Roles of p38- and c-jun NH2-terminal kinase-mediated pathways in 2-methoxyestradiol-induced p53 induction and apoptosis

As 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, has been established to cause apoptosis of prostate cancer cells, the downstream effectors of the signaling remain unclear. In the current study, we investigated molecular mechanisms by which 2-ME induces apoptosis in human prostate cancer cell line, LNCaP. It was found that 2-ME mediates apoptosis through p53 induction. Nuclear factor kappaB (NFkappaB) was activated by 2-ME and closely regulated by the mitogen-activated protein kinase, p38. Inhibition of p38 or NFkappaB resulted in suppression of p53 induction and apoptosis. Moreover, we demonstrated that 2-ME activates the c-jun NH2-terminal kinase (JNK)/activation protein (AP)-1 pathway. Interestingly, inhibition of JNK strongly reduced Bcl-2 phosphorylation by 2-ME as well as p53 induction, and almost completely suppressed 2-ME-induced apoptosis. Androgen stimulation with dihydrotestosterone, a major endogenous metabolite of testosterone, also significantly inhibited p38/NFkappaB and JNK/AP-1 activation and apoptosis. The results suggest that not only p53 induction through p38/JNK-dependent NFkappaB/AP-1 activation but also JNK-dependent Bcl-2 phosphorylation are required for 2-ME-induced apoptosis; moreover, inhibition of these pathways may be involved in androgen-mediated resistance to apoptosis.     

The significance of thymidine phosphorylase/platelet-derived endothelial cell growth factor activity in renal cell carcinoma

BACKGROUND: Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PDECGF) and has angiogenic activity. Little is known about the significance of TP/PDECGF activity in patients with renal cell carcinoma (RCC). The authors examined the enzymatic activity of TP in 66 RCC specimens and investigated the association between the level of TP activity and the stage/grade status of patients with RCC. Furthermore, the authors examined the correlation between TP/PDECGF activity and prognosis. METHODS: TP activity levels in nonfixed, fresh-frozen RCC specimens and in specimens of normal kidney were determined using a thin-layer chromatography assay. RESULTS: The activity of TP was approximately 3.5-fold greater in RCC specimens compared with normal kidney specimens. TP activity in patients with Stage III-IV RCC was 2.6-fold greater compared with TP activity in patients with Stage I-II RCC. In addition, the level of TP activity was correlated with a higher grade of RCC. Patients who had RCC with low TP activity had a longer postoperative disease-specific survival compared with patients who had RCC with high TP activity in the 5-year follow-up. CONCLUSIONS: The current study is the first to demonstrate a correlation between levels of TP activity and both disease progression and a higher grade of RCC. It also is the first to show that elevated TP activity in patients with RCC predicts a poor prognosis. The results suggest that high TP/PDECGF activity may be associated with the malignant potential of RCC and that TP/PDECGF may be a molecular therapeutic target in patients with RCC.     

Inhibitory effects of triterpenes isolated from Hoelen on free radical-induced lysis of red blood cells

Hoelen, sclederma of Poria cocos Wolf, has long been used as a sedative and diuretic in traditional medicine. Formerly, we demonstrated that Hoelen in vitro protects red blood cells from AAPH-induced hemolysis. In this study, tests were carried out to identify the main ingredient of Hoelen that has the scavenging effect on free-radicals. Triterpene carboxylic acids isolated from the methanol extract of Hoelen, i.e. pachymic acid, polyporenic acid, 3-epidehydrotumulosic acid, 3beta-hydroxylanosta-7,9(11), 24-trien-21-oic acid and 3-o-acetyl-16 alpha -hydroxytrametenolic acid, were found to have inhibitory activities against AAPH-induced lysis of red blood cells.     

An association of Bcl-2 phosphorylation and Bax localization with their functions after hyperthermia and paclitaxel treatment

Apoptosis is induced by many kinds of therapy-related inducers, such as hyperthermia and chemotherapeutic agents. However, differences in apoptotic pathways between these inducers remain unclear, although knowing the differences is important to map out a therapeutic strategy. Therefore, we focused on the localization and phosphorylation of Bcl-2 and Bax, key mediators of the apoptotic pathway, after hyperthermia and paclitaxel treatment of PC-10 squamous cell carcinoma cells that excessively expressed Bcl-2 and Bax in the cytoplasm. Paclitaxel treatment markedly induced qualitative changes in Bcl-2, whereas hyperthermia did only quantitative changes in Bax. The levels of Bax increased gradually with the duration of hyperthermia, whereas Bcl-2 levels slightly decreased. On the other hand, paclitaxel treatment induced dose- and time-dependent phosphorylation of Bcl-2. Interestingly, phosphorylated Bcl-2 was observed in the specific subcellular sites, mitochondria- and lysosome-rich fractions. Both treatments disturbed the heterodimerization of Bax with Bcl-2. Hyperthermia, but not paclitaxel treatment, induced a gradual Bax translocation from the cytoplasm to the nucleus. Although both treatments induced a prominent cell cycle disturbance in the G2M phase, paclitaxel treatment induced typical apoptosis, and hyperthermia hardly induced apoptosis. Our results suggest that the subcellular redistribution of Bax and the phosphorylation of Bcl-2 depend on the type of apoptosis inducers, such as hyperthermia and paclitaxel, and Bcl-2 has a central role in the decision of apoptotic outcome. Our data may afford new insights in apoptosis from the aspect of an association of Bcl-2 phosphorylation with intracellular Bax localization.     

Allelotype analysis in relapsed childhood acute lymphoblastic leukemia

We performed for the first time the allelotype of relapsed childhood acute lymphoblastic leukemia (ALL). A total of 38 cases were screened for loss of heterozygosity (LOH) using 71 markers. In all, 26 (68%) patients showed LOH on at least one chromosomal arm, indicating that LOH is a frequent event at relapse. The most frequent loss was found on chromosomal arm 9p at the p16/INK4a locus (39%). LOH at the TEL gene locus on chromosomal arm 12p also occurred often (25%). Frequent loss was observed on chromosome arms 4q (20%), 6q (21%), and 17q (20%). Sequential analysis (i.e. samples obtained from both initial diagnosis and relapse) shows that some patients (63%) have the identical LOH status at both phases, suggesting the presence of the same clone. Other samples (37%) showed distinct LOH alterations, indicating clonal evolution at relapse. Despite the heterogeneous and complex changes, some shared LOH loci occurred in these matched samples, suggesting that many of the same tumor-suppressor genes are aberrant at both phases. In summary, novel tumor-suppressor genes on chromosome arms 4q, 6q, and 17q, as well as the p16 and TEL genes, have an important role in the relapse of childhood ALL.     

Genetic pathways of 'de novo' colorectal carcinomas with reference to fetal-type glycogen phosphorylase positive foci

'De novo' carcinogenesis has been advocated besides 'adenoma carcinoma sequence' as another dominant pathway leading to the colorectal carcinoma. Our previous study demonstrated that brain (fetal)-type glycogen phosphorylase (BGP) positive foci in the transitional mucosa (BGP foci) have frequent p53 mutations and that the distribution of BGP foci has a close relationship with the location of 'de novo' carcinoma. The aims of the present study were to investigate further genetic alterations in the BGP foci and to clarify the mechanism of 'de novo' carcinogenesis. Twenty-eight colorectal carcinomas with invasion into submucosa or superficial muscularis propria without any adenoma component expressing immunoreactive p53 protein were selected from 168 resected specimens. Investigations of the p53, K-ras and APC mutations was performed in the BGP foci, BGP negative colorectal mucosa and 'de novo' carcinoma using PCR-SSCP and DNA squencing. In all 28 cases, immunoreactive BGP was positive in the carcinomas and the BGP foci were observed sporadically in the mucosa adjacent to the carcinoma. No K-ras mutation was observed in either carcinoma or BGP foci in any of the cases. Mutations of p53 and APC were 14 (50.0%) and 9 (32.1%) in 'de novo' carcinomas, and 11 (39.3%) and 1 (3.6%) in BGP foci, respectively. Both p53 and APC mutations were detected in 8 and 1, p53 mutation alone in 6 and 10, APC mutation alone in 1 and 0 out of 28 carcinomas and BGP positive foci, respectively. These results suggest that the BGP foci may play a very important role in the 'de novo' colorectal carcinogenesis from the frequent genetic alterations of p53, and that there may be two major pathways, i.e., the p53-APC pathway and the p53 alone pathway, from the chain of genetic alterations between BGP foci and 'de novo' carcinoma.     

High serum soluble E-selectin levels are associated with postoperative haematogenic recurrence in esophageal squamous cell carcinoma patients

E-selectin has been reported to be associated with haematogenic metastasis in various cancer patients. In order to evaluate the risk of postoperative haematogenic recurrence of esophageal squamous cell carcinoma (SCC) patients, we examined the preoperative serum levels of soluble E-selectin and clinicopathological data of the patients. Preoperative serum was obtained from 135 esophageal SCC patients who received esophagectomies from 1990 to 1998. Serum soluble E-selectin levels were measured by means of enzyme linked immunoreactive synthesis assay (ELISA). The expression of sialyl Lewis A and X antigens were evaluated in 58 out of 135 patients. Thirty-five patients (25.9%) had haematogenic recurrence in their postoperative course. Serum soluble E-selectin levels of the haematogenic recurrence group (mean 55.6 ng/ml) were significantly higher than those of the non-haematogenic recurrence group (mean 41.1 ng/ml). When the cut-off level of soluble E-selectin was 56 ng/ml, the logistic regression analysis showed that high serum soluble E-selectin levels, lymph node metastasis and intraepithelial spread were associated with postoperative haematogenic recurrence of the esophageal SCC patients (OR 2.99, p=0.047, OR 4.94, p=0.009 and OR 5.0, p=0.019. respectively). Univariate analysis revealed that the patients with a high serum soluble E-selectin level tended to have poor survival (p=0.078) and Cox multivariate analysis revealed that a high serum soluble E-selectin level was a prognostic factor in esophageal SCC patients (RR 1.84, p=0.065). The patients with a high serum soluble E-selectin concomitant with expression of sialyl Lewis antigens had a significant risk of postoperative haematogenic recurrence (p=0.005). These results indicated that preoperative high serum soluble E-selectin was a risk factor in the development of postoperative haematogenic recurrence and was a prognostic factor in esophageal SCC patients.     

Correlation between serum resistin level and adiposity in obese individuals

OBJECTIVE: Resistin is associated with insulin resistance in mice and may play a similar role in humans. The aim of our study was to examine the relationship of serum resistin level to body composition, insulin resistance, and related obesity phenotypes in humans. RESEARCH METHODS AND PROCEDURES: Sixty-four young (age 32 +/- 10 years), obese (BMI 32.9 +/- 5.6), nondiabetic subjects taking no medication, and 15 lean (BMI 21.1 +/- 1.3) volunteers were studied cross-sectionally. Thirty-five of the subjects were also reevaluated after 1.5 years on a weight reduction program entailing dieting and exercise; changes of serum resistin were compared with changes of BMI, body composition, fat distribution, and several indices of insulin sensitivity derived from plasma glucose and serum insulin levels measured during 75-g oral glucose tolerance test. RESULTS: In a cross-sectional analysis, serum resistin was significantly higher in obese subjects than in lean volunteers (24.58 +/- 12.93 ng/mL; n = 64 vs. 12.83 +/- 8.30 ng/mL; n = 15; p < 0.01), and there was a correlation between resistin level and BMI, when the two groups were combined (rho = 0.35, p < 0.01). Although cross-sectional analysis in obese subjects revealed no correlation between serum resistin and parameters related to adiposity or insulin resistance, longitudinal analysis revealed change in serum resistin to be positively correlated with changes in BMI, body fat, fat mass, visceral fat area, and mean glucose and insulin (rho = 0.39, 0.40, 0.44, 0.50, 0.40, and 0.50; p = 0.02, 0.03, 0.02, <0.01, 0.02, and <0.01, respectively). DISCUSSION: Resistin appears to be related to human adiposity and to be a possible candidate factor in human insulin resistance.     

Tissue-specific induction of cytochromes P450 1A1 and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in engineered C57BL/6J mice of arylhydrocarbon receptor gene

Tissue-specific induction of mRNA of cytochrome P450 (P450 or CYP) 1A1 and 1B1 by polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) was investigated in wild and arylhydrocarbon receptor (AhR)-deficient C57BL/6J mice. Ratios of mRNA expression of CYP1A1 or CYP1B1 over beta-actin were determined and used to compare levels of expression and induction of these P450s by PAHs and PCBs in various organs. CYP1A1 mRNA was detected in control mice at very low levels in liver, lung, heart, kidney, intestine, thymus, testis, uterus, ovary, and brain and was highly induced in these organs by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. In AhR(+/+) and AhR(-/-) mice, CYP1B1 mRNA was found to be constitutively expressed at significant levels in heart (the ratio of mRNAs of CYP1B1 to beta-actin was approximately 0.6), kidney ( approximately 0.8), intestine ( approximately 0.3), testis ( approximately 0.9), thymus ( approximately 0.4), uterus ( approximately 0.3), ovary ( approximately 1.4), and brain ( approximately 0.4), whereas it was low in liver and lung (the mRNA ratio to beta-actin was <0.2 in these cases). CYP1B1 in the latter two organs was highly induced by PAHs and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. The induction of CYP1B1 by PAHs and PCBs was more extensive in organs in which the constitutive expression of CYP1B1 was low. For example, CYP1B1 was induced 9-fold and 10-fold by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in livers of male and female mice, respectively, whereas in testis and ovary, the fold induction of CYP1B1 by two inducers was only 1.1 and 1.4, respectively. Liver microsomal xenobiotic oxidation activities were induced by these PAHs and PCBs in male and female AhR(+/+) mice. These results suggest that CYP1A1 and CYP1B1 are differentially regulated in their expression in extrahepatic organs of mice and could be induced by PAHs and PCBs with different extents of induction depending on the inducers used and the organs examined in AhR(+/+) mice. The findings of significant levels of constitutive expression of CYP1B1 in AhR(-/-) mice as well as AhR(+/+) mice in several organs including heart, kidney, thymus, testis, ovary, and brain in AhR(-/-) mice as well as AhR(+/+) mice are of importance in understanding the basis of toxicity and carcinogenesis by chemicals that are metabolized by CYP1B1.     

Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections (2001). III. Secular changes in susceptibility

The bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa) isolated from patients diagnosed as urinary tract infections (UTIs) in 10 institutions in Japan were supplied between September and December, 2001. Then, the susceptibilities of these bacteria to various antimicrobial agents were examined, and the results were compared with those obtained between 1992 and 2000. Comparison was made by classifying strains isolated from patients into those in uncomplicated UTIs and those in complicated UTIs (including with or without indwelling catheter). The drug sensitivity of S. aureus in this year was comparable to those in up to the previous year, and S. aureus showed the best susceptibility to vancomycin (VCM). E. faecalis showed good susceptibility to ampicillin and imipenem, and the MIC90s were 2 micrograms/mL. The susceptibility of E. faecalis to VCM was also good. E. coli showed good susceptibility to the drugs except penicillins. Among cephems, the susceptibility to cefozopran (CZOP) was better (MIC90: < or = 0.125 microgram/mL). Just as the last report, the decreases in susceptibility of E. coli to quinolones were also observed in the patients with complicated UTIs. The susceptibility of Klebsiella spp. to all the test drugs did not significantly change in 2001 and was generally good but not to penicillins. Among cephems, Klebsiella spp. showed good susceptibility to flomoxef, cefpirome, cefixime, and CZOP with < or = 0.125 microgram/mL of MIC90s either in uncomplicated or complicated UTIs. Although the drug sensitivity of P. aeruginosa was generally low, the detection of the strains that showed good susceptibility to quinolones and carbapenems (MIC: < or = 0.125-2 micrograms/mL) were relatively frequent.