FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer

Resistance to chemotherapy and metastases are the major causes of breast cancer-related mortality. Moreover, cancer stem cells (CSC) play critical roles in cancer progression and treatment resistance. Previously, it was found that CSC-like cells can be generated by aberrant activation of epithelial–mesenchymal transition (EMT), thereby making anti-EMT strategies a novel therapeutic option for treatment of aggressive breast cancers. Here, we report that the transcription factor FOXC2 induced in response to multiple EMT signaling pathways as well as elevated in stem cell-enriched factions is a critical determinant of mesenchymal and stem cell properties, in cells induced to undergo EMT- and CSC-enriched breast cancer cell lines. More specifically, attenuation of FOXC2 expression using lentiviral short hairpin RNA led to inhibition of the mesenchymal phenotype and associated invasive and stem cell properties, which included reduced mammosphere-forming ability and tumor initiation. Whereas, overexpression of FOXC2 was sufficient to induce CSC properties and spontaneous metastasis in transformed human mammary epithelial cells. Furthermore, a FOXC2-induced gene expression signature was enriched in the claudin-low/basal B breast tumor subtype that contains EMT and CSC features. Having identified PDGFR-β to be regulated by FOXC2, we show that the U.S. Food and Drug Administration-approved PDGFR inhibitor, sunitinib, targets FOXC2-expressing tumor cells leading to reduced CSC and metastatic properties. Thus, FOXC2 or its associated gene expression program may provide an effective target for anti-EMT-based therapies for the treatment of claudin-low/basal B breast tumors or other EMT-/CSC-enriched tumors.

Paclitaxel resistance and multicellular spheroid formation are induced by kallikrein-related peptidase 4 in serous ovarian cancer cells in an ascites mimicking microenvironment

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.

Combined expression of KLK4, KLK5, KLK6, and KLK7 by ovarian cancer cells leads to decreased adhesion and paclitaxel-induced chemoresistance.

OBJECTIVE: Chemoresistance is a critical feature of advanced ovarian cancer with only 30% of patients surviving longer than 5 years. We have previously shown that four kallikrein-related (KLK) peptidases, KLK4, KLK5, KLK6 and KLK7 (KLK4-7), are implicated in peritoneal invasion and tumour growth, but underlying mechanisms were not identified. We also reported that KLK7 overexpression confers chemoresistance to paclitaxel, and cell survival via integrins. In this study, we further explored the functional consequenses of overexpression of all four KLKs (KLK4-7) simultaneously in the ovarian cancer cell line, OV-MZ-6, and its impact on integrin expression and signalling, cell adhesion and survival as contributors to chemoresistance and metastatic progression. METHODS: Quantitative gene and protein expression analyses, confocal microscopy, cell adhesion and chemosensitivity assays were performed. RESULTS: Expression of α5β1/αvβ3 integrins was downregulated upon combined stable KLK4-7 overexpression in OV-MZ-6 cells. Accordingly, the adhesion of these cells to vitronectin and fibronectin, the extracellular matrix binding proteins of α5β1/αvβ3 integrins and two predominant proteins of the peritoneal matrix, was decreased. KLK4-7-transfected cells were more resistant to paclitaxel (10-100 nmol/L: 38-54%), but not to carboplatin, which was associated with decreased apoptotic stimuli. However, the KLK4-7-induced paclitaxel resistance was not blocked by the MEK1/2 inhibitor, U0126. CONCLUSIONS: This study demonstrates that combined KLK4-7 expression by ovarian cancer cells promotes reduced integrin expression with consequently less cell-matrix attachment, and insensitivity to paclitaxel mediated by complex integrin and MAPK independent interactions, indicative of a malignant phenotype and disease progression suggesting a role for these KLKs in this process.

Tenofovir-associated proteinuria

Proteinuria was observed in 27% of 153 patients taking tenofovir for more than 1 year. Concomitant protease inhibitor therapy and cumulative tenofovir exposure were independently associated with proteinuria in this cohort. Proteinuria was reversible in 11 of 12 patients who ceased tenofovir because of proteinuria without altering other medications. Clinicians should be aware that tenofovir can cause reversible proteinuria in patients with HIV.

The ionic products from bredigite bioceramics induced cementogenic differentiation of periodontal ligament cells via activation of the Wnt/β-catenin signalling pathway

Periodontitis results from the destructive inflammatory reaction of the host elicited by a bacterial biofilm adhering to the tooth surface and if left untreated, may lead to the loss of the teeth and the surrounding tissues, including the alveolar bone. Cementum is a specialized calcified tissue covering the tooth root and an essential part of the periodontium which enables the attachment of the periodontal ligament to the root and the surrounding alveolar bone. Periodontal ligament cells (PDLCs) represent a promising cell source for periodontal tissue engineering. Since cementogenesis is the critical event for the regeneration of periodontal tissues, this study examined whether inorganic stimuli derived from bioactive bredigite (Ca7MgSi4O16) bioceramics could stimulate the proliferation and cementogenic differentiation of PDLCs, and further investigated the involvement of the Wnt/β-catenin signalling pathway during this process via analysing gene/protein expression of PDLCs which interacted with bredigite extracts. Our results showed that the ionic products from bredigite powder extracts led to significantly enhanced proliferation and cementogenic differentiation, including mineralization–nodule formation, ALP activity and a series of bone/cementum-related gene/protein expression (ALP, OPN, OCN, BSP, CAP and CEMP1) of PDLCs in a concentration dependent manner. Furthermore, the addition of cardamonin, a Wnt/β-catenin signalling inhibitor, reduced the pro-cementogenesis effect of the bredigite extracts, indicating the involvement of the Wnt/β-catenin signalling pathway in the cementogenesis of PDLCs induced by bredigite extracts. The present study suggests that an entirely inorganic stimulus with a specific composition of bredigite bioceramics possesses the capacity to trigger the activation of the Wnt/β-catenin signalling pathway, leading to stimulated differentiation of PDLCs toward a cementogenic lineage. The results indicate the therapeutic potential of bredigite ceramics in periodontal tissue engineering application.

The N550K/H mutations in FGFR2 confer differential resistance to PD173074, dovitinib and ponatinib ATP-competitive inhibitors

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified fourteen dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2mutant endometrial cancers (EC). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors. Whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2mutant cell lines, 3/7 showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2N550K in JHUEM-2 cells (FGFR2C383R) conferred resistance (~5 fold) to PD173074, providing independent data that FGFR2N550K can be associated with drug resistance. Biochemical in vitro kinase analyses also shows ponatinib is more effective than dovitinib at inhibiting FGFR2N550K. We propose tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

Delivery of dimethyloxallyl glycine in mesoporous bioactive glass scaffolds to improve angiogenesis and osteogenesis of human bone marrow stromal cells

Development of hypoxia-mimicking bone tissue engineering scaffolds is of great importance in stimulating angiogenesis for bone regeneration. Dimethyloxallyl glycine (DMOG) is a cell-permeable, competitive inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH), which can stabilize hypoxia-inducible factor 1α (HIF-1α) expression. The aim of this study was to develop hypoxia-mimicking scaffolds by delivering DMOG in mesoporous bioactive glass (MBG) scaffolds and to investigate whether the delivery of DMOG could induce a hypoxic microenvironment for human bone marrow stromal cells (hBMSC). MBG scaffolds with varied mesoporous structures (e.g. surface area and mesopore volume) were prepared by controlling the contents of mesopore-template agent. The composition, large-pore microstructure and mesoporous properties of MBG scaffolds were characterized. The effect of mesoporous properties on the loading and release of DMOG in MBG scaffolds was investigated. The effects of DMOG delivery on the cell morphology, cell viability, HIF-1α stabilization, vascular endothelial growth factor (VEGF) secretion and bone-related gene expression (alkaline phosphatase, ALP; osteocalcin, OCN; and osteopontin, OPN) of hBMSC in MBG scaffolds were systematically investigated. The results showed that the loading and release of DMOG in MBG scaffolds can be efficiently controlled by regulating their mesoporous properties via the addition of different contents of mesopore-template agent. DMOG delivery in MBG scaffolds had no cytotoxic effect on the viability of hBMSC. DMOG delivery significantly induced HIF-1α stabilization, VEGF secretion and bone-related gene expression of hBMSC in MBG scaffolds in which DMOG counteracted the effect of HIF-PH and stabilized HIF-1α expression under normoxic condition. Furthermore, it was found that MBG scaffolds with slow DMOG release significantly enhanced the expression of bone-related genes more than those with instant DMOG release. The results suggest that the controllable delivery of DMOG in MBG scaffolds can mimic a hypoxic microenvironment, which not only improves the angiogenic capacity of hBMSC, but also enhances their osteogenic differentiation.

Nutritional status of Ugandan women living with HIV/AIDS : anthropometry and body composition assessment

Human immunodeficiency virus (HIV) that leads to acquired immune deficiency syndrome (AIDs) reduces immune function, resulting in opportunistic infections and later death. Use of antiretroviral therapy (ART) increases chances of survival, however, with some concerns regarding fat re-distribution (lipodystrophy) which may encompass subcutaneous fat loss (lipoatrophy) and/or fat accumulation (lipohypertrophy), in the same individual. This problem has been linked to Antiretroviral drugs (ARVs), majorly, in the class of protease inhibitors (PIs), in addition to older age and being female. An additional concern is that the problem exists together with the metabolic syndrome, even when nutritional status/ body composition, and lipodystrophy/metabolic syndrome are unclear in Uganda where the use of ARVs is on the increase. In line with the literature, the overall aim of the study was to assess physical characteristics of HIV-infected patients using a comprehensive anthropometric protocol and to predict body composition based on these measurements and other standardised techniques. The other aim was to establish the existence of lipodystrophy, the metabolic syndrome, andassociated risk factors. Thus, three studies were conducted on 211 (88 ART-naïve) HIV-infected, 15-49 year-old women, using a cross-sectional approach, together with a qualitative study of secondary information on patient HIV and medication status. In addition, face-to-face interviews were used to extract information concerning morphological experiences and life style. The study revealed that participants were on average 34.1±7.65 years old, had lived 4.63±4.78 years with HIV infection and had spent 2.8±1.9 years receiving ARVs. Only 8.1% of participants were receiving PIs and 26% of those receiving ART had ever changed drug regimen, 15.5% of whom changed drugs due to lipodystrophy. Study 1 hypothesised that the mean nutritional status and predicted percent body fat values of study participants was within acceptable ranges; different for participants receiving ARVs and the HIV-infected ART-naïve participants and that percent body fat estimated by anthropometric measures (BMI and skinfold thickness) and the BIA technique was not different from that predicted by the deuterium oxide dilution technique. Using the Body Mass Index (BMI), 7.1% of patients were underweight (<18.5 kg/m2) and 46.4% were overweight/obese (≥25.0 kg/m2). Based on waist circumference (WC), approximately 40% of the cohort was characterized as centrally obese. Moreover, the deuterium dilution technique showed that there was no between-group difference in the total body water (TBW), fat mass (FM) and fat-free mass (FFM). However, the technique was the only approach to predict a between-group difference in percent body fat (p = .045), but, with a very small effect (0.021). Older age (β = 0.430, se = 0.089, p = .000), time spent receiving ARVs (β = 0.972, se = 0.089, p = .006), time with the infection (β = 0.551, se = 0.089, p = .000) and receiving ARVs (β = 2.940, se = 1.441, p = .043) were independently associated with percent body fat. Older age was the greatest single predictor of body fat. Furthermore, BMI gave better information than weight alone could; in that, mean percentage body fat per unit BMI (N = 192) was significantly higher in patients receiving treatment (1.11±0.31) vs. the exposed group (0.99±0.38, p = .025). For the assessment of obesity, percent fat measures did not greatly alter the accuracy of BMI as a measure for classifying individuals into the broad categories of underweight, normal and overweight. Briefly, Study 1 revealed that there were more overweight/obese participants than in the general Ugandan population, the problem was associated with ART status and that BMI broader classification categories were maintained when compared with the gold standard technique. Study 2 hypothesized that the presence of lipodystrophy in participants receiving ARVs was not different from that of HIV-infected ART-naïve participants. Results showed that 112 (53.1%) patients had experienced at least one morphological alteration including lipohypertrophy (7.6%), lipoatrophy (10.9%), and mixed alterations (34.6%). The majority of these subjects (90%) were receiving ARVs; in fact, all patients receiving PIs reported lipodystrophy. Period spent receiving ARVs (t209 = 6.739, p = .000), being on ART (χ2 = 94.482, p = .000), receiving PIs (Fisher’s exact χ2 = 113.591, p = .000), recent T4 count (CD4 counts) (t207 = 3.694, p = .000), time with HIV (t125 = 1.915, p = .045), as well as older age (t209 = 2.013, p = .045) were independently associated with lipodystrophy. Receiving ARVs was the greatest predictor of lipodystrophy (p = .000). In other analysis, aside from skinfolds at the subscapular (p = .004), there were no differences with the rest of the skinfold sites and the circumferences between participants with lipodystrophy and those without the problem. Similarly, there was no difference in Waist: Hip ratio (WHR) (p = .186) and Waist: Height ratio (WHtR) (p = .257) among participants with lipodystrophy and those without the problem. Further examination showed that none of the 4.1% patients receiving stavudine (d4T) did experience lipoatrophy. However, 17.9% of patients receiving EFV, a non-nucleoside reverse transcriptase inhibitor (NNRTI) had lipoatrophy. Study 2 findings showed that presence of lipodystrophy in participants receiving ARVs was in fact far higher than that of HIV-infected ART-naïve participants. A final hypothesis was that the prevalence of the metabolic syndrome in participants receiving ARVs was not different from that of HIV-infected ART-naïve participants. Moreover, data showed that many patients (69.2%) lived with at least one feature of the metabolic syndrome based on International Diabetic Federation (IDF, 2006) definition. However, there was no single anthropometric predictor of components of the syndrome, thus, the best anthropometric predictor varied as the component varied. The metabolic syndrome was diagnosed in 15.2% of the subjects, lower than commonly reported in this population, and was similar between the medicated and the exposed groups (χ 21 = 0.018, p = .893). Moreover, the syndrome was associated with older age (p = .031) and percent body fat (p = .012). In addition, participants with the syndrome were heavier according to BMI (p = .000), larger at the waist (p = .000) and abdomen (p = .000), and were at central obesity risk even when hip circumference (p = .000) and height (p = .000) were accounted for. In spite of those associations, results showed that the period with disease (p = .13), CD4 counts (p = .836), receiving ART (p = .442) or PIs (p = .678) were not associated with the metabolic syndrome. While the prevalence of the syndrome was highest amongst the older, larger and fatter participants, WC was the best predictor of the metabolic syndrome (p = .001). Another novel finding was that participants with the metabolic syndrome had greater arm muscle circumference (AMC) (p = .000) and arm muscle area (AMA) (p = .000), but the former was most influential. Accordingly, the easiest and cheapest indicator to assess risk in this study sample was WC should routine laboratory services not be feasible. In addition, the final study illustrated that the prevalence of the metabolic syndrome in participants receiving ARVs was not different from that of HIV-infected ART-naïve participants.

Matrix metalloproteinase biosensor based on a porous silicon reflector

Matrix metalloproteinases (MMPs) are proteolytic enzymes important to wound healing. In non-healing wounds, it has been suggested that MMP levels become dysfunctional, hence it is of great interest to develop sensors to detect MMP biomarkers. This study presents the development of a label-free optical MMP biosensor based on a functionalised porous silicon (pSi) thin film. The biosensor is fabricated by immobilising a peptidomimetic MMP inhibitor in the porous layer using hydrosilylation followed by amide coupling. The binding of MMP to the immobilised inhibitor translates into a change of effective optical thickness (EOT) over the time. We investigate the effect of surface functionalisation on the stability of pSi surface and evaluate the sensing performance. We successfully demonstrate MMP detection in buffer solution and human wound fluid at physiologically relevant concentrations. This biosensor may find application as a point-of-care device that is prognostic of the healing trajectory of chronic wounds.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Lysine residues of IGF-I are substrates for transglutaminases and modulate downstream IGF-I signalling

Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.

An assessment of MMP and TIMP gene expression in cell lines and stroma : tumour differences in microdissected breast cancer biopsies

To examine matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) mRNA levels in archival breast cancer biopsies, we employed microdissection to separate tumour tissue from the surrounding breast tissue, or stroma and RT-PCR to determine gross qualitative and small quantitative differences in the patterns of expression. In this study, a significant correlation (p < 0.05, by Mann-Whitney U analysis) between TIMP-2 expression and lymph node involvement was identified, while MMP-11 and TIMP-1 expression patterning also significantly (p < 0.05) differed between those tumours showing calcification and those that did not. When compared by Spearmans’ ρ correlation analysis, a significant association (p < 0.05, ρ = 0.404) was identified in the pattern of MMP-2 and MMP-9 gene expression. In this study, the use of microdissection and a systematic strategy of RT-PCR analysis have allowed us to investigate localized MMP and MMP inhibitor expression within breast tumours. We have identified patterns of gene expression that may further reveal aspects of breast carcinogenesis, and a robust method for examining changes in clinically important genes using archival biopsies and across stroma-tumour boundaries.

Modulation of in vitro platelet 5-HT release by species of Erythrina and Cymbopogon

Extracts of Australian plants were screened to detect constituents affecting adenosine di-phosphate (ADP) induced platelet aggregation and [14C]5-hydroxytryptamine (5-HT) release. Extracts of four tested plants including, Eremophila gilesii, Erythrina vespertilio, Cymbopogon ambiguus, and Santalum acuminatum, were found to cause significant inhibition of platelet 5-HT release. Inhibition levels ranged from 56-98%, and was not due to the non-specific effects of protein binding tannins. These extracts, and those we have previously identified as being active, were examined further to determine if they affect epinephrine (EPN), arachidonic acid (A.A) or collagen stimulated platelet aggregation and 5-HT release. Among those extracts investigated, we found that both the methanolic extract of E. vespertilio and the dichloromethane (DCM) extract of C. ambiguus were most potent and caused significant inhibition of platelet activation induced by EPN, A.A and to a lesser extent by collagen. Inhibition of ADP induced platelet 5-HT release by both of these extracts, was dose-dependent, with IC50 values for E. vespertilio and C. ambiguus estimated to be 20.4 microl (1.855 mg/ml) and 8.34 microl (0.758 mg/ml), respectively. Overall, C. ambiguus exhibited most activity and also caused dose-dependent inhibition of A.A induced platelet activation. These results indicate that inhibition may occur specifically at a site within the A.A pathway, and suggest the presence of a cyclo-oxygenase inhibitor. Both E. vespertilio and C. ambiguus are reported to be traditional headache treatments, with the present study providing evidence that they affect 5-HT release.

Current status of pharmacogenomics testing for anti-tumor drug therapies : approaches to non-melanoma skin cancer

Skin cancer is one of the most commonly occurring cancer types, with substantial social, physical, and financial burdens on both individuals and societies. Although the role of UV light in initiating skin cancer development has been well characterized, genetic studies continue to show that predisposing factors can influence an individual's susceptibility to skin cancer and response to treatment. In the future, it is hoped that genetic profiles, comprising a number of genetic markers collectively involved in skin cancer susceptibility and response to treatment or prognosis, will aid in more accurately informing practitioners' choices of treatment. Individualized treatment based on these profiles has the potential to increase the efficacy of treatments, saving both time and money for the patient by avoiding the need for extensive or repeated treatment. Increased treatment responses may in turn prevent recurrence of skin cancers, reducing the burden of this disease on society. Currently existing pharmacogenomic tests, such as those that assess variation in the metabolism of the anticancer drug fluorouracil, have the potential to reduce the toxic effects of anti-tumor drugs used in the treatment of non-melanoma skin cancer (NMSC) by determining individualized appropriate dosage. If the savings generated by reducing adverse events negate the costs of developing these tests, pharmacogenomic testing may increasingly inform personalized NMSC treatment.

GEF-H1-RhoA signaling pathway mediates LPS-induced NF-κB transactivation and IL-8 synthesis in endothelial cells

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the roles of GEF-H1 (Guanine-nucleotide exchange factor-H1)-RhoA signalling in LPS-induced interleukin-8 (IL-8, CXCL8) production in endothelial cells. First, we observed that GEF-H1 expression was upregulated in a dose- and time-dependent manner as consistent with TLR4 (Toll-like receptor 4) expression after LPS stimulation. Afterwards, Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activities, reduced LPS-induced NF-κB phosphorylation. Inhibition of GEF-H1 and RhoA expression reduced LPS-induced NF-κB and p38 phosphorylation. TLR4 knockout blocked LPS-induced activity of RhoA, however, MyD88 knockout did not impair the LPS-induced activity of RhoA. Nevertheless, TLR4 and MyD88 knockout both significantly inhibited transactivation of NF-κB. GEF-H1-RhoA and MyD88 both induced significant changes in NF-κB transactivation and IL-8 synthesis. Co-inhibition of GEF-H1-RhoA and p38 expression produced similar inhibitory effects on LPS-induced NF-κB transactivation and IL-8 synthesis as inhibition of p38 expression alone, thus confirming that activation of p38 was essential for the GEF-H1-RhoA signalling pathway to induce NF-κB transactivation and IL-8 synthesis. Taken together, these results demonstrate that LPS-induced NF-κB activation and IL-8 synthesis in endothelial cells are regulated by the MyD88 pathway and GEF-H1-RhoA pathway.