-converting enzyme inhibitor (ACEI) were compared on renal function, renal histology, mononuclear cell infiltration, and gene expression profile. Results. CsA induced a decline in glomerular filtration and developed characteristic striped fibrosis that were mostly evident at day 28. Mg attenuated CsA-induced impaired renal function, whereas ACEI did not. Interstitial inflammation as evidenced by monocyte/macrophage infiltration preceded the renal fibrosis and increased progressively with the CsA treatment period. Concomitantly, CsA markedly up-regulated expression of chemoattractant proteins, osteopontin, and monocyte chemoattractant protein-1. These changes were abolished by Mg but were only partially affected with ACEI. CsA promoted renal mRNA expression of fibrogenic molecules and extracellular matrices that were almost completely abolished by Mg but partially suppressed by ACEI. Similarly, CsA-induced chronic fibrotic lesion was markedly attenuated by Mg supplementation but was partially attenuated by ACEI. Conclusion. Mg supplementation abolished CsA-induced precedent interstitial inflammation possibly via inhibition of chemoattractants expression and consequently attenuated tubulointerstitial fibrosis. In this protective mechanism, factors independent of the renin-angiotensin system appears to be mainly involved.

Chemopreventive effects of bovine lactoferrin on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced rat bladder carcinogenesis.

Chemopreventive effects of bovine lactoferrin (bLF), which is found at high concentrations in colostrum, on rat bladder carcinogenesis were investigated using a rat bladder medium‐term bioassay. In experiment 1, a total of 80 F344 male rats, 6 weeks old, were divided into 5 groups. Groups 1 and 2 were treated with 0.05%N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) in the drinking water for 8 weeks and after a 1‐week interval, received dietary supplementation with 2% and 0.2% bLF, respectively. Group 3 received 0.05% BBN for 8 weeks and then no treatment. Group 4 was administered 2% bLF alone from week 9, without prior carcinogen exposure. Group 5 was maintained without any treatment throughout the experiment. All rats were killed at the end of week 36. Group 1 demonstrated a significantly decreased multiplicity of the bladder tumors (carcinomas and papillomas) as compared with group 3, Maximum cut surface areas of bladder tumors were also significantly decreased in groups 1 and 2 compared with group 3. No bladder tumors were observed in groups 4 or 5. In experiment 2, a total of 60 rats were divided into two groups (30 rats each); both were treated with 0.05% BBN for 4 weeks and after a 1‐week interval, one received 2% bLF (group 1) and the other, basal diet (group 2) for 4 weeks. Group 1 demonstrated a tendency for decrease of the 5‐bromo‐2′‐deoxyuridine (BrdU) labeling index. bLF was detected in the urine of rats fed bLF by ELISA as well as western blot analysis. The findings indicate that 2% bLF can inhibit BBN‐induced rat bladder carcinogenesis, and that this may be due to bLF in the urine.

Cytotoxic activity of pierisin, from the cabbage butterfly, Pieris rapae, in various human cancer cell lines.

Pierisin, a protein purified from pupae of the cabbage butterfly, Pieris rapae, exhibits cytotoxic effects against the human gastric cancer TMK-1 cell line, inducing apoptosis. The present study was performed to determine whether pierisin might exert a similar influence on nine other human cancer cell lines and human umbilical vein endothelial cells (HUVECs). Pierisin showed cytotoxic effects in all the human cells tested, with IC50 values ranging from 0.043 ng/ml to 150 ng/ml. Among the target cells, the cervical carcinoma cell line, HeLa, was the most sensitive to pierisin, showing a 1000-fold less IC50 value than that of HUVECs. While pierisin clearly induced apoptotic cell death in most cancer cell lines and HUVECs, the pathway appeared to be probably different from that involving anti-Fas, TNF-alpha and p53. Pierisin may thus be a promising new candidate for cancer therapy.

Serum hepatocyte growth factor levels in patients with chronic renal failure.

The serum levels of hepatocyte growth factor (HGF) were determined in chronic renal failure (CRF) patients. Nondialysis patients with renal insufficiency had significantly higher serum HGF than normal subjects (0.34 +/- 0.10 ng/ml, n = 21 vs. 0.19 +/- 0.05 ng/ml, n = 15; p < 0.001), and the elevated serum HGF correlated with their serum creatinine levels. Hemodialysis (HD) patients treated for 5-10 years showed higher serum HGF than those receiving HD for 1 year or less (0.45 +/- 0.14 ng/ml, n = 8 vs. 0.33 +/- 0.11 ng/ml, n = 9; p < 0.05). Continuous ambulatory peritoneal dialysis patients also showed elevated serum HGF levels comparable to those of HD patients. There was no difference in serum HGF levels in HD patients with or without acquired cystic disease of kidney. Consequently, serum HGF is elevated in CRF, which may be attributed to the increased production of HGF in response to the chronic renal injury, the effect of heparin, or reduced removal of serum HGF in CRF patients.

“Decoy” of androgen‐responsive element induces apoptosis in LNCaP cells

In an androgen‐dependent manner, the androgen receptor (AR) binds to the androgen‐responsive element (ARE) in the regulatory region of target genes. We hypothesize that an “ARE decoy,” a double‐stranded oligonucleotide containing the same DNA sequence as ARE, can inhibit prostatic proliferation by competitive inhibition of AR transcriptional activity.

Clinical Effects of Long-Term Use of Neutralized Dialysate for Continuous Ambulatory Peritoneal Dialysis

The long-term effects of neutralized dialysate used in continuous ambulatory peritoneal dialysis (CAPD) were evaluated in 8 well-controlled patients. Twelve milliliters of 8.4% sodium bicarbonate was added to Dianeal PD-1 immediately before every administration. The final pH was 6.8 and the concentration of sodium bicarbonate was 6 mmol/l. The final sodium level was 138 mEq/l. This dialysate was used for 5 months. For 2 months before and 3 months after this period, Dianeal PD-2 was used as the dialysate for comparison. Blood bicarbonate levels significantly improved during the use of the neutralized dialysate. Blood sodium, chloride and magnesium levels and the effluent volume significantly increased. Sodium balance improved during the period when neutralized dialysate was used. Total leukocyte counts in the effluent decreased, and leukocyte viability increased. Abdominal distention, abdominal pain during instillation, nausea and headache improved. No side effects, including peritonitis, occurred during the trial of neutralized dialysate. The results suggest that this dialysate was less irritating to the peritoneal membrane than the control dialysate and that the therapeutic effects were satisfactory.

Trace element concentrations in hair, fingernails and plasma of patients with chronic renal failure on hemodialysis and hemofiltration.

Trace element concentrations in hair and fingernails and plasma of nondialyzed, hemodialyzed and hemofiltered patients with chronic renal failure and healthy volunteers were measured by atomic and flameless atomic absorption spectrophotometry and neutron activation analysis. Plasma aluminum concentrations in all three groups of patients were higher than in the controls. Aluminum levels in plasma and red blood cells were higher in hemofiltered patients than in dialyzed patients. The aluminum concentrations in the hair of both nondialyzed and dialyzed patients was higher than in the controls, while that in the hemofiltered patients it was not. These elevated aluminum concentrations appear to be mainly caused by the use of aluminum-contaminated dialysate. Calcium concentrations in the hair of nondialyzed and dialyzed patients were higher than in the controls. Plasma zinc concentrations in all the patients were lower than the controls, and the concentrations in hemofiltered patients were lower than in the dialyzed patients.