The presence and impact of estrogen metabolism on the biology of triple-negative breast cancer.

PURPOSE: While triple-negative breast cancer (TNBC) is negative for estrogen receptor alpha, a substantial proportion of carcinomas express estrogen receptor beta (ERß); consequently, estrogen actions and metabolism may be relevant in this cancer subtype. METHODS: A cohort of 81 TNBC patients from Tohoku University Hospital, Japan were characterised with regard to the expression of estrogen receptor beta and enzymes known to modulate levels of estrogens in breast and other tissues (Aromatase, 17-beta- Hydroxysteroid dehydrogenases 1, 2 and 6). This was done at the protein level by means of immunohistochemistry. As this cohort has been previously characterised for androgens, this also allows for comparison between the expressions of estrogen-related proteins and of androgen-related proteins. Preliminary mechanistic studies in cell culture were also undertaken. RESULTS: 17ßHSD2 was detected in the highest number of cases followed by 17ßHSD1, 17ßHSD6 and aromatase. When comparing the expression of ERß with that of the enzymes, it was positively correlated with the expression of 17ßHSD6 (p < 0.05) and trended towards correlation with dual expression of 17ßHSD1 and 2 (p < 0.07). 17ßHSD1 was associated with significantly reduced tumour volume (p = 0.0025), while ERß was associated with a trend towards reduced lymphovascular invasion, (p < 0.061). Interestingly, in survival analysis, 17ßHSD6 expression was the only one of these five factors that influenced survival, with positive samples being associated with longer disease-free survival compared to those that were negative for 17ßHSD6 (p < 0.05). In assessing associations with expression of proteins in the androgenic pathway, expression of aromatase appeared to be associated with androgenic pathways in TNBC patients (p < 0.05). Due to this association and the potential relevance to androgen-directed therapies in TNBC, we evaluated this interaction in vitro. We observed androgen-dependent upregulation of aromatase and ERß in a subset of AR expressing TNBC cell lines (MDA-MB-453, SUM-185-PE and MFM-223). CONCLUSION: Overall this study suggests the presence of, and a potential protective effect of estrogens in TNBC.

Effects of cytokines derived from cancer-associated fibroblasts on androgen synthetic enzymes in estrogen receptor-negative breast carcinoma.

PURPOSE: The tumor microenvironment plays pivotal roles in promotion of many malignancies. Cancer-associated fibroblasts (CAFs) have been well-known to promote proliferation, angiogenesis, and metastasis but mechanistic understanding of tumor-stroma interactions is not yet complete. Recently, estrogen synthetic enzymes were reported to be upregulated by co-culture with stromal cells in ER positive breast carcinoma (BC) but effects of co-culture on androgen metabolism have not been extensively examined. Therefore, we evaluated roles of CAFs on androgen metabolism in ER-negative AR-positive BC through co-culture with CAFs. METHODS: Concentrations of steroid hormone in supernatant of co-culture of MDA-MB-453 and primary CAFs were measured using GC-MS. Cytokines derived from CAFs were determined using Cytokine Array. Expressions of androgen synthetic enzymes were confirmed using RT-PCR and Western blotting. Correlations between CAFs and androgen synthetic enzymes were analyzed using triple-negative BC (TNBC) patient tissues by immunohistochemistry. RESULTS: CAFs were demonstrated to increase expressions and activities of 17ßHSD2, 17ßHSD5, and 5α-Reductase1. IL-6 and HGF that were selected as potential paracrine mediators using cytokine array induced 17ßHSD2, 17ßHSD5, and 5α-Reductase1 expression. Underlying mechanisms of IL-6 paracrine regulation of 17ßHSD2 and 17ßHSD5 could be partially dependent on phosphorylated STAT3, while phosphorylated ERK could be involved in HGF-mediated 5α-Reductase1 induction. α-SMA status was also demonstrated to be significantly correlated with 17ßHSD2 and 17ßHSD5 status in TNBC tissues, especially AR-positive cases. CONCLUSIONS: Results of our present study suggest that both IL-6 and HGF derived from CAFs could contribute to the intratumoral androgen metabolism in ER-negative BC patients.

How far have we come in terms of estrogens in breast cancer? [Review].

Major advances in breast cancer treatment have almost always been linked to the actions of estrogen. Therefore, this review focused on estrogen actions in the breast, particularly the developments of the past 20 years, the present understanding of disease biology and possible future developments. Within these areas have focused on what is known about the underlying molecular biology and in particular integration of the bioinformatics revolution of the last 15 years with other facets of research. In addition, there will be an emphasis on the understanding brought about by a greater appreciation for the intracrinology of the breast.

On-demand killing of adherent cells on photo-acid-generating culture substrates.

As a powerful tool of cell screening and cell purification, we developed a novel method to kill adherent cells as cultured on a substrate by micro-projection of incoherent visible light. To kill the cells by the mild light irradiated by electrically controllable micro-projection systems currently available, we introduced the assist of the photo-responsive culture substrates functionalized with a photo-acid-generating polymer. In clear contrast to the existing laser-based methods requiring point scanning, areal micro-projection of blue light with the wavelength 436 nm killed many CHO-K1 cells at a time in the irradiated area on the substrate. The effect of the photo-generated acid was so confined that selective killing of targeted cells was achieved without critical damage to the neighboring cells. Further, we demonstrated the photo-selective killing of the adherent cells after preliminarily patterning through the photo-induced removal of cell adhesion-inhibiting polymer.

Validity of "One-size-fits-all" Approaches for the National Health Screening and Education Program: A Large-scale Cohort Study of Corporate Insurance Beneficiaries.

Objective Metabolic syndrome represents a unified condition of atherosclerotic diseases caused by abdominal obesity. The aims of this study were to examine the applicability of the prevalent fixed cut-off values of the abdominal circumference (AC) and body mass index (BMI) to age and gender groups and to identify suitable lifestyle modification factors. Methods We defined an outcome as having ≥ 2 risk components that are necessary to diagnose metabolic syndrome and examined the cross-sectional association of the AC and BMI with the outcome. We also assessed the effects of time-updated lifestyle information on metabolic traits using longitudinal data. Patients We enrolled 22,953 beneficiaries of a corporate health insurance scheme who underwent annual health examinations between January 2004 and December 2014. Results The AC [per 5-cm increase, odds ratio (OR) 1.17, 95% confidence interval (CI) 1.12-1.24] and BMI (OR 1.10, 95% CI 1.07-1.13) were significantly associated with the outcome, adjusted for age, gender, current smoking status, drinking habits, and other lifestyle information. The association between the outcome and AC was modified by gender (p for interaction = 0.033), and the association between the outcome and BMI was modified by age group (p for interaction = 0.049). In the longitudinal analysis, current smoking, drinking habits, and unhealthy eating habits were associated with an increased AC and BMI, whereas regular physical activity was associated with a decreased AC and BMI. Conclusion We showed that the association between the AC or BMI and metabolic syndrome was modified by gender or age group. Further studies will be needed to customize the national health screening and education programs.

Stepwise assembly of micropatterned co-cultures using photoresponsive culture surfaces and its application to hepatic tissue arrays.

Cell micropatterning, a method to place cells at arbitrary regions, is becoming an essential tool to conduct cell biology and tissue engineering. Conventional cell patterning techniques usually allow only single patterning with single cell type on the same culture surface. However, biomedical research today requires even sophisticated fabrication methods that require spatiotemporal control of multiple cell arrangements. Here we introduce in situ cell micropatterning system which enables stepwise cell patterning using a photoresponsive cell culture surface (PRCS) whose cell adhesiveness could be altered by the UV irradiation. To demonstrate an application to tissue engineering, a liver-mimic tissue array was fabricated and liver-specific gene expressions were quantified with real time PCR. Patterned co-culture systems composed of HepG2 spheroids with Balb/3T3 were fabricated, and the optimum spheroid diameter, which yielded the highest cellular functions, was determined to be 150 microm. After 20 days of patterned co-culture of HepG2 spheroids and Balb/3T3, CYP3A4 expression increased 50-fold higher than conventionally cultured HepG2; CYP3A4 expression was 20% higher than randomly co-cultured HepG2 and Balb/3T3. Thus the combination of PRCS and the photomask-free irradiation apparatus showed the versatility of experimental setups and proved to be a powerful tool for biomedical studies.

Cross-contamination of bacteria-colonized pierced earring holes and fingers in nurses is a potential source of health care-associated infections.

BACKGROUND: In recent years, the wearing of pierced earrings for personal adornment has increased among health care workers in Japan. However, the transmission dynamics between bacteria in pierced earring holes and fingers has not been clearly shown. METHODS: Earlobes and fingers of 200 nurses (128 nurses with pierced earlobes and 72 nurses with unpierced earlobes) working at a university hospital were sampled to determine whether cross-transmission of bacteria-colonized pierced earring holes and fingers in nurse is possible. RESULTS: Of 128 nurses who had pierced earring holes, Staphylococcus aureus was recovered from earlobes of 24 nurses (18.8%) compared with 7 of 72 nurses without pierced earring holes (9.7%) (P = .09). Of those 15 nurses yielding S aureus from both earlobes and fingers, 12 were from nurses who had pierced earring holes compared with 3 nurses without pierced earring holes. Excluding 1 nurse, antimicrobial susceptibility patterns and genotypes of S aureus from both earlobe and fingers of each nurse were identical. CONCLUSION: Pierced earlobes can be a source of health care-associated infection via cross-transmission of bacteria from earlobe holes to fingers.

S100P and Ezrin promote trans-endothelial migration of triple negative breast cancer cells.

PURPOSE: Triple negative breast cancer (TNBC) patients generally have an adverse clinical outcome because their tumors often recur and metastasize to distant sites in the first 3 years after surgery. Therefore, it has become pivotal to identify potential factors associated with metastasis. Here, we focused on the effects of S100P and Ezrin on the trans-endothelial migration (TEM) of TNBC cells, as they have both been suggested to play a role in this process in other malignancies. METHODS: The expression of S100P and Ezrin was examined by immunohistochemistry in 58 primary TNBC samples. The mRNA and protein levels of S100P and Ezrin were assessed in breast cancer-derived cell lines using qRT-PCR and Western blotting, respectively. Proliferation and migration assays were performed using TNBC-derived MFM-223 and SUM-185-PE cells transfected with S100P and Ezrin siRNAs. Two different timeframes were employed for TEM assays using TNBC-derived cells and human umbilical vein endothelial-derived cells, respectively. Correlations between the status of EzrinThr-567 expression and various clinicopathological features were analyzed by immunohistochemistry. RESULTS: We found that S100P and Ezrin double negative TNBC cases were significantly associated with a better disease-free survival. We also found that single and double siRNA-mediated knockdown of S100P and Ezrin in TNBC-derived cells significantly inhibited their TEM and destabilized the intercellular junctions of endothelial cells. In addition, we found that EzrinThr-567 immunoreactivity significantly correlated with vascular invasion in TNBC patients. CONCLUSIONS: From our data we conclude that S100P, Ezrin and EzrinThr-567 are involved in the trans-endothelial migration of TNBC cells and that they may serve as potential targets in TNBC patients.

Pressure-driven perfusion culture microchamber array for a parallel drug cytotoxicity assay.

This article reports a pressure-driven perfusion culture chip developed for parallel drug cytotoxicity assay. The device is composed of an 8 x 5 array of cell culture microchambers with independent perfusion microchannels. It is equipped with a simple interface for convenient access by a micropipette and connection to an external pressure source, which enables easy operation without special training. The unique microchamber structure was carefully designed with consideration of hydrodynamic parameters and was fabricated out of a polydimethylsiloxane by using multilayer photolithography and replica molding. The microchamber structure enables uniform cell loading and perfusion culture without cross-contamination between neighboring microchambers. A parallel cytotoxicity assay was successfully carried out in the 8 x 5 microchamber array to analyze the cytotoxic effects of seven anticancer drugs. The pressure-driven perfusion culture chip, with its simple interface and well-designed microfluidic network, will likely become an advantageous platform for future high-throughput drug screening by microchip.

Association between kidney function and genetic polymorphisms in atherosclerotic and chronic kidney diseases: A cross-sectional study in Japanese male workers.

BACKGROUND: Several single nucleotide polymorphisms (SNPs) have been implicated in the predisposition to chronic kidney disease (CKD). Atherosclerotic disease is deeply involved in the incidence of CKD; however, whether SNPs related to arteriosclerosis are involved in CKD remains unclear. This study aimed to identify SNPs associated with CKD and to examine whether risk allele accumulation is associated with CKD. METHODS: We conducted a cross-sectional study using data of 4814 male workers to examine the association between estimated glomerular filtration rate (eGFR) and 59 candidate polymorphisms (17 CKD, 42 atherosclerotic diseases). We defined the genetic risk score (GRS) as the total number of risk alleles that showed a significant association in this analysis and examined the relationship with CKD (eGFR < 60 ml/min/1.73m2). Multivariate logistic regression, discrimination by area under the receiver operating characteristic curve, integrated discrimination improvement (IDI), and category-free net reclassification improvement (cNRI) were evaluated. RESULTS: In total, 432 participants were categorized as having CKD. We found eight candidate SNPs with P value < 0.05 (CX3CR1 rs3732379, SHROOM3 rs17319721, MTP rs1800591, PIP5K1B rs4744712, APOA5 rs662799, BRAP rs3782886, SPATA5L1 rs2467853, and MCP1 rs1024611) in the multivariate linear regression adjusted for age, body mass index, systolic blood pressure, and fasting blood glucose. Among these eight SNPs, BRAP rs3782886 and SPATA5L1 rs2467853 were significantly associated with eGFR (false discovery rate < 0.05). GRS was significantly associated with CKD (odds ratio, 1.17; 95% confidence interval, 1.09-1.26). C-statisics improved from 0.775 to 0.780 but showed no statistical significance. However, adding GRS significantly improved IDI and cNRI (0.0057, P = 0.0028, and 0.212, P < 0.001, respectively). CONCLUSIONS: After adjustment for clinical factors, kidney function was associated with BRAP rs3782886 and SPATA5L1 rs2467853 and the GRS for CKD that we developed was associated CKD.

High Proteolytic Resistance of Spider-Derived Inhibitor Cystine Knots.

Proteolytic stability in gastrointestinal tract and blood plasma is the major obstacle for oral peptide drug development. Inhibitor cystine knots (ICKs) are linear cystine knot peptides which have multifunctional properties and could become promising drug scaffolds. ProTx-I, ProTx-II, GTx1-15, and GsMTx-4 were spider-derived ICKs and incubated with pepsin, trypsin, chymotrypsin, and elastase in physiological conditions to find that all tested peptides were resistant to pepsin, and ProTx-II, GsMTx-4, and GTx1-15 showed resistance to all tested proteases. Also, no ProTx-II degradation was observed in rat blood plasma for 24 hours in vitro and ProTx-II concentration in circulation decreased to half in 40 min, indicating absolute stability in plasma and fast clearance from the system. So far, linear peptides are generally thought to be unsuitable in vivo, but all tested ICKs were not degraded by pepsin and stomach could be selected for the alternative site of drug absorption for fast onset of the drug action. Since spider ICKs are selective inhibitors of various ion channels which are related to the pathology of many diseases, engineered ICKs will make a novel class of peptide medicines which can treat variety of bothering symptoms.

The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system.

Kv2.1, the voltage-gated ion channel, is ubiquitously expressed in variety of tissues and dysfunction of this ion channel is responsible for multiple diseases. Electrophysiological properties of ion channels are so far characterized with eukaryotic cells using the manual patch clamp which requires skilful operators and expensive equipments. In this research, we created a simple and sensitive drug screen method using bacterial giant spheroplasts and the automated patch clamp which does not require special skills. We expressed a eukaryotic voltage-gated ion channel Kv2.1 in Escherichia coli using prokaryotic codon, and prepared giant spheroplasts large enough for the patch clamp. Human Kv2.1 currents were successfully recorded from giant spheroplasts with the automated system, and Kv2.1-expressed E. coli spheroplasts could steadily reacted to the dose-response assay with TEA and 4-AP. Collectively, our results indicate for the first time that the bacterial giant spheroplast can be applied for practical pharmaceutical assay using the automated patch clamp.

11ß-Prostaglandin F2α, a bioactive metabolite catalyzed by AKR1C3, stimulates prostaglandin F receptor and induces slug expression in breast cancer.

Prostaglandins are a group of lipid compounds involved in inflammation and cancer. We focused on PGF2α and its stereoisomer 11ß-PGF2α and examined the expression and functions of their cognate receptor (FP receptor) and metabolizing enzymes (AKR1B1 and AKR1C3 respectively) in breast cancer. In immunohistochemical analysis FP receptor status associated with adverse clinical outcome only in the AKR1C3 positive cases. Therefore, we studied FP receptor-mediated functions of 11ß-PGF2α using FP receptor expressed MCF-7 cell line (MCF-FP). 11ß-PGF2α treatment phosphorylated ERK and CREB and induced Slug expression through FP receptor in MCF-FP, and MCF-FP cells demonstrated decreased chemosensitivity compared to parental controls. Finally, the correlation between FP receptor and Slug was also confirmed immunohistochemically in breast cancer cases. Overall these results indicated that the actions of AKR1C3 can produce FP receptor ligands whose activation results in carcinoma cell survival in breast cancer.

Transient in vivo reporter gene assay for ecdysteroid action in the Bombyx mori silk gland.

To analyze the molecular mechanisms underlying hormone-regulated gene expression during molt and metamorphosis, we developed a transient reporter gene assay system using the silkworm anterior silk gland. Reporter plasmids were delivered into dissected anterior silk glands by particle bombardment and bombarded glands transplanted into other larvae, to which hormones were then administered. When the green fluorescent protein gene, coupled with the constitutive cytoplasmic actin gene A3 promoter, was introduced into the anterior silk gland, strong green fluorescence was observed a few days later. Bombarded silk glands transplanted into other larvae showed the same morphological changes as intrinsic glands after 20-hydroxyecdysone (20E) alone or 20E plus juvenile hormone (JH) treatment, indicating that the transplanted gland received hormonal signals properly. When a 20E-responsive reporter construct containing four tandemly repeated pal-1 ecdysone response elements upstream from the luciferase gene was delivered into the gland, an approximately 50-fold increase in luciferase activity was detected 30 h after 20E injection. This induction was comparable to that in an ecdysteroid-responsive Bombyx cell line. This in vivo reporter assay system is thus a rapid, effective tool for analyzing gene expression regulated by 20E and probably by JH.

Effects of density changes in the chest on lung stereotactic radiotherapy.

To experimentally and theoretically evaluate dose distribution during lung stereotactic radiotherapy, we investigated the relative electron densities in lung and tumor tissues using X-ray computed tomography images obtained from 30 patients in three breathing states: free breathing, inspiration breath-hold, and expiration breath-hold. We also calculated dose distribution using Monte Carlo simulation for lung tissue with two relative electron densities. The effect of changes in relative electron density on dose distribution in lung tissue was evaluated using calculated differential and integral dose volume histograms. The relative electron density of lung tissue was 0.22 in free breathing, 0.23 in shallow expiration, and 0.17 in shallow inspiration, and there was a tendency for relative electron density to decrease with age. The relative electron density of tumor tissue was approximately 0.9, with little variation due to differences in breathing state. As the relative electron density of lung tissue decreases, the low-dose region expands and leads to changes in the marginal dose.

Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori.

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.