Carotenoid supplementation and GnRH challenges influence female endocrine physiology, immune function, and egg-yolk characteristics in Japanese quail (Coturnix japonica)

Androgens and carotenoids circulating in plasma affect the physiology and behavior of vertebrates. Much is known about control mechanisms and functions of each of these substances, yet their interactive effects are not well understood. Here we examine possible additive, multiplicative, and interactive effects of testosterone and carotenoids on female endocrine physiology, immunocompetence, and investment in eggs by simultaneously manipulating levels of testosterone [via gonadotropin releasing hormone (GnRH) challenges] and carotenoids (via diet supplementation) in captive female Japanese quail (Coturnix japonica). Females were randomly assigned to one of four treatments: carotenoid supplementation, GnRH challenge, GnRH challenge?+?carotenoid supplementation, or control. Carotenoid supplementation significantly increased circulating plasma carotenoid levels and acquired immune system performance, but not innate immunity. GnRH challenges elevated circulating testosterone and carotenoid levels, and induced immunosuppression in females. However, females in the GnRH challenge?+?carotenoid supplementation treatment had higher cell-mediated immune responses than control females and similar responses to those of carotenoid-supplemented females. Hence, availability of carotenoids in female quail seemed to counteract immunosuppressive effects of GnRH challenges. Our results provide further evidence for synergistic effects of carotenoids and testosterone on endocrine physiology and immune function in female birds. Elevated plasma testosterone or carotenoids levels resulted in increased deposition of those compounds to eggs, respectively. Furthermore, because we found that concentrations of testosterone and carotenoids in yolks were correlated within each treatment group, differential deposition of hormones and carotenoids in eggs may not only respond to surrounding social and environmental conditions, but also to other components of the egg.     

Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

Polysaccharide peptides from Coriolus versicolor competitively inhibit model cytochrome P450 enzyme probe substrates metabolism in human liver microsomes

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy or health supplement in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited rat CYP2C11-mediated tolbutamide 4-hydroxylation and in human CYP2C9. In this study, the effects of the water extractable fraction of PSP on the metabolism of model CYP1A2, CYP2D6, CYP2E1 and CYP3A4 probe substrates were investigated in pooled human liver microsomes. PSP (1.25-20μM) dose-dependently decreased CYP1A2-mediated metabolism of phenacetin to paracetamol (IC(50) 19.7μM) and CYP3A4-mediated metabolism of testosterone to 6β-hydroxytestosterone (IC(20) 7.06μM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (K(i)=18.4μM). Inhibition of testosterone to 6β-hydroxytestosterone was also competitive and concentration-dependent (K(i)=31.8μM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC(20) values at 15.6μM and 11.9μM, respectively. This study demonstrated that PSP competitively inhibited the CYP1A2- and CYP3A4-mediated metabolism of model probe substrates in human liver microsomes in vitro. The relatively high K(i) values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb-drug interaction related to these CYP isoforms.     

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC(50)=1.06μM) and CYP2C9 (IC(50)=3.89μM), minimal inhibition on CYP3A4 (IC(20)=11.94μM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12μM, 2.00μM and 95.03μM, respectively. HM-1 competitively inhibited testosterone 6β-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.     

Small molecule inhibitors of the HPV16-E6 interaction with caspase 8

High-risk strains of human papillomaviruses (HPVs) cause nearly all cases of cervical cancer as well as a growing number of head and neck cancers. The oncogenicity of these viruses can be attributed to the activities of their two primary oncoproteins, E6 and E7. The E6 protein has among its functions the ability to prevent apoptosis of infected cells through its binding to FADD and caspase 8. A small molecule library was screened for candidates that could inhibit E6 binding to FADD and caspase 8. Flavonols were found to possess this activity with the rank order of myricetin > morin > quercetin > kaempferol = galangin ? (apigenin, 7-hydroxyflavonol, rhamnetin, isorhamnetin, geraldol, datiscetin, fisetin, 6-hydroxyflavonol). Counter screening, where the ability of these chosen flavonols to inhibit caspase 8 binding to itself was assessed, demonstrated that myricetin, morin and quercetin inhibited GST-E6 and His-caspase 8 binding in a specific manner. The structure–activity relationships suggested by these data are unique and do not match prior reports on flavonols in the literature for a variety of anticancer assays.     

BRCA1-IRIS overexpression promotes formation of aggressive breast cancers

Introduction

Women with HER2⁺ or triple negative/basal-like (TN/BL) breast cancers succumb to their cancer rapidly due, in part to acquired Herceptin resistance and lack of TN/BL-targeted therapies. BRCA1-IRIS is a recently discovered, 1399 residue, BRCA1 locus alternative product, which while sharing 1365 residues with the full-length product of this tumor suppressor gene, BRCA1/p220, it has oncoprotein-like properties. Here, we examine whether BRCA1-IRIS is a valuable treatment target for HER2⁺ and/or TN/BL tumors.

Methodology/Principal Findings

Immunohistochemical staining of large cohort of human breast tumor samples using new monoclonal anti-BRCA1-IRIS antibody, followed by correlation of BRCA1-IRIS expression with that of AKT1, AKT2, p-AKT, survivin and BRCA1/p220, tumor status and age at diagnosis. Generation of subcutaneous tumors in SCID mice using human mammary epithelial (HME) cells overexpressing TERT/LT/BRCA1-IRIS, followed by comparing AKT, survivin, and BRCA1/p220 expression, tumor status and aggressiveness in these tumors to that in tumors developed using TERT/LT/Ras^V12-overexpressing HME cells. Induction of primary and invasive rat mammary tumors using the carcinogen N-methyl-N-nitrosourea (NMU), followed by analysis of rat BRCA1-IRIS and ERα mRNA levels in these tumors.High BRCA1-IRIS expression was detected in the majority of human breast tumors analyzed, which was positively correlated with that of AKT1-, AKT2-, p-AKT-, survivin, but negatively with BRCA1/p220 expression. BRCA1-IRIS-positivity induced high-grade, early onset and metastatic HER2⁺ or TN/BL tumors. TERT/LT/BRCA1-IRIS overexpressing HME cells formed invasive subcutaneous tumors that express high AKT1, AKT2, p-AKT and vimentin, but no CK19, p63 or BRCA1/p220. NMU-induced primary and invasive rat breast cancers expressed high levels of rat BRCA1-IRIS mRNA but low levels of rat ERα mRNA.

Conclusion/Significance

BRCA1-IRIS overexpression triggers aggressive breast tumor formation, especially in patients with HER2⁺ or TN/BL subtypes. We propose that BRCA1-IRIS inhibition may be pursued as a novel therapeutic option to treat these aggressive breast tumor subtypes.     

Proteomic identification of predictive biomarkers of resistance to neoadjuvant chemotherapy in luminal breast cancer: a possible role for 14-3-3 theta/tau and tBID?

Introduction

Chemotherapy resistance is a major obstacle in effective neoadjuvant treatment for estrogen receptor-positive breast cancer. The ability to predict tumour response would allow chemotherapy administration to be directed towards only those patients who would benefit, thus maximising treatment efficiency. We aimed to identify putative protein biomarkers associated with chemotherapy resistance, using fresh tumour samples with antibody microarray analysis and then to perform pilot clinical validation experiments.

Materials and methods

Chemotherapy resistant and chemotherapy sensitive tumour samples were collected from breast cancer patients who had received anthracycline based neoadjuvant therapy consisting of epirubicin with cyclophosphamide followed by docetaxel. A total of 5 comparative proteomics experiments were performed using invasive ductal carcinomas which demonstrated estrogen receptor positivity (luminal subtype). Protein expression was compared between chemotherapy resistant and chemotherapy sensitive tumour samples using the Panorama XPRESS Profiler725 antibody microarray containing 725 antibodies from a wide variety of cell signalling and apoptosis pathways. A pilot series of archival samples was used for clinical validation of putative predictive biomarkers.

Results

AbMA analysis revealed 38 differentially expressed proteins which demonstrated at least 1.8 fold difference in expression in chemotherapy resistant tumours and 7 of these proteins (Zyxin, 14-3-3 theta/tau, tBID, Pinin, Bcl-xL, RIP and MyD88) were found in at least 2 experiments. Clinical validation in a pilot series of archival samples revealed 14-3-3 theta/tau and tBID to be significantly associated with chemotherapy resistance.

Conclusions

For the first time, antibody microarrays have been used to identify proteins associated with chemotherapy resistance using fresh breast cancer tissue. We propose a potential role for 14-3-3 theta/tau and tBID as predictive biomarkers of neoadjuvant chemotherapy resistance in breast cancer. Further validation in a larger sample series is now required.     

Pilot and feasibility study: comparative proteomic analysis by 2-DE MALDI TOF/TOF MS reveals 14-3-3 proteins as putative biomarkers of response to neoadjuvant chemotherapy in ER-positive breast cancer

Neoadjuvant chemotherapy is used to treat oestrogen receptor-positive breast cancer however chemo-resistance is a major obstacle in this molecular subtype. The ability to predict tumour response would allow chemotherapy administration to be directed towards patients who would most benefit, thus maximising treatment efficacy. We aimed to identify protein biomarkers associated with response to neoadjuvant chemotherapy, in a pilot study using comparative 2-DE MALDI TOF/TOF MS proteomic analysis of breast tumour samples. A total of 3 comparative proteomic experiments were performed, comparing protein expression between chemotherapy-sensitive and chemotherapy-resistant oestrogen receptor-positive invasive ductal carcinoma tissue samples. This identified a list of 132 unique proteins that were significantly differentially expressed (≥ 2 fold) in chemotherapy resistant samples, 57 of which were identified in at least two experiments. Ingenuity? Pathway Analysis was used to map the 57 DEPs onto canonical signalling pathways. We implicate several isoforms of 14-3-3 family proteins (theta/tau, gamma, epsilon, beta/alpha and zeta/delta), which have previously been associated with chemotherapy resistance in breast cancer. Extensive clinical validation is now required to fully assess the role of these proteins as putative markers of chemotherapy response in luminal breast cancer subtypes.     

In Vitro and In Vivo Isolation and Characterization of Duvenhage Virus

A fatal human case of Duvenhage virus (DUVV) infection in a Dutch traveller who had returned from Kenya was reported in 2007. She exhibited classical symptoms of rabies encephalitis with distinct pathological findings. In the present study we describe the isolation and characterization of DUVV in vitro and its passage in BALB/c mice. The virus proved to be neuroinvasive in both juvenile and adult mice, resulting in about 50% lethality upon peripheral infection. Clinical signs in infected mice were those of classical rabies. However, the distribution of viral antigen expression in the brain differed from that of classical rabies virus infection and neither inclusion bodies nor neuronal necrosis were observed. This is the first study to describe the in vitro and in vivo isolation and characterization of DUVV.