Mechanistic insights of epithelial protein lost in neoplasm in prostate cancer metastasis.

EPLIN is frequently downregulated or lost in various cancers. The purpose of this study was to evaluate the importance of EPLIN in prostate cancer progression, with particular focus on the mechanistic implications to elucidate EPLIN's tumor suppressive function in cancer. EPLIN expression was evaluated in prostate cancer cell lines and tissues. PC-3 and LNCaP EPLINα overexpression models were generated through transfection with EPLINα sequence and EPLIN knockdown was achieved using shRNA in CA-HPV-10 cells. Functional assays were performed to evaluate cellular characteristics and potential mechanisms were evaluated using a protein microarray, and validated using western blot analysis. EPLIN expression was reduced in clinical prostate cancer sections, including hyperplasia (p ≤ 0.001) and adenocarcinoma (p = 0.005), when compared to normal prostate tissue. EPLINα overexpression reduced cell growth, migration and invasion, and influenced transcript, protein and phosphoprotein expression of paxillin, FAK and Src. EPLIN knockdown increased the invasive and migratory nature of CA-HPV-10 cells and also induced changes to FAK and Src total and/or phospho expression. Functional characterization of cellular migration and invasion in addition to FAK and Src inhibition demonstrated differential effects between control and EPLINα overexpression and EPLIN knockdown cell lines. This study highlights that EPLIN expression in prostate cancer is able to influence several aspects of cancer cell characteristics, including cell growth, migration and invasion. The mechanism of the tumor suppressive action of EPLIN remains to be fully elucidated; and this study proposes a role for EPLIN's ability to regulate the aggressive characteristics of prostate cancer cells partially through regulating FAK/Src signaling.

Safety and feasibility of platelet transfusion through long catheters in the neonatal intensive care unit: an in vitro study.

OBJECTIVE: To assess the safety and feasibility of platelet transfusion through small-bore long lines used in the neonatal intensive care unit (NICU), including double-lumen umbilical venous catheters (UVCs) and 24 G and 28 G peripherally inserted central catheters (PICCs). DESIGN: Prospective in vitro controlled study. SETTING: Blood transfusion service laboratory. METHODS: In vitro platelet transfusions were set up as per NICU practice. Transfusion line pressure was monitored. Post-transfusion swirling, presence of aggregates, pH analysis and automated cell count in vitro activation response by flow cytometry assessing CD62P expression were assessed. MAIN OUTCOME MEASURES: All transfusions completed successfully. The rate of infusion was reduced in 5 of 16 transfusions through 28 G lines due to 'pressure high' alarms. There was no difference in swirling values or transfusion aggregate formation, CD62P expression levels, platelet count, platelet distribution width, mean platelet volume, plateletcrit or platelet to large cell ratio across transfusions post-transfusion. CONCLUSIONS: This study showed that in vitro platelet transfusion performed through 24 G and 28 G neonatal PICC lines and double-lumen UVCs is non-inferior to 24 G short cannulas, using outcome measures of platelet clumping, platelet activation and line occlusion. This suggests that where available these lines can be used if necessary for platelet transfusion.

Mimicking the tumour microenvironment: three different co-culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells.

Interactions in the tumour microenvironment can promote chronic lymphocytic leukaemia (CLL) cell survival, proliferation and drug resistance. A detailed comparison of three co-culture systems designed to mimic the CLL lymph node and vascular microenvironments were performed; two were mouse fibroblast cell lines transfected with human CD40LG or CD31 and the third was a human microvascular endothelial cell line, HMEC-1. All three co-culture systems markedly enhanced CLL cell survival and induced a consistent change in CLL cell phenotype, characterized by increased expression of CD38, CD69, CD44 and ITGA4 (CD49d); this phenotype was absent following co-culture on untransfected mouse fibroblasts. In contrast to HMEC-1 cells, the CD40LG and CD31-expressing fibroblasts also induced ZAP70 expression and marked CLL cell proliferation as evidenced by carboxyfluorescein succinimidyl ester labelling and increased Ki-67 expression. Taken together, our data show that co-culture on different stroma induced a remarkably similar activation phenotype in CLL cells but only the CD40LG and CD31-expressing fibroblasts increased ZAP70 expression and CLL cell proliferation, indicating that ZAP70 may play a critical role in this process. This comparative study reveals a number of striking similarities between the co-culture systems tested but also highlights important differences that should be considered when selecting which system to use for in-vitro investigations.

Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38.

Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. The mean percentage of positive cells expressing CD38 was 87%+/-8.5% and the mean cell viability 74%+/-17%. This high level of transduction of all the CLL cell samples tested demonstrates the utility of this technique which should prove applicable for the introduction and analysis of other genes in these non-dividing cells.

Importance of activated leukocyte cell adhesion molecule (ALCAM) in prostate cancer progression and metastatic dissemination.

Activated Leukocyte Cell Adhesion Molecule (ALCAM) has been linked to the progression of numerous human cancers, where it appears to play a complex role. The current study aims to further assess the importance of ALCAM in prostate cancer and the prognostic potential of serum ALCAM as a biomarker for prostate cancer progression. Here we demonstrate enhanced levels of tissue ALCAM are associated with metastasis. Additionally, elevated serum ALCAM is indicative of progression and poorer patient outlook, and demonstrates comparable prognostic ability to PSA in terms of metastasis and prostate cancer survival. ALCAM suppression enhanced proliferation and invasiveness in PC-3 cells and motility/migration in PC-3 and LNCaP cells. ALCAM suppressed PC-3 cells were generally less responsive to HGF and displayed reduced MET transcript expression. Furthermore a recombinant human ALCAM-Fc chimera was able to inhibit LNCaP cell attachment to HECV and hFOB1.19 cells. Taken together, ALCAM appears to be a promising biomarker for prostate cancer progression, with enhanced serum expression associated with poorer prognosis. Suppression of ALCAM appears to impact cell function and cellular responsiveness to certain micro environmental factors.

Src kinase promotes adhesion-independent activation of FAK and enhances cellular migration in tamoxifen-resistant breast cancer cells.

Src kinase is intimately involved in the control of matrix adhesion and cell migration through its ability to modulate the activity of focal adhesion kinase (FAK). In light of our previous observations that acquisition of tamoxifen resistance in breast cancer cells is accompanied by elevated Src kinase activity, we wish to investigate whether FAK function is also altered in these cells and if this leads to an enhanced migratory phenotype. In in vitro adhesion assays, tamoxifen-resistant (TamR) MCF7 cells had a greater affinity for the matrix proteins fibronectin, laminin, vitronectin and collagen and subsequently demonstrated a much greater migratory capacity across these substrates compared to their weakly-migratory, endocrine-sensitive counterparts. Additionally, elevated levels of activated Src in TamR cells promoted an increase in FAK phosphorylation at Y861 and Y925 and uncoupled FAK activation from an adhesion-dependent process. Inhibition of Src activity using the Src/Abl inhibitor AZD0530 reduced FAK activity, suppressed cell spreading on matrix-coated surfaces and significantly inhibited cell migration. Our data thus suggest that Src kinase plays a central role in the enhanced migratory phenotype that accompanies endocrine resistance through its modulation of FAK signalling and demonstrates the potential use of Src inhibitors as potent suppressors of tumour cell migration.

In vitro significance of SOCS-3 and SOCS-4 and potential mechanistic links to wound healing.

Wound healing and the management of chronic wounds represent a significant burden on the NHS. Members of the suppressor of cytokine signalling (SOCS) family have been implicated in the regulation of a range of cellular processes. The current study aims to explore the importance of SOCS-3 and SOCS-4 in regulating cellular traits associated with wound healing. SOCS-3 over-expression and SOCS-4 knockdown mutant lines were generated and verified using q-PCR and western blotting in human keratinocytes (HaCaT) and endothelial cells (HECV). Over-expression of SOCS-3 resulted in a significantly reduced proliferative rate in HaCaT keratinocytes and also enhanced the tubule formation capacity of HECV cells. SOCS-4 knockdown significantly reduced HaCaT migration and HECV cell tubule formation. Suppression of SOCS-4 influenced the responsiveness of HaCaT and HECV cells to EGF and TGFß and resulted in a dysregulation of phospho-protein expression in HaCaT cells. SOCS-3 and SOCS-4 appear to play regulatory roles in a number of keratinocyte and endothelial cellular traits associated with the wound healing process and may also be able to regulate the responsiveness of these cells to EGF and TGFß. This implies a potential regulatory role in the wound healing process and, thus highlights their potential as novel therapies.

Potential roles of suppressor of cytokine signaling in wound healing.

Wound healing is a dynamic process comprising three overlapping, highly orchestrated stages known as inflammation, proliferation and re-epithelialization, and tissue remodeling. This complex process is regulated by numerous cytokines, with dysregulation of cytokine-induced signaling leading to impaired wound healing. Suppressor of cytokine signaling (SOCS) proteins are a family of eight intracellular proteins which may hold the potential to maintain homeostasis during wound healing through their negative feedback inhibition of cytokine signaling. To date, the roles of SOCS proteins in inflammation, autoimmunity and cancer have been comprehensively illustrated; however, only a limited number of studies focused on their role in wound healing. This review demonstrates the possible links between SOCS proteins and wound healing, and also highlights the potential importance of this family in a variety of other aspects of regenerative medicine.

Tamoxifen resistance in breast cancer cells is accompanied by an enhanced motile and invasive phenotype: inhibition by gefitinib ('Iressa', ZD1839).

Despite an initial response to antihormonal therapies, the development of resistance will occur in a significant number of breast cancer patients. The mechanisms that underlie acquired resistance are not yet clear. Using a previously established in vitro cell model of tamoxifen resistance in MCF7 cells, shown to display autocrine epidermal growth factor receptor (EGFR) signalling, we assessed how resistance might modulate their metastatic phenotype in vitro, as metastatic disease is the single most important factor affecting the mortality of cancer patients. Furthermore, we investigated the effect of the EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib ('Iressa', ZD1839; AstraZeneca), on this behaviour. The acquisition of tamoxifen resistance in MCF7 cells was accompanied by a dramatic and significant increase in their invasive and motile nature. The affinity of these cells for matrix components was also enhanced. Inhibition of EGFR signalling with gefitinib reduced both basal and TGF-alpha-stimulated invasion and motility and reduced cell-matrix adhesion. In conclusion, we demonstrate here that resistance to tamoxifen in breast cancer cells is accompanied by a significant increase in their basal motile and invasive activity, properties associated with increased metastatic potential. Inhibition of EGFR signalling by gefitinib significantly inhibited cell motility and invasion thus suggesting a role for the EGF receptor in the aggressive phenotype of tamoxifen-resistant breast cancer cells.

The influence of leptin on trabecular architecture and marrow adiposity in GH-deficient rats.

The relationship between the degree of GH deficiency and impaired bone integrity is not simple and may be influenced by related endocrine variables. To test the hypothesis that elevated adiposity and hyperleptinaemia are contributory factors, we quantified femoral trabecular organisation in two models of GH deficiency with divergent degrees of adiposity - the moderately GH-deficient/hyperleptinaemic transgenic growth retarded (Tgr) rat and the profoundly GH-deficient/hypoleptinaemic dw/dw rat. Trabecular density (bone volume/total volume) and surface were reduced by 16% in dw/dw males, with a more fragmented trabecular lattice. This impairment was more pronounced in Tgr rats, with trabecular number and density further reduced (by an additional 21%) and relative surface (bone surface/bone volume), trabecular convexity (structural modal index) and fragmentation (pattern factor) increased. To establish whether the presence of obesity/hyperleptinaemia exacerbates bone impairment in GH deficiency, trabecular structure was assessed in dw/dw rats following diet-induced obesity (DIO). DIO had minimal effect on trabecular architecture, the increased concavity of trabecular surfaces being the only observable effect. Similarly, infusion of leptin into the tibial bone marrow cavity had no effect on trabecular organisation or tibial growth in wild-type rats. However, while this procedure also failed to affect trabecular architecture or osteoclast number in dw/dw rats, distal osteoblast surface was increased by 23%, marrow adipocyte number and epiphyseal plate width being reduced (by 40 and 5% respectively), without increasing caspase-3 immunoreactivity. These findings suggest that while leptin may directly inhibit adipocyte differentiation and favour osteoblast production, hyperleptinaemia makes only a minimal contribution to the impairment of bone structure in GH deficiency.

Elevated Src kinase activity attenuates Tamoxifen response in vitro and is associated with poor prognosis clinically.

Activated Src kinase may contribute to the progression and spread of breast cancers and recent in vitro evidence suggests a role for Src in acquired endocrine resistance. The purpose of this study was to investigate whether modulation of Src activity in endocrine-sensitive and endocrine-resistant breast cancer cells directly affected their phenotype and sensitivity to 4-hydroxy Tamoxifen (tamoxifen) and to determine whether Src activity in breast cancer tissue affected patient outcome. Expression of constitutively active Src in ER-positive, endocrine-sensitive MCF7 breast cancer cells resulted in the development of an aggressive phenotype, akin to that previously observed in cell models of Tamoxifen resistance, and, significantly, attenuated their response to tamoxifen. Conversely, expression of dominant negative-Src in tamoxifen-resistant MCF7 cells resensitized them to tamoxifen. An exploratory immunohistochemical study of an archival primary breast tumor series (n = 75) with parallel clinicopathological data and in normal breast tissues (n = 19) revealed higher levels of activated Src in the cytoplasm (p < 0.01) and lower levels of nuclear Src (p < 0.01) in tumor tissue compared with normal tissue. Whereas elevated levels of activated-Src in the cytoplasm of tumors was significantly associated with reduced survival in ER+ patients (p = 0.031), elevated levels of activated Src within the nucleus appeared to associate with an improved hormonal response. Together these data are further suggestive of a role for Src in breast cancer where it may alter response to endocrine therapy.

SRC as a therapeutic target in breast cancer.

Src kinase plays a key role in multiple signalling pathways regulating diverse cell functions from proliferation and survival to invasion and angiogenesis. In breast cancer, both Src protein levels and kinase activity are frequently elevated and its important role in these oncogenic processes make it a potential target for therapeutic intervention. Importantly, recent evidence has revealed a role for Src in mediating anti-hormone action and in acquired endocrine resistance. A number of small molecule inhibitors of Src kinase have been developed with preclinical data demonstrating their effectiveness at suppressing breast cancer growth and invasion in vitro whilst inhibiting disease spread in vivo. Significantly, there appears to be added benefit when these agents are given in combination with anti-oestrogens. These new findings suggest that Src inhibitors might have therapeutic value in breast cancer patients.

Elevated Src activity promotes cellular invasion and motility in tamoxifen resistant breast cancer cells.

Src kinase plays a central role in growth factor signalling, regulating a diverse array of cellular functions including proliferation, migration and invasion. Recent studies have demonstrated that Src activity is frequently elevated in human tumours and correlates with disease stage. We have previously demonstrated that, upon acquisition of tamoxifen resistance, MCF7 cells display increased epidermal growth factor receptor (EGFR) activation and a more aggressive phenotype in vitro. Since tumours exhibiting elevated EGFR signalling may possess elevated levels of Src activity, we wished to investigate the role of Src in our MCF7 model of endocrine resistance. Src kinase activity was significantly elevated in tamoxifen-resistant (TamR) cells in comparison to wild type MCF7 cells. This increase was not due to elevated Src protein or gene expression. Treatment of TamR cells with the novel Src inhibitor, AZD0530, significantly reduced the amount of activated Src detectable in both cell types whilst having no effect on total Src levels. AZD0530 significantly suppressed the motile and invasive nature of TamR cells in vitro, reduced basal levels of activated focal adhesion kinase (FAK) and paxillin and promoted elongation of focal adhesions. Furthermore, the use of this compound in conjunction with the EGFR inhibitor, gefitinib, was markedly additive towards inhibition of TamR cell motility and invasion. These observations suggest that Src plays a pivotal role in mediating the motile and invasive phenotype observed in endocrine-resistant breast cancer cells. The use of Src inhibitors in conjunction with EGFR inhibitors such as gefitinib may provide an effective method with which to prevent cancer progression and metastasis.

Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation.

We have previously demonstrated that, following acquisition of endocrine resistance, breast cancer cells display an altered growth rate together with increased aggressive behaviour in vitro. Since dysfunctional cell-cell adhesive interactions can promote an aggressive phenotype, we investigated the integrity of this protein complex in our breast cancer model of tamoxifen resistance. In culture, tamoxifen-resistant MCF7 (TamR) cells grew as loosely packed colonies with loss of cell-cell junctions and demonstrated altered morphology characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT). Neutralising E-cadherin function promoted the invasion and inhibited the aggregation of endocrine-sensitive MCF7 cells, whilst having little effect on the behaviour of TamR cells. Additionally, TamR cells had increased levels of tyrosine-phosphorylated beta-catenin, whilst serine/threonine-phosphorylated beta-catenin was decreased. These cells also displayed loss of association between beta-catenin and E-cadherin, increased cytoplasmic and nuclear beta-catenin and elevated transcription of beta-catenin target genes known to be involved in tumour progression and EMT. Inhibition of EGFR kinase activity in TamR cells reduced beta-catenin tyrosine phosphorylation, increased beta-catenin-E-cadherin association and promoted cell-cell adhesion. In such treated cells, the association of beta-catenin with Lef-1 and the transcription of c-myc, cyclin-D1, CD44 and COX-2 were also reduced. These results suggest that homotypic adhesion in tamoxifen-resistant breast cancer cells is dysfunctional due to EGFR-driven modulation of the phosphorylation status of beta-catenin and may contribute to an enhanced aggressive phenotype and transition towards a mesenchymal phenotype in vitro.

Phytoestrogen concentrations in serum from Japanese men and women over forty years of age.

Asian individuals have much lower incidences of prostate and breast cancer than populations from Western developed countries. They also consume a lower fat, higher fiber diet, with a large intake of phytoestrogens. These phytoestrogens may protect against hormone-dependent cancers and other diseases. Our study used established gas chromatography-mass spectrometry (GC-MS) methodologies to measure the concentrations of four phytoestrogens (daidzein, genistein, equol and enterolactone) in serum samples obtained from Japanese men (n = 102) and women (n = 125) > 40 y old. The results were compared with those obtained with samples from the UK. The Japanese men and women had higher (P < 0.001) concentrations of circulating daidzein, genistein and equol than individuals from the UK. The mean concentration of genistein in Japanese men, for example, was 492.7 nmol/L, compared with 33.2 nmol/L in men from the UK. The two populations, however, had similar serum concentrations of enterolactone. Furthermore, 58% of the Japanese men and 38% of the Japanese women had equol concentrations > 20 nmol/L, compared with none of the UK men and 2.2% of the UK women. These results support previously published GC-MS results from studies with low numbers of samples.