A Tyr-W-MIF-1 analog containing D-Pro2 acts as a selective mu2-opioid receptor antagonist in the mouse

The antagonistic properties of Tyr-d-Pro-Trp-Gly-NH(2) (d-Pro(2)-Tyr-W-MIF-1), a Tyr-Pro-Trp-Gly-NH(2)(Tyr-W-MIF-1) analog, on the antinociception induced by the mu-opioid receptor agonists Tyr-W-MIF-1, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), Tyr-Pro-Trp-Phe-NH(2) (endomorphin-1), and Tyr-Pro-Phe-Phe-NH(2) (endomorphin-2) were studied in the mouse paw-withdrawal test. d-Pro(2)-Tyr-W-MIF-1 injected intrathecally (i.t.) had no apparent effect on the thermal nociceptive threshold. d-Pro(2)-Tyr-W-MIF-1 (0.1-0.4 nmol) coadministered i.t. showed a dose-dependent attenuation of the antinociception induced by Tyr-W-MIF-1 without affecting endomorphin- or DAMGO-induced antinociception. However, higher doses of d-Pro(2)-Tyr-W-MIF-1 (0.8-1.2 nmol) significantly attenuated endomorphin-1- or DAMGO-induced antinociception, whereas the antinociception induced by endomorphin-2 was still not affected by d-Pro(2)-Tyr-W-MIF-1. Pretreatment i.t. with various doses of naloxonazine, a mu(1)-opioid receptor antagonist, attenuated the antinociception induced by Tyr-W-MIF-1, endomorphin-1, endomorphin-2, or DAMGO. Judging from the ID(50) values for naloxonazine against the antinociception induced by the mu-opioid receptor agonists, the antinociceptive effect of Tyr-W-MIF-1 is extremely less sensitive to naloxonazine than those of endomorphin-1 or DAMGO. In contrast, endomorphin-2-induced antinociception is extremely sensitive to naloxonazine. The present results clearly suggest that d-Pro(2)-Tyr-W-MIF-1 is the selective antagonist to be identified for the mu(2)-opioid receptor in the mouse spinal cord. d-Pro(2)-Tyr-W-MIF-1 may also discriminate between Tyr-W-MIF-1-induced antinociception and the antinociception induced by endomorphin-1 or DAMGO, all of which show a preference for the mu(2)-opioid receptor in the spinal cord.     

ARID3B induces malignant transformation of mouse embryonic fibroblasts and is strongly associated with malignant neuroblastoma

ARID3B, a member of the AT-rich interaction domain (ARID) family of proteins, plays an essential role in the survival of neural crest during embryogenesis. Here, we report evidence that ARID3B is involved in the development of malignant neuroblastoma, a childhood tumor derived from neural crest. (a) ARID3B is expressed by all five cell lines derived from neuroblastoma tested by us. (b) Analysis of published DNA microarray data of fresh neuroblastoma tumors showed that ARID3B is expressed in 80% of stage IV tumors, whereas only in 9% of stage I-III+IVs tumors. (c) In vitro growth of several neuroblastoma cell lines is suppressed significantly by antisense as well as siRNA treatment. (d) An increase of the ARID3B expression level by transfection in the SY5Y neuroblastoma cell line enhances the malignancy in tumor growth assays in nu/nu mice. (e) ARID3B by itself can immortalize mouse embryonic fibroblasts (MEFs) in vitro and confers malignancy to MEF when transfected together with MYCN, the best characterized oncogene for neuroblastoma. Thus, ARID3B seems to play a key role in the malignant transformation of neuroblastoma and may serve not only as a marker of malignancy but also as a potential target for cancer therapy of stage IV neuroblastoma for which there is currently no effective treatment available.     

Prognostic impact of orotate phosphoribosyl transferase among 5-fluorouracil metabolic enzymes in resectable colorectal cancers treated by oral 5-fluorouracil-based adjuvant chemotherapy

Orotate phosphoribosyl transferase (OPRT) is the main enzyme that involves in phosphoribosylation of 5-fluorouracil (5-FU), an essential step that leads to tumor growth inhibition. In our study, the prognostic relevance of OPRT, thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in resectable colorectal cancer (CRC) patients treated by oral 5-FU were compared to further clarify the prognostic value of OPRT. Tumor tissue was collected from 90 CRC patients and the patients were followed for 5.2 years (Median). TS, DPD and OPRT activities in the extract of tumor tissue were determined enzymatically. The cut-off value of OPRT (0.147 nmol/(min mg), TS (0.044 pmol/mg) and DPD (72.10 pmol/(min mg) were determined by maximal chi(2) method. Among these 5-FU metabolic enzymes, only high OPRT group demonstrated significantly better disease-free survival (DFS) (p = 0.0152) and better overall survival (p = 0.0078). In Cox regression analysis, node status (p < 0.0005) and OPRT (p = 0.044) were significant factors for DFS. OPRT activity in tumor tissue was a predictor of prognosis in resectable CRC patients treated by oral 5-FU-based adjuvant chemotherapy, and was useful to pick-up high risk patients independent from known prognosis factors.     

Lung disorders of workers exposed to rush smear dust in China

The aim of this study was to evaluate the lung disorders of the workers exposed to rush smear dust. A cross sectional study was carried out on 1,709 current workers (788 male, 921 female) in 80 factories. All subjects were asked by questionnaire, and health examination including chest X-ray was conducted for 661 workers in 35 factories. Lung function test was also examined for 119 non-smoking males among 661 subjects. Dust samplings were collected and total and respirable dust concentrations at 127 spots in 35 factories were measured. The geometric mean dust concentration in the workshops was up to 20.00 mg/m(3), and the geometric mean respirable dust concentration reached 8.22 mg/m(3). The mean quartz concentration of accumulated dust was 29.2%. The prevalence of radiographic small opacities profusion category > or = 1/0, according to the ILO 1980 Classification System, was 2.6% among 661 employees. One worker was found to have pneumoconiotic findings of 2/2 profusion accompanied with large opacity. The prevalence of pneumoconiosis (1/0 or more) correlated with cumulative dust exposure (r=0.192, p<0.0001). The similar relationship was found between the prevalence rate of cough or sputa and worksite dust concentration. In non-smokers, a positive association was found between the prevalence of cough and occupational exposure duration (r=0.080, p=0.004). Approximately 19.3% and 34.5% of employees suffered from respiratory impairment for FVC and FEV1.0, respectively. This is the first report of "rush" pneumoconiosis in China. Rush mat workers were found to be at high risk for pneumoconiosis, a preventable disease. Our results showed a dose-response relationship between rush-mat dust level and the prevalence of pneumoconiosis. Similar relationship between the prevalence of cough and sputum and the work duration was found for non-smoking workers but not for smoking workers.     

The relationship between 5-fluorouracil sensitivity and single nucleotide polymorphisms of the orotate phosphoribosyl transferase gene in colorectal cancer

Orotate phosphoribosyl transferase (OPRT) is an enzyme playing an important role in exertion of the effect of 5-fluorouracil (5-FU). A type of gene polymorphism, single nucleotide polymorphism (SNP), is considered to be a factor affecting individual differences in exertion of drug effects, and its analysis has recently made progress. We investigated the correlation between SNP of OPRT and 5-FU sensitivity in colon and rectal cancers. The subjects were 31 patients with colorectal cancer who underwent surgical excision between December 2003 and July 2004 at our department. Of SNP of OPRT, 638G/C, 1050T/A, and 1336A/G located in the coding region were analyzed by invader assay. The growth inhibition rate (% IR) of colorectal cancer by 5-FU was obtained by the CDDST method, and 5-FU sensitivity was compared among strains (wild-, homo-, and hetero-types) of each polymorphism. There was no relationship between the strains and 5-FU sensitivity in any of the SNPs. The investigated SNPs of OPRT may have no major influence on 5-FU sensitivity. However, there are many unknown factors in the relationship between SNP of OPRT and 5-FU sensitivity, and SNP analysis of other regions is necessary.     

Fbxw7 contributes to tumor suppression by targeting multiple proteins for ubiquitin-dependent degradation

Fbxw7 (also known as Sel-10, hCdc4 or hAgo) is the F-box protein component of a Skp1-Cul1-F-box protein (SCF) ubiquitin ligase. Fbxw7 contributes to the ubiquitin-mediated degradation of cyclin E, c-Myc, Aurora-A, Notch and c-Jun, all of which appear to function as cell-cycle promoters and oncogenic proteins. Loss of Fbxw7 results in elevated expression of its substrates, which may lead to oncogenesis. However, it remains largely unclear which accumulating substrate is most related to cancer development in Fbxw7-mutant cancer cells. In the present study, we examined the abundance of cyclin E, c-Myc and Aurora-A in seven cancer cell lines, which harbor wild-type (three lines) or mutant (four lines) Fbxw7. Although these three substrates accumulated in the Fbxw7-mutant cells, the extent of increase in the expression of these proteins varied in each line. Forced expression of Fbxw7 reduced the levels of cyclin E, c-Myc and Aurora-A in the Fbxw7-mutant cells. In contrast, a decrease in the expression of cyclin E, c-Myc or Aurora-A by RNA interference significantly suppressed the rate of proliferation and anchorage-independent growth of the Fbxw7-mutant cells. These findings thus suggest that the loss of Fbxw7 results in accumulation of cyclin E, c-Myc and Aurora-A, all of which appear to be required for growth promotion of cancer cells. Fbxw7 seems to regulate the levels of multiple targets to suppress cancer development.     

Cross talk between apoptosis and invasion signaling in cancer cells through caspase-3 activation

In solid tumors, cancer cells are exposed to various microenvironmental stresses such as hypoxia, nutritional depletion, and low pH. Cancer cells adapt to these stresses and circumvent cell death. When the antiapoptotic signals overcome the stress, cancer cells might acquire physiologic functions, such as invasiveness, instead of cell death. Here, we report that tumor cells acquire an invasive capacity from apoptotic signals through caspase activation. We treated rat ascites hepatoma MM1 cells with an apoptosis-inducing drug, etoposide, or hypoxia, and assessed the invasion capacity with an in vitro bioassay. Although MM1 cells hardly showed invasiveness in serum-free medium, under stress conditions, invasive capacity accompanied with morphologic change was induced with caspase-3 activation. Such stress-induced invasion as well as morphologic change was suppressed by blocking caspase-3 activity with caspase inhibitors or by RNA interference of caspase-3. In contrast, lysophosphatidic acid-induced invasiveness was not affected by caspase-3 inhibition. These results suggest that caspase-3 activation contributes to the stress-induced invasive capacity of these cancer cells.     

STI571 (Glivec) inhibits the interaction between c-KIT and heat shock protein 90 of the gastrointestinal stromal tumor cell line, GIST-T1

The gastrointestinal stromal tumor cell line, GIST-T1, has a heterogenic 57-base pair deletion in exon 11 of the c-kit mutation, and the c-KIT protein in the GIST-T1 cells constitutively activated. We report that STI571 (Glivec; Novartis, Basel, Switzerland), a specific inhibitor of c-KIT, inhibits the clustering of c-KIT at the cell membrane of the GIST-T1 cells. Furthermore, STI571 prevents the interaction between c-KIT and the molecular chaperone, heat shock protein 90 (Hsp90). Geldanamycin, an inhibitor of Hsp90, also prevents interaction between c-KIT and Hsp90, and inhibits tyrosine phosphorylation of c-KIT. Our results indicate that c-KIT molecules are assembled on the cell surface of the GIST-T1 cells, and that the interaction between c-KIT and Hsp90 plays an important role in c-KIT activation.     

Evaluation of 5-fluorouracil applicability by multi-point collagen gel droplet embedded drug sensitivity test

The drug sensitivity of tumor cells is one of key issues to explore individualized therapy for cancer patients. One of such methods is in vitro anticancer drug sensitivity test which is generally based on one drug concentration and contact time. In this study, 5-fluorouracil (5-FU) sensitivity of cancer cells from colorectal cancer patients was evaluated by collagen gel droplet embedded drug sensitivity test (CD-DST) under multiple drug concentrations and contact durations. Cancer cells from 19 patients were measured for 9 drug concentration/contact time conditions (cohort 1) and from 34 patients were measured for 2 drug concentration/contact time conditions (cohort 2) using CD-DST. There was not significant difference in growth inhibition rate for 1.0 microg/ml for 24 h and 0.2 microg/ml for 120 h, which gives the same area under the curve (AUC) (p=0.832) in all 53 patients (cohort 1 and 2). In cohort 1, 9 conditions were successfully measured in 18 of 19 cohort 1 patients (94.7%). The drug concentrations and growth inhibition rate approximated to logarithmic curve for all 3 contact times and 50% inhibitory concentration (IC50) values at 3 contact times could be calculated in these 18 patients. Growth inhibition rate and AUC also approximated to logarithmic curve. These values varied several orders of magnitude among patients. In vitro antitumor effect of 5-FU depended on AUC in colorectal tumor and it might support the use of continuous infusion or oral therapy which generates significant AUC with manageable toxicity. Some patients demonstrating low 5-FU sensitivity could not be indicated for 5-FU based therapy, and non-5-FU therapy should be explored for them.     

Impact of orotate phosphoribosyl transferase activity as a predictor of lymph node metastasis in gastric cancer

Orotate phosphoribosyl transferase (OPRT) is an essential nucleotide metabolic enzyme for cell proliferation and also a key enzyme for conversion of 5-FU to its active form in tumor tissue. The association between tumor OPRT activity and pathophysiological status, including lymph node metastasis [pN+], and the impact of OPRT for predicting pN+ were investigated in gastric cancer. The lymph node status of 73 resectable gastric cancer patients was analyzed preoperatively by computed tomography (CT), ultrasonography and magnetic resonance, and the OPRT activity of collected tumor tissue was measured. Then these data were compared with pathological observation of a surgical lymph node specimen. OPRT activity in the tumor tissue decreased as the depth of invasion increased. An OPRT test demonstrated superior sensitivity and comparable accuracy and sensitivity for predicting pN+, against current imaging diagnoses. Furthermore, the analysis of node negative patients by CT revealed that 80% of false negative patients were retrieved by this OPRT test. Thus, OPRT activity in tumor tissue was a powerful predictor of pN+ in resectable gastric cancer, and the preoperative OPRT test, when it becomes possible, would provide a basis for accurate evaluation of disease status, which is indispensable for the planning of personalized therapy.