Neoadjuvant Endocrine Therapy Versus Neoadjuvant Chemotherapy in Node-Positive Invasive Lobular Carcinoma.

BACKGROUND: Neoadjuvant chemotherapy (NACT) is often recommended for patients with node-positive invasive lobular carcinoma (ILC) despite unclear benefit in this largely hormone receptor-positive (HR+) group. We sought to compare overall survival (OS) between patients with node-positive ILC who received neoadjuvant endocrine therapy (NET) and those who received NACT. METHODS: Women with cT1-4c, cN1-3 HR+ ILC in the National Cancer Data Base (2004-2014) who underwent surgery following neoadjuvant therapy were identified. Kaplan-Meier curves and Cox proportional hazards modeling were used to estimate unadjusted and adjusted overall survival (OS), respectively. RESULTS: Of the 5942 patients in the cohort, 855 received NET and 5087 received NACT. NET recipients were older (70 vs. 54 years) and had more comorbidities (Charlson-Deyo score ≥ 1: 21.1% vs. 11.5%), lower cT classification (cT3-4: 44.2% vs. 51.0%), lower rates of mastectomy (72.5% vs. 82.2%), lower rates of pathologic complete response (0% vs. 2.5%), and lower rates of postlumpectomy (73.2% vs. 91.0%) and postmastectomy (60.0% vs. 80.8%) radiation versus NACT recipients (all p < 0.001). NACT recipients had higher unadjusted 10-year OS versus NET recipients (57.9% vs. 36.0%), but after adjustment, there was no significant difference in OS between the two groups (p = 0.10). CONCLUSIONS: Patients with node-positive ILC who received NET presented with smaller tumors, older age, and greater burden of comorbidities versus NACT recipients but had similar adjusted OS. While there is evidence from clinical trials supporting efficacy of NET in HR+ breast cancer, our findings suggest the need for further, histology-specific investigation regarding the optimal inclusion and sequence of endocrine therapy and chemotherapy in ILC.

Long-term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets.

Pig islets are an alternative source for islet transplantation to treat type 1 diabetes (T1D), but reproducible curative potential in the pig-to-nonhuman primate (NHP) model has not been demonstrated. Here, we report that pig islet grafts survived and maintained normoglycemia for >6 months in four of five consecutive immunosuppressed NHPs. Pig islets were isolated from designated pathogen-free (DPF) miniature pigs and infused intraportally into streptozotocin-induced diabetic rhesus monkeys under pretreatment with cobra venom factor (CVF), anti-thymocyte globulin (ATG) induction and maintenance with anti-CD154 monoclonal antibody and low-dose sirolimus. Ex vivo expanded autologous regulatory T cells were adoptively transferred in three recipients. Blood glucose levels were promptly normalized in all five monkeys and normoglycemia (90-110 mg/dL) was maintained for >6 months in four cases, the longest currently up to 603 days. Intravenous glucose tolerance tests during the follow-up period showed excellent glucose disposal capacity and porcine C-peptide responses. Adoptive transfer of autologous regulatory T cells was likely to be associated with more stable and durable normoglycemia. Importantly, the recipients showed no serious adverse effects. Taken together, our results confirm the clinical feasibility of pig islet transplantation to treat T1D patients without the need for excessive immunosuppressive therapy.

Budding yeast Cdc20: a target of the spindle checkpoint.

The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.

The regulation of Cdc20 proteolysis reveals a role for APC components Cdc23 and Cdc27 during S phase and early mitosis.

BACKGROUND: In eukaryotic cells, a specialized proteolysis machinery that targets proteins containing destruction-box sequences for degradation and that uses a ubiquitin ligase known as the anaphase-promoting complex/cyclosome (APC) plays a key role in the regulation of mitosis. APC-dependent proteolysis triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Recently, two highly conserved WD40-repeat proteins, Cdc20 and Cdh1/Hct1, have been identified as substrate-specific regulators for APC-dependent proteolysis in the budding yeast Saccharomyces cerevisiae. Here, we have investigated the cell cycle regulation of Cdc20 and Cdh1/Hct1. RESULTS: Whereas the levels CDH1/HCT1 RNA and Cdh1/Hct1 protein are constant throughout the cell cycle, CDC20 RNA and Cdc20 protein are present only during late S phase and mitosis and Cdc20 protein is unstable throughout the entire cell cycle. The instability of Cdc20 depends on CDC23 and CDC27, which encode components of the APC. During the G1 phase, a destruction box within Cdc20 mediates its instability, but during S phase and mitosis, although Cdc20 destruction is still dependent on CDC23 and CDC27, it does not depend on the Cdc20 destruction box. CONCLUSIONS: There are remarkable differences in the regulation of Cdc20 and Cdh1/Hct1. Furthermore, the APC activator Cdc20 is itself a substrate of the Cdc27 have a role in the degradation of Cdc20 during S Phase and early mitosis that is not mediated by its destruction box.

Activation of the endogenous p53 growth inhibitory pathway in HeLa cervical carcinoma cells by expression of the bovine papillomavirus E2 gene.

We previously showed that expression of the bovine papillomavirus (BPV) E2 gene results in a dramatic inhibition of the proliferation of several human cervical carcinoma cell lines, including HeLa cells which contain human papillomavirus (HPV) type 18 DNA. We have assessed the status of endogenous G1 cell cycle regulatory proteins, including the tumor suppressor proteins, p53 and p105Rb, in order to investigate growth regulatory pathways in HeLa cells following E2 expression. The p53 tumor suppressor protein is stabilized following the introduction of the E2 gene into HeLa cells. This results in the induction of the p53-responsive gene encoding the cyclin dependent kinase (cdk) inhibitor, p21/WAF1, complex formation between p21/WAF1 and cdk2 and reduction of in vitro cdk2/cyclin E kinase activity. The reduced cdk kinase activity is accompanied by the accumulation of the growth inhibitory hypophosphorylated form of the tumor suppressor protein, p105Rb. The level of the p105Rb-regulated transcription factor, E2F1, is reduced, as is transcription of a variety of E2F1-regulated genes, including B-myb. Thus, the p53 growth inhibitory pathway has evidently not accumulated mutations in HeLa cells but rather appears intact. However, this pathway remains dormant, until it is mobilized by appropriate manipulations, such as the expression of the BPV E2 protein.

Identification of a variant estrogen receptor lacking exon 4 and its coexpression with wild-type estrogen receptor in ovarian carcinomas.

By means of reverse transcription-PCR we have identified an alternatively spliced mRNA coding for a variant estrogen receptor (ER) that lacks exon 4 (ERDelta4) and is coexpressed with the wild-type ER mRNA in ovarian carcinomas. Furthermore, Western blot analysis revealed the expression of the ERDelta4 protein in normal as well as neoplastic ovarian tissues along with the wild-type ER, although the relative amounts of the wild-type ER and ERDelta4 proteins varied. The trans-activational properties of this variant were studied in ER-negative COS1 cell lines by cotransfection of the ERDelta4 expression vector and a reporter gene containing the estrogen response element. The ERDelta4 protein was not able to activate transcription of a reporter gene. However, it inhibited estrogen-dependent transcriptional activation in a dominant negative fashion when it was cotransfected with the wild-type ER and reporter plasmid. Because it has been shown that ERDelta4 is not able to bind to its response element, the observed inhibitory effect probably occurs through protein-protein interactions. Although several variants of the ER have been described from cancerous cells, none has been identified in ovarian tissues, and ERDelta4 is the only isoform detected in normal tissues. These results may have implications for understanding the physiological role of ERDelta4 in normal cells, because it may affect the function of the wild-type ER, depending on the level of the variant ER protein relative to that of the wild-type ER.

Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration.

Tumors are stiff and data suggest that the extracellular matrix stiffening that correlates with experimental mammary malignancy drives tumor invasion and metastasis. Nevertheless, the relationship between tissue and extracellular matrix stiffness and human breast cancer progression and aggression remains unclear. We undertook a biophysical and biochemical assessment of stromal-epithelial interactions in noninvasive, invasive and normal adjacent human breast tissue and in breast cancers of increasingly aggressive subtype. Our analysis revealed that human breast cancer transformation is accompanied by an incremental increase in collagen deposition and a progressive linearization and thickening of interstitial collagen. The linearization of collagen was visualized as an overall increase in tissue birefringence and was most striking at the invasive front of the tumor where the stiffness of the stroma and cellular mechanosignaling were the highest. Amongst breast cancer subtypes we found that the stroma at the invasive region of the more aggressive Basal-like and Her2 tumor subtypes was the most heterogeneous and the stiffest when compared to the less aggressive luminal A and B subtypes. Intriguingly, we quantified the greatest number of infiltrating macrophages and the highest level of TGF beta signaling within the cells at the invasive front. We also established that stroma stiffness and the level of cellular TGF beta signaling positively correlated with each other and with the number of infiltrating tumor-activated macrophages, which was highest in the more aggressive tumor subtypes. These findings indicate that human breast cancer progression and aggression, collagen linearization and stromal stiffening are linked and implicate tissue inflammation and TGF beta.

Regulation of c-fos gene transcription and myeloid cell differentiation by acute myeloid leukemia 1 and acute myeloid leukemia-MTG8, a chimeric leukemogenic derivative of acute myeloid leukemia 1.

Both acute myeloid leukemia 1 and c-Fos are regulatory factors of hematopoietic cell differentiation. We identified that the c-fos promoter contains an acute myeloid leukemia 1 binding site at nucleotide positions -6-+14. c-fos promoter activity was induced by transient overexpression of acute myeloid leukemia 1 in Jurkat T-cells, but not by that of the short form of acute myeloid leukemia 1-MTG8, a chimeric acute myeloid leukemia 1 protein. In 32Dcl3 myeloid cells, stable overexpression of acute myeloid leukemia 1-MTG8 blocked the c-fos gene transcription and cell differentiation, but that of acute myeloid leukemia did not. These data suggest that acute myeloid leukemia 1 and acute myeloid leukemia 1-MTG8 reciprocally regulate the myeloid cell differentiation, possibly by the way of regulating c-fos gene transcription.

Intraperitoneal gene therapy with adenoviral-mediated p53 tumor suppressor gene for ovarian cancer model in nude mouse.

In an effort to develop a method for better local control of advanced ovarian cancers, we have established a peritoneal tumor model of ovarian cancer in the nude mouse and applied intraperitoneal gene therapy with the recombinant adenoviral-mediated wild-type p53 tumor suppressor gene (Avp53). The results indicate that: (a) the recombinant adenoviral vector system effectively infected the tumor and normal cells in the peritoneal cavity; and (b) Avp53 treatment effectively suppressed the growth of peritoneal tumors and prolonged the survival of the treated group, especially when the tumor burden was less. These results suggest that intraperitoneal gene therapy using Avp53 is potentially useful as an adjuvant therapeutic modality in human ovarian cancer.

Reactivity of antibodies against 10-amino acid residue and pro-domain of stromelysin-3.

Stromelysin-3 (ST3) has two highly conserved domains in the pro-domain. In particular, an unusual 10-amino acid residue sandwiched between the pro-domain and the catalytic domain of ST3 exists in ST3 but not in other matrix metalloproteinases (MMPs). To specifically detect ST3 expression in human tumors, we have made two kinds of ST3-specific polyclonal antibodies. One was raised against the synthetic 10-amino acid residue (88GLSARNRQKR97) specific to ST3, and the other against recombinant ST3 pro-domain (62APATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKR97) containing the decapeptide and PRCGVPD sequence obtained by expression in Escherichia coli. Two protein species, 59 kDa and 45 kDa which were consistent with those expected for pro-ST3 and the mature form of ST3, were specifically detected in 100-fold concentrated conditioned media of fetal lung fibroblast by Western blot analysis. Immunohistochemical staining indicated that in infiltrating ductal breast carcinoma and squamous cell carcinoma of the uterine cervix, reactivity of those antibodies was found not only in fibroblastic cells surrounding cancer cells but also in neoplastic cells. However, reactivity of two ST3 antibodies was inhibited by excess of the synthetic peptide (10-amino acid residue) not only in fibroblastic cells but also in neoplastic cells. These findings suggest that antibodies against the ST3 specific region may cross react with the recently known membrane type-metalloproteinase (MT-MMP), which have RXKR sequences between the pro- and catalytic domain.

Genomic alterations in tubular breast carcinomas.

Tubular carcinoma of the breast is a well-differentiated variant of invasive ductal carcinoma and has been shown to have an exceptionally favorable prognosis, as manifested by a low incidence of lymph node metastases and an excellent overall survival. It is unknown whether this subtype represents an early step along the continuum of development to a more aggressive, poorly differentiated ductal cancer, or whether these cancers are destined to remain well differentiated with limited metastatic potential. We undertook an analysis of 18 pure tubular carcinomas of the breast using comparative genomic hybridization to evaluate the chromosomal changes in these tumors. An average of 3.6 chromosomal alterations of the genome were identified per case. The most frequent change involved loss of 16q (in 78% of tumors) and gain of 1q (in 50% of tumors). All but one case with 1q gain also exhibited a concomitant 16q loss. Other frequent changes involved 16p gain in 7 of 18 cases (39%) and distal 8p loss in 5 of 18 cases (28%). Comparison with known genomic alterations in a mixed group of invasive cancers shows tubular cancer to have fewer overall chromosomal changes per tumor (P <.01), higher frequency of 16q loss (P <.001), and lower frequency of 17p loss (P =.007). These results strongly suggest that tubular carcinomas are a genetically distinct group of breast cancers.

Antiviral effects of 28-deacetylsendanin on herpes simplex virus-1 replication.

The compound purified from the fruit of Melia azedarach exerted an antiviral effect on herpes simplex virus-1 (HSV-1) in Vero cells. It was identified as 28-deacetylsendanin (28-DAS). The 50% inhibitory concentration (IC50) of 28-DAS was 1.46 microg/ml without cytotoxicity at 400 microg/ml on Vero cells. Electron microscopy showed that low electron-dense cores of newly synthesized nucleocapsids remained in swollen nuclei and no extracellular virus particles were observed at 15 h p.i. Consistent with this result, it was confirmed by a plaque assay that few infectious progeny viruses were released from the 28-DAS-treated virus-infected cells at 24 h p.i. Intracellular viruses in 28-DAS-treated virus-infected cells were 23% of untreated and infected cells. The synthesis of thymidine kinase (TK) was reduced by 28-DAS at early stage. In conclusion, 28-DAS inhibited the replication of HSV-1, reduced the synthesis of HSV-1 TK, and led to the formation of defective nucleocapsids.

Epitope analysis of a sperm acrosomal antigen defined by HSA-5 monoclonal antibody.

Among the monoclonal antibodies recommended by the WHO Sperm Antigen Workshop for immunocontraceptive vaccine development, HSA-5 showed a high degree of sperm specificity and significantly inhibited in vitro fertilization in both humans and mice. Using a Western blot assay, HSA-5 was found to recognize a sperm antigen designated as HSAg-5 (human) or MSAg-5 (mouse) which ranged in molecular weight from 18 to 100 kDa. This monoclonal antibody was used as the probe for the immunoscreening of mouse testis cDNA libraries constructed in the lambda gt-11 expression vector. One of the positive cDNA clones was shown to have a cDNA insert of approximately 1 kb and to encode a recombinant fusion protein containing 77 amino acid residues in the C-terminal region of MSAg-5. This 1 kb cDNA insert was engineered in a pGEX vector to express a recombinant glutathione S-transferase fusion protein (GST-5). Using an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, both anti-GST-5 sera and the monoclonal antibody were shown to react with GST-5. The Northern blot of a mouse testis RNA preparation revealed that the isolated cDNA probe hybridized with a 4.0 kb mRNA. Several oligopeptides were synthesized based on the predicted C-terminal hydrophilic regions of the recombinant fusion protein. Using ELISA and a dot blot assay, peptide regions containing the immunogenic epitopes recognized by HSA-5 monoclonal antibody were identified.

Management of ductal carcinoma in situ.

The dramatic increase in the incidence of ductal carcinoma in situ (DCIS) of the breast has made it imperative for all clinicians to develop a better understanding of this disease. Although this preinvasive form of breast cancer is not life-threatening, treatment options may include mastectomy, breast-conserving surgery, radiotherapy, or tamoxifen. Current treatment modalities may be overly aggressive because many cases of DCIS may not recur or progress to invasive cancer. Until we are better able to identify those patients at low risk for progression, it is unlikely that current treatment will change. The adequate understanding of risk assessment is fundamental to the treatment planning for DCIS, and physicians are encouraged to include patients in the decision-making process.

The antibody response to HPV proteins and the genomic state of HPVs in patients with cervical cancer.

Human papillomavirus (HPV) DNAs are often found to be integrated into the human genome in high-grade cervical intraepithelial neoplasia (CIN) as well as in invasive cervical cancers. Investigation of the relationship between the genomic status of specific HPV genes and their antibody responses to the virus-like particles (VLPs) of HPV-16 L1/L2 proteins and the in vitro translated HPV-16 E6 and E7 proteins may help to illustrate the mechanism of HPV-related cervical carcinogenesis and host immune response. Cervical cancer tissues obtained from 39 patients were studied to evaluate the physical status of HPV genes by Southern blotting, DNA-PCR, and RT-PCR of E2. The antibody response against the HPV-16 L1/L2 VLPs of serum specimens were tested by ELISA and the antibody response against the HPV-16 E6 and E7 proteins were tested by radioimmunoprecipitation assay (RIPA), respectively. Integrated forms of HPV-16 DNA were found in 23 of the 38 patients (60.5%). The HPV-16 positive cervical cancer patients showed a significantly higher prevalence rate (39.5%; 15/38) of antibodies to HPV-16 L1/L2 VLPs than that of the control group (8.7%; 2/28) (P < 0.05). Antibodies to HPV-16 L1/L2 VLPs were more commonly detectable in cervical cancer patients having the episomal form of HPV-16 DNA (pure episomal and mixed forms) (60%; 9/15) than in those who had only the integrated forms of HPV-16 DNA (26.1%; 6/23) (P < 0.05). Antibodies to E6 and E7 proteins were positive in 36.8% (14/38) and 50% (19/38) of the patients with HPV-16 positive cervical cancer, respectively. These were significantly higher than the positive rates for the control group (8.3% and 2.8%) (P < 0.05). The differences between sero-reactivities to E6 and E7 proteins in the patients with episomal forms of HPV-16 DNA and those with integrated forms of HPV-16 DNA were not statistically significant (P > 0.05). Integrated forms of HPV-16 DNA were prevalent in most patients with cervical cancer in Korea. Antibodies to HPV-16 L1/L2 VLPs, in vitro translated HPV-16 E6 and E7 proteins, appeared in a significantly larger proportion of the HPV-associated cervical cancer patients than in the controls. Antibodies to HPV-16 L1/L2 VLPs were more often detected in cervical cancer patients having the episomal form of HPV-16 DNA than in those having only integrated forms of HPV-16 DNA. Antibody responses to HPV-16 E6 and E7 proteins were not influenced by the different viral states.

The effects of the adenovirus-mediated wild-type p53 delivery in human epithelial ovarian cancer cell line in vitro and in vivo.

Hwang ES, Kim J, Kim JS, Kao C, Ko S-C, Chung L, Lee J-H. The effects of the adenovirus-mediated wild-type p53 delivery in human epithelial ovarian cancer cell lines in vitro and in vivo. Int J Gynecol Cancer 1998; 8: 27-36. The effect of p53 overexpression on the proliferation of various ovarian cancer cell lines was tested by using an adenovirus vector, Avp53, that expresses wild-type human p53. Cell lines SKOV3, 2774, and OVCAR3, which bear mutations in the endogenous p53 gene, were all affected by Avp53 treatment, undergoing growth suppression and apoptosis at a dose that had little effect on the growth of normal fibroblasts. In these cells, p21WAF1/CIP1 was readily induced and the hypophosphorylated pRb protein accumulated by the treatment of Avp53, suggesting that the growth inhibitory pathway can be activated in these cells by the expression of wild-type p53. However, in PA-1 cell line which endogenously expresses wild-type p53, p21WAF1/CIP1 was not induced by p53 transduction, although p53 was found transcriptionally active. These results indicate that the tested ovarian cancer cell lines bear defects either in p53 itself or in the responsiveness to p53. The cytocidal effect of Avp53 was also examined in vivo against tumors developed in the peritoneal cavity of nude mice. Avp53 administered intraperitoneally eradicated microscopic and small-sized tumor nodules, demonstrating that the intraperitoneal administration of Avp53 may serve as an effective adjuvant therapy for ovarian cancers.

Ozone and hydrogen peroxyacetic acid treatment to reduce or remove EBDCs and ETU residues in a solution.

Laboratory studies were conducted in a model system to determine the effects of ozone (1 and 3 ppm) and hydrogen peroxyacetic acid (HPA) (5 and 50 ppm) at pH 4.6, 7.0, and 10.7 and at 10 and 21 degrees C on the degradation of mancozeb in solution over a 30 min period. All samples were analyzed for residues by GLC and HPLC. Ozonation and HPA treatment were effective in degrading mancozeb in solution. Rate of mancozeb degradation was dependent on pH, with the fastest rate at pH 7.0. Ethylenethiourea (ETU) residue concentrations in the mancozeb solutions were monitored over 60 min. Under controlled conditions, the ETU residue concentrations increased during the 15 min reaction time and then decreased for all three pH values. At 3 ppm of ozone treatment, no ETU residues were detected at all three pH ranges after 15 min of reaction time. Degradation of ETU by HPA was greatest at pH 4.6, and no ETU residues remained after 5 min at either 5 or 50 ppm. The results showed that ozone and HPA gave excellent degradation of pesticide residues depending on pH and temperature. These experiments indicated the potential for the removal of pesticide residues on fruit and in processed products.

Postharvest treatments for the reduction of mancozeb in fresh apples.

The objective of this study was to determine the effectiveness of chlorine, chlorine dioxide, ozone, and hydrogen peroxyacetic acid (HPA) treatments on the degradation of mancozeb and ethylenethiourea (ETU) in apples. This study was based on model experiments at neutral pH and temperature. Fresh apples were treated with two different levels of mancozeb (1 and 10 microg/mL). Several of the treatments were effective in reducing or removing mancozeb and ETU residues on spiked apples. Mancozeb residues decreased 56-99% with chlorine and 36-87% with chlorine dioxide treatments. ETU was completely degraded by 500 ppm of calcium hypochlorite and 10 ppm of chlorine dioxide at a 1 ppm spike level. However, at a 10 ppm spike level, the effectiveness of ETU degradation was lower than observed at 1 ppm level. Mancozeb residues decreased 56-97% with ozone treatment. At 1 and 3 ppm of ozone, no ETU residue was detected at 1 ppm of spiked mancozeb after both 3 and 30 min. HPA was also effective in degrading the mancozeb residues, with 44-99% reduction depending on treatment time and HPA concentrations. ETU was completely degraded at 500 ppm of HPA after 30 min of reaction time. These treatments indicated good potential for the removal of pesticide residues on fruit and in processed products.

Evaluation of urinary N-acetyl cysteinyl allyl isothiocyanate as a biomarker for intake and bioactivity of Brussels sprouts.

Isothiocyanates (ITC), glucosinolate hydrolysis products from Brussels sprouts (BS) and other cruciferous vegetables, are considered to protect the body from cancer by induction of detoxification enzymes such as quinone reductase (QR). Urinary N-acetyl-cysteine (NAC) conjugates of ITC have been proposed as biomarkers of crucifer intake. Here we asked if dietary intake and induction of detoxification enzymes are dose-related to urinary NAC conjugate appearance. Male F344 rats (4/group) received an AIN 76B-40 diet containing 0, 10 or 20% freeze-dried BS for 6 days. A human subject ingested 500 g BS. Urinary AITC-NAC was identified in human and rat urine. Ten and 20% BS diets caused a 1.4- and 2.3-fold induction of QR in the pancreas, a 1.5- and 2.5-fold induction in liver and a 3.1- and 3.6-fold induction in colonic epithelium, respectively. Liver and pancreatic QR induction was dose-related, whereas induction of QR in colon was less different between the two doses. Excretion of the conjugate was dose-related only on day 1, and unrelated to dose after day 2. These results suggest that urinary NAC-AITC is a qualitative biomarker for ingestion and bioactivity of BS, but that it may not be dose-related when rats are fed continuously for 2 or more days.

Canonical Wnt signalling activates TAZ through PP1A during osteogenic differentiation.

TAZ, a transcriptional modulator, has a key role in cell proliferation, differentiation and stem cell self-renewal. TAZ activity is regulated by several signalling pathways, including Hippo, GPCR and Wnt signalling, but the regulatory mechanisms of TAZ activation are not yet clearly understood. In this report, we show that TAZ is regulated by canonical Wnt signalling during osteogenic differentiation. Wnt3a increases TAZ expression and an inhibitor of GSK3ß, a downstream effector of Wnt signalling, induces TAZ. Wnt3a facilitates the dephosphorylation of TAZ, which stabilises TAZ and prevents it from binding 14-3-3 proteins, thus inducing the nuclear localisation of TAZ. Dephosphorylation of TAZ occurs via PP1A, and depletion of PP1A blocks Wnt3a-induced TAZ stabilisation. Wnt3a-induced TAZ activates osteoblastic differentiation and siRNA-induced TAZ depletion decreases Wnt3a-induced osteoblast differentiation. Taken together, these results show that TAZ mediates Wnt3a-stimulated osteogenic differentiation through PP1A, suggesting that the Wnt signal regulates the Hippo pathway.