Critical role of TRPC1-mediated Ca²âº entry in decidualization of human endometrial stromal cells.

Decidualization is an ovarian steroid-induced remodeling/differentiation process of uterus essential for embryo implantation and placentation. Here, we investigated the possible involvement of enhanced Ca²âº dynamics in the decidualization process in human endometrial stromal cells (hESC) in its connection with a recently emerging nonvoltage-gated Ca²âº entry channel superfamily, the transient receptor potential (TRP) protein. Combined application of 17ß-estradiol (E2) (10 nM) and progesterone (P4) (1 µM) for 7-14 d resulted in morphological changes of hESC characteristic of decidualization (i.e. cell size increase), whereas sole application of E2 exerted little effects. A 7- to 14-d E2/P4 treatment greatly increased the expression level of decidualization markers IGF binding protein-1 (IGFBP-1) and prolactin and also up-regulated the expression of TRPC1, a canonical TRP subfamily member that has been implicated in store-operated Ca²âº influx (SOC) in other cell types. In parallel with this up-regulation, SOC activity in hESC, the nuclear translocation of phosphorylated cAMP responsive element binding protein (p-CREB) and the expression of Forkhead box protein 01 were enhanced significantly. Small interfering RNA knockdown of TRPC1 counteracted the E2/P4-induced up-regulation of IGFBP-1 and prolactin and enhancement of SOC activity together with the inhibition of hESC size increase, p-CREB nuclear translocation, and FOXO1 up-regulation. Coadministration of SOC inhibitors SK&F96365 or Gd³âº with E2/P4 also suppressed the up-regulation of IGFBP-1 and hESC size increase. Similar inhibitory effects were observed with extracellularly applied TRPC1 extracellular loop 3-directed antibody, which is known to bind a near-pore domain of TRPC1 channel and block its Ca²âº transporting activity. These results strongly suggest that up-regulation of TRPC1 protein and consequent enhancement of SOC-mediated Ca²âº influx may serve as a crucial step for the decidualization process of hESC probably via p-CREB-dependent transcriptional activity associated with FOXO1 activation.

Assessment of HB-EGF levels in peritoneal fluid and serum of ovarian cancer patients using ELISA.

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a rational target for ovarian cancer therapy. The aim of this study was to examine HB-EGF levels in the peritoneal fluid and serum of ovarian cancer (OVCA) patients. PATIENTS AND METHODS: Samples were collected from six healthy women, 21 OVCA patients, and 21 ovarian cyst patients. HB-EGF levels were measured using a sandwich ELISA kit and calculated using a parallel line assay. RESULTS: No significant difference between the slopes of the standard and sample curves was observed at an anti-HB-EGF antibody concentration of 1.6 µg/ml. HB-EGF levels in the peritoneal fluid and serum of OVCA patients were significantly higher than those in patients with ovarian cysts or controls. Serum HB-EGF levels were also significantly correlated with levels in peritoneal fluid in OVCA patients. CONCLUSION: We developed an assay for the exact measurement of HB-EGF levels in peritoneal fluid and serum.

Nasal instillation of nanoparticle-rich diesel exhaust particles slightly affects emotional behavior and learning capability in rats.

In the present study, in order to reveal novel adverse effects of ultrafine particles (UFP) on the central nervous system, the effects of nanoparticle-rich diesel exhaust particles (NRDEP; count mode diameter, 21.45 nm) on emotional behavior, learning capability and brain neurotransmitter levels were studied in rats by intranasal instillation (iNI). NRDEP (10 and 50 µg/rat) was instilled into 2-week old infant, male rats once a week for 4 weeks. Spontaneous motor activity measured was observed to be inverse to the dose level. In active avoidance tests using a shuttle box, NRDEP-treated animals showed a lower avoidance performance than control animals given air-instillation. The levels of dopamine and its metabolite (DOPAC) in the medial mammillary nucleus of the brain tended to be lower in the NRDEP-treated animals. From these results, although the effects of NRDEP by iNI on the emotionality and the brain neurotransmitter levels were not fully clear, the results obtained by avoidance testing suggested involvement of UFP in learning capability.

Emerging strategies for ErbB ligand-based targeted therapy for cancer.

ErbB receptors are crucial for development and evolution and have been intensely pursued as targets for cancer therapeutics. Inhibiting the signaling activity of individual receptors in this family has advanced human cancer treatment. However, actual curative effects of the existing anti-ErbB therapeutics are still insufficient. A large percentage of patients who are initially responsive to ErbB receptor-targeted therapies later become resistant. Mechanisms responsible for tumor resistance to ErbB-targeted agents are as follows: many epidermal growth factor receptor (EGFR)- and HER2-targeted therapies cannot inhibit signaling through the ErbB receptor heterodimer, and anti-EGFR agents can suppress extracellular signal-related kinase (ERK) signal proliferation but not protein kinase B/Akt survival signals. ErbB ligand-based targeted therapy against HB-EGF or amphiregulin may overcome such obstacles. Here we discuss the efficacy of CRM197, a specific inhibitor of HB-EGF, and its possible clinical adaptation in combination with conventional chemotherapeutic agents in cancer therapy.

HB-EGF inhibition in combination with various anticancer agents enhances its antitumor effects in gastric cancer.

Advanced gastric cancer (GC) is one of the most lethal malignancies. Although many anticancer agents exist for the treatment of GC, its prognosis remains extremely poor. Therefore, further development of targeted therapies is required for patients with GC. To assess the role of heparin-binding epidermal growth factor-like growth factor (HB-EGF) as a target for GC therapy, the expression of EGF receptor ligands in GC cell lines, and the antitumor effects of an HB-EGF inhibitor (CRM197) as a single agent and in combination with other anticancer agents was assessed in GC cells. HB-EGF was the predominantly expressed ligand among EGF receptor ligands in all the cells. CRM197 induced significant cell apoptosis. Anticancer agents augmented the secretion of HB-EGF into the medium and simultaneously induced cell apoptosis. Combination of CRM197 with other anticancer agents significantly enhanced cell apoptosis. Additionally, co-administration of CRM197 and paclitaxel resulted in synergistic antitumor effects. These results suggested that HB-EGF is a rational target for GC therapy.

Primary leiomyosarcoma of the fallopian tube.

Primary sarcoma of the fallopian tube is a very rare neoplasm. We report the case of a 69-year-old woman affected with leiomyosarcoma of the left fallopian tube. Her chief complaint was lower abdominal pain. The preoperative diagnosis was a left adnexal malignant tumor based on pelvic examination, abdominal computed tomography, and magnetic resonance imaging. Following a laparotomy, she was ultimately diagnosed with a FIGO IIc fallopian tube leiomyosarcoma. She was treated with total abdominal hysterectomy, bilateral salpingo-oophorectomy, pelvic lymph node dissection, partial omentectomy, and low anterior resection for rectal invasion. The patient subsequently received adjuvant chemotherapy with pirarubicin and ifosfamide. Thirty months after the first therapy, a computed tomography scan revealed metastasis of the liver, lung, and supraclavicular lymph node. The patient died of the disease 39 months after the initial treatment.

Change in cervical length in cases resulting in threatened preterm labor.

PURPOSE: To determine the predisposing changes in cervical length (CL) and the critical range of CL in which significant uterine contractions emerge resulting in threatened preterm labor (TPL). METHODS: Sixty-eight uncomplicated singleton pregnancies where the CL was <25 mm before 31 weeks were divided into cases with TPL (n = 23) or without (n = 45). CL and uterine contractions were monitored sequentially starting between 16 and 20 weeks. The gestational ages when a CL of <25 or <15 mm was first observed, the interval between these two measurements, and the CL value at TPL diagnosis were analyzed retrospectively. RESULTS: (1) The gestational ages when a CL of <25 and <15 mm was first detected were lower in the TPL group (25 (median); 18-30 (range) and 28; 25-33 weeks, respectively) than in the non-TPL group (27; 20-30 and 33; 26-35 weeks; P = 0.030 and P < 0.001). (2) The interval between the two measurements was shorter in the TPL group (2.5; 0-15 weeks) than in the non-TPL group (5.5; 0-13 weeks, P = 0.034). (3) The CL value at TPL diagnosis was 13 mm (median), ranging from 7 to 18 mm. CONCLUSION: Cases with early onset and subsequent rapid CL shortening before 31 weeks resulted in TPL when CL decreased below the range 7-18 mm.

Efficacy of ligand-based targeting for the EGF system in cancer.

Although drugs inhibiting ErbB receptors such as epidermal growth factor receptor (EGFR) and HER2 have been developed as anticancer agents targeting the EGF family, they are not effective for all types of cancer and instead target only certain types. We propose the following four main reasons for these observations: (i) although seven EGFR ligands exist, effective inhibition of specific EGFR ligands may occur because their expression levels differ in different malignancies; (ii) suppressing EGFR ligands inhibits aggregation of EGFR and other ErbB receptors and activation of ERK and Akt signals; (iii) EGFR ligands may have various combinations for signal transduction through the EGFR pathway and other receptor signals; and (iv) the intracellular C-terminals of EGFR ligands move into the nucleus and strongly regulate cell proliferation. In this review, we describe important implications for targeted cancer therapy against EGFR ligands and describe the current situation in the development of ligand-based therapies for cancer.

MR diagnosis of steroid cell tumor of the ovary: value of chemical shift imaging.

A 53-year-old asymptomatic woman was found to have a pelvic mass at medical examination. Magnetic resonance (MR) imaging revealed a 4-cm solid mass at the right adnexal region, which showed marked hyperintensity on T(2)-weighted imaging and marked enhancement on post-contrast T(1)-weighted imaging. Chemical-shift imaging showed slight but significant signal loss on out-of-phase images, which suggested the presence of intratumoral lipid. The resected specimen exhibited typical features of steroid cell tumor, and Oil Red O stain was positive for cytoplasmic lipid.

Monitoring of endometrial K-ras mutation in tamoxifen-treated patients with breast cancer.

INTRODUCTION: A high incidence of endometrial K-ras mutations has been reported in tamoxifen (TAM)-treated patients with breast cancer. We examined the changes in the frequency of the endometrial K-ras mutations after the cessation of TAM treatment. METHODS: DNA was extracted from fresh cytological or polypectomy samples of the endometrium in 28 patients who had undergone TAM treatment of breast cancer. Mutations were detected by an enriched polymerase chain reaction-enzyme-linked minisequence assay (Sumitomo Metal Industry, Inc, Tokyo, Japan). K-ras codon 12 mutations were monitored in these 28 patients. RESULTS: An initial examination detected endometrial K-ras mutations in 13 of the 28 patients. However, repeated examinations performed after cessation of TAM treatment did not detect endometrial K-ras mutations in any of these 13 patients. No endometrial K-ras mutation has been detected in the repeated examinations performed for these patients for more than 2 years since the cessation of TAM treatment. In addition, the 15 patients who did not have endometrial K-ras mutations in the initial examination did not demonstrate them in repeat examinations. CONCLUSIONS: The cessation of TAM treatment may reduce the risk of developing endometrial cancers through K-ras mutations.

New approach to cancer therapy: heparin binding-epidermal growth factor-like growth factor as a novel targeting molecule.

Heparin binding-epidermal growth factor-like growth factor (HB-EGF) is one of the EGF receptor ligands and possesses several functional domains. It is involved in diverse biological processes, including wound healing, blast implantation, atherosclerosis and tumor formation, through its interactions with various molecules. We have reported that HB-EGF gene expression is significantly elevated in human ovarian cancer, and further demonstrated that HB-EGF plays key roles in the acquisition of malignant phenotypes, such as cell survival in peritoneal fluid, cell adhesion on extracellular matrices, invasion, angiogenesis, tumorigenicity, and chemoresistance in ovarian cancer. Thus, HB-EGF was considered as a promising target for cancer therapy. In vitro as well as in vivo experiments have revealed that cross-reacting material 197 (CRMI97), a specific inhibitor of HB-EGF, or a small interfering RNA for HB-EGF can block each step involved in peritoneal dissemination. According to these pieces of evidence, the development of targeting tools against HB-EGF, such as CRM197, could allow us to improve the prognosis of cancer patients.

Influence of di-(2-ethylhexyl)phthalate on fetal testicular development by oral administration to pregnant rats.

Influence of di-(2-ethylhexyl)phthalate (DEHP) on testicular development was studied by oral administration of DEHP at doses of 500 and 1000 mg/kg/day to pregnant rats on gestational days (G) 7 to 18. Ethinyl estradiol (EE) at dose levels of 0.25 and 0.5 mg/kg/day was used as a reference substance. Each 5-6 pregnant rats were sacrificed and their fetuses were examined on G12, 14, 16, 18 and 20. Fetal deaths averaging 20-36% were observed at every examination in the group receiving 1000 mg/kg of DEHP. Increases of fetal deaths over 50% were also observed in the reference group that received 0.5 mg/kg of EE. Microscopic examination of the fetal testis in groups treated with DEHP revealed degeneration of germ cells in G16 fetuses and localized proliferation or hyperplasia of interstitial cells in G18 and 20 fetuses. Germ cells having more than two nuclei were observed in a few cases including the control testes of G14 fetuses. These multinucleated cells were observed frequently in G20 fetuses treated with DEHP. Examination of testes of naturally delivered offspring of dams treated with 1000 mg/kg of DEHP at 7 weeks of age revealed scattered atrophy or dilatation of seminiferous tubules. Another experiment was carried out to confirm the dose of DEHP affecting testicular development and spermatogenesis. DEHP was given to pregnant rats at doses of 125, 250 and 500 mg/kg/day during G7-18. Similar histopathological changes were observed in fetal testes of the group exposed to 500 and 250 mg/kg of DEHP, but not in those exposed to 125 mg/kg. In postnatal examinations, however, no abnormality was found in the testes at 5 and 10 weeks after birth in any of the treated groups. Furthermore, no abnormal findings were observed in the function of sperm, sperm counts and sperm morphology in the offspring of the group treated with DEHP during the fetal period at 10 weeks of age. Thus, 125 mg/kg/day is considered the no-observed-effect-level of DEHP on testicular development of rats by exposure in utero during the period of organogenesis.

Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells.

Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phase of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.

K-ras mutation in tamoxifen-related endometrial polyps.

BACKGROUND: K-ras mutation is thought to occur at an early stage of neoplastic progression in the endometrium. The authors investigated mutations in codon 12 of K-ras in tamoxifen (TAM)-related endometrial polyps. METHODS: DNA was extracted from 11 frozen endometrial polyps from TAM-treated patients with breast carcinoma. Mutations were detected using the mutant allele-specific amplification method. The results subsequently were analyzed for correlations with immunohistochemical data that were obtained using antibodies against estrogen receptors (ERs; alpha and beta forms), progesterone receptors (PRs; A and B forms), and Ki-67. RESULTS: Mutations in codon 12 of K-ras were observed in 7 of 11 TAM-related endometrial polyps. Expression levels of ER-alpha and PR-B were high in the glandular epithelium and low in the stroma. PR-A expression was high in both the glandular epithelium and the stroma. In the glandular epithelium, expression of ER-beta appeared to be lower than expression of ER-alpha. The Ki-67 index in the glandular epithelium ranged from 2 to 38, whereas the index ranged from 0 to 4 in the stroma (P < 0.01). CONCLUSIONS: The incidence of mutations in codon 12 of K-ras in TAM-related endometrial polyps (64%) was greater than the incidence of these same mutations in sporadic endometrial hyperplasias (4.5-23%). High expression levels of ER-alpha, PR-A, and PR-B in the glandular epithelium were observed in all polyps, regardless of K-ras codon 12 mutation status and Ki-67 index. The authors' findings may support the hypothesis that the polyp-carcinoma sequence partly indicates the development of endometrial carcinoma in postmenopausal women who have been treated with TAM.

Contribution of estrogen receptor alpha to oncogenic K-Ras-mediated NIH3T3 cell transformation and its implication for escape from senescence by modulating the p53 pathway.

We previously reported that enhanced transcriptional activation of estrogen receptor alpha (ERalpha) contributed to [(12)Val]K-Ras-mediated NIH3T3 cell transformation. Functional inactivation of ERalpha by a dominant negative mutant of ERalpha (DNER) in the presence of activated K-Ras 4B mutant arrested the cell cycle at G(0)/G(1), subsequently provoking replicative cell senescence, finally abrogating tumorigenic potential. p53-dependent up-regulation of p21 was implicated in this cell senescence induction. Alterations in the MDM2 protein in response to DNER accounted for this p21-mediated cell senescence induction. An oncogenic K-Ras 4B mutant significantly increased MDM2 proteins coprecipitated with p53, and suppressed p53 transcriptional activity. In turn, DNER exerted its function to decrease MDM2 proteins coprecipitated with p53, followed by the stimulation of p53 activity in the presence of the oncogenic K-Ras 4B mutant. In addition, overexpression of wild type ERalpha in NIH3T3 cells resulted in the significant increase in the MDM2 protein level and the resultant suppression of p53 transcriptional activity. Finally, we demonstrated that c-Jun expression overcame the suppression and resultant enhancement of p21 protein level in response to DNER. The data imply that the ERalpha-AP1 pathway activated by oncogenic K-Ras 4B mutant contributes to the NIH3T3 cells' transformation by modulating p53 transcriptional activity through MDM2.