Development of acquired resistance to lapatinib may sensitise HER2-positive breast cancer cells to apoptosis induction by obatoclax and TRAIL.

BACKGROUND: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. METHODS: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. RESULTS: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. CONCLUSIONS: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.

Renal function and novel urinary biomarkers in infants with neonatal encephalopathy.

AIM: Perinatal asphyxia is associated with multi-organ injury including acute kidney injury (AKI). New urinary biomarkers may detect more subtle renal injury. METHODS: Urinary biomarkers (albumin, beta-2 microglobulin, cystatin-C, epidermal growth factor, neutrophil gelatinase-associated lipocalin, osteopontin, uromodulin) were serially measured from days 1 to 7 in term infants with perinatal asphyxia and controls and compared to 'Kidney Disease Improving Global Outcome' scoring of renal injury and to encephalopathy grade. RESULTS: A total of 255 urine samples were taken from infants exposed to perinatal asphyxia (n = 82) and term controls (n = 10). Thirty-nine infants underwent therapeutic hypothermia, four died and 30 infants had acute kidney injury. Infants with acute kidney injury had significantly higher levels of urinary albumin (day 2), cystatin-C (days 1, 2, 3 and 7), neutrophil gelatinase-associated lipocalin (days 2, 3 and 7) and osteopontin (days 2, 3 and 7) and lower epidermal growth factor and uromodulin (day 1) compared to those without AKI. Day 2 cystatin-C predicted AKI with an area under receiver operating characteristic curve of 0.89, p < 0.001, cut-off 9.8 × 104  pg/mL. NE grade II/III infants had significantly elevated levels of urinary cystatin-C, neutrophil gelatinase-associated lipocalin and decreased EGF compared to grade 0/I infants. CONCLUSION: Asphyxiated infants who develop acute kidney injury have significantly altered urinary biomarkers postnatally. Validation of neonatal AKI urinary biomarkers in a large prospective study is required. Long-term follow-up of infants post-asphyxial insult for chronic renal injury is advised.

Identification of transcription factors associated with castration-resistance: is the serum responsive factor a potential therapeutic target?

BACKGROUND: Advanced prostate cancer is treated by hormone ablation therapy. However, despite an initial response, the majority of men relapse to develop castration-resistant disease for which there are no effective treatments. We have previously shown that manipulating individual proteins has only minor alterations on the resistant phenotype so we hypothesize that targeting the central transcription factors (TFs) would represent a better therapeutic approach. METHODS: We have undertaken a transcriptomic analysis of gene expression differences between the androgen-dependent LNCaP parental cells and its castration-resistant Abl and Hof sublines, revealing 1,660 genes associated with castration-resistance. Using effective bioinformatic techniques, these transcriptomic data were integrated with TF binding sites resulting in a list of TFs associated with the differential gene expression observed. RESULTS: Following validation of the gene-chip results, the serum response factor (SRF) was chosen for clinical validation and functional analysis due to its recent association with prostate cancer progression. SRF immunoreactivity in prostate tumor samples was shown for the first time to be associated with castration-resistance. SRF inhibition by siRNA and the small molecule inhibitor CCG-1423 resulted in decreased proliferation. CONCLUSION: SRF is a key TF by which resistant cells survive with depleted levels of androgens representing a target for therapeutic manipulation.

Selective modulation of monocyte and neutrophil responses with activated protein C in preterm infants.

BACKGROUND: Inflammation is associated with many disorders of preterm infants including periventricular leukomalacia, chronic lung disease, and necrotizing enterocolitis. Activated protein c (APC) has shown positive immunomodulatory effects. OBJECTIVES: We aimed to study neutrophil and monocyte function in response to lipopolysaccharide (LPS) and APC stimulation ex vivo in preterm infants <32 weeks gestation over the first week of life compared to neonatal and adult controls. METHODS: Peripheral blood was taken on day 1, 3, and 7 and stimulated with LPS in the absence or presence of APC. Expression of toll-like receptor 4 (TLR4) and CD11b and reactive oxygen intermediate (ROI) release from neutrophils and monocytes was examined by flow cytometry. RESULTS: LPS induced neutrophil ROI in adults and preterm infants and was significantly reduced by APC. Baseline and LPS-induced monocyte ROI production in preterm neonates was increased compared to adult and term controls. Neutrophil TLR4 baseline expression was higher in term controls compared to preterm infants. CONCLUSION: Increased systemic ROI release in preterm infants may mediate tissue damage, ROI was reduced by APC. However, due to the high risk of hemorrhage further examination of APC mutant forms with anti-inflammatory but decreased anticoagulant properties is merited.

Exploring the Effectiveness of Digital Games in Producing pro-Environmental Behaviors when Played Collaboratively and Individually: A Mixed Methods Study in India.

Environmental sustainability education should create eco-awareness and produce pro-environmental behaviors. Traditional instructional methods create eco-awareness but do not make people act. Purposefully designed digital games for attitudinal instruction provide cognitive knowledge, engage learners emotionally by showing the consequences of harmful behaviors, and encourage correct behaviors. Most studies involving games in different subjects showed that knowledge acquisition was greater in collaborative learning than individual game play. However, a similar comparison with respect to attitudinal learning involving a socio-scientific topic has not been conducted before. This mixed methods study conducted in a high school in India, examined the attitudinal learning among students who played a game individually (n = 45) and collaboratively (n = 44). Also, differences between students who played the game and a control group (n = 42) was examined. Surveys based on the Theory of Planned Behavior (TPB) and the Attitudinal Learning Instrument (ALI), showed that attitudinal learning from games was similar for collaborative and individual players. Also, attitudinal learning from games was higher compared to traditional instructional methods. Interviews explained the learning experiences of game players and how it produced pro-environmental behaviors.

Neonatal Encephalopathy Is Associated With Altered IL-8 and GM-CSF Which Correlates With Outcomes.

Aim: To investigate the relationship between cytokines associated with innate immune cell activation and brain injury and outcome in infants with NE compared to neonatal controls. Methods: Serum and CSF biomarkers associated with activated neutrophils and monocytes [Interleukin-8 (IL-8) and Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF)] were serially measured using duplex immunoassays on days 1, 3 and 7 in term newborns with NE and controls. Results were compared to grade of encephalopathy, seizures, MRI brain imaging, mortality and Bayley Score of Infant and Toddler Development (Bayley-III) at 2 years of age. Results: Ninety-four infants had serum samples collected with 34 CSF samples. NE Grade II/III was significantly associated with elevated on day 2 serum IL-8. Mortality was best predicted by elevated day 1 IL-8. GM-CSF was initially elevated on day 1 and abnormal MRI imaging was associated with decreased day 2 GM-CSF. Elevated GM-CSF at day of life 6-7 correlated negatively with composite cognitive, language and motor Bayley-III scores at 2 years. Conclusion: Moderate or severe NE and mortality was associated with elevated IL-8. Day 2 GM-CSF could predict abnormal MRI results in NE and Bayley-III. Therefore, these cytokines are altered in NE and may predict early outcomes and further implicate inflammatory processes in NE.

The CHK1 inhibitor MU380 significantly increases the sensitivity of human docetaxel-resistant prostate cancer cells to gemcitabine through the induction of mitotic catastrophe.

As treatment options for patients with incurable metastatic castration-resistant prostate cancer (mCRPC) are considerably limited, novel effective therapeutic options are needed. Checkpoint kinase 1 (CHK1) is a highly conserved protein kinase implicated in the DNA damage response (DDR) pathway that prevents the accumulation of DNA damage and controls regular genome duplication. CHK1 has been associated with prostate cancer (PCa) induction, progression, and lethality; hence, CHK1 inhibitors SCH900776 (also known as MK-8776) and the more effective SCH900776 analog MU380 may have clinical applications in the therapy of PCa. Synergistic induction of DNA damage with CHK1 inhibition represents a promising therapeutic approach that has been tested in many types of malignancies, but not in chemoresistant mCRPC. Here, we report that such therapeutic approach may be exploited using the synergistic action of the antimetabolite gemcitabine (GEM) and CHK1 inhibitors SCH900776 and MU380 in docetaxel-resistant (DR) mCRPC. Given the results, both CHK1 inhibitors significantly potentiated the sensitivity to GEM in a panel of chemo-naïve and matched DR PCa cell lines under 2D conditions. MU380 exhibited a stronger synergistic effect with GEM than clinical candidate SCH900776. MU380 alone or in combination with GEM significantly reduced spheroid size and increased apoptosis in all patient-derived xenograft 3D cultures, with a higher impact in DR models. Combined treatment induced premature mitosis from G1 phase resulting in the mitotic catastrophe as a prestage of apoptosis. Finally, treatment by MU380 alone, or in combination with GEM, significantly inhibited tumor growth of both PC339-DOC and PC346C-DOC xenograft models in mice. Taken together, our data suggest that metabolically robust and selective CHK1 inhibitor MU380 can bypass docetaxel resistance and improve the effectiveness of GEM in DR mCRPC models. This approach might allow for dose reduction of GEM and thereby minimize undesired toxicity and may represent a therapeutic option for patients with incurable DR mCRPC.

Pediatric Intensive Care: Immunomodulation With Activated Protein C ex vivo.

Objective: Sepsis is major cause of morbidity and mortality in the Pediatric Intensive Care Unit (PICU). PICU patients may develop transient immune deficiency during sepsis. Activated Protein C (APC) has significant anti-inflammatory and cytoprotective effects. Clinical trials of APC in adult sepsis initially showed improved outcome but recent trials showed no benefit in adults or children. We aimed to assess the effects of APC treatment on innate immune responses in children. Design and Subjects: We compared neutrophil and monocyte responses to lipopolysaccharide (LPS) with and without APC treatment in PICU patients at the time of evaluation for sepsis compared with healthy adults and age-matched pediatric controls. We used flow cytometry to examine cell activation (CD11b expression), function [intracellular reactive oxygen intermediate (ROI) release] and LPS recognition [Toll like Receptor 4 (TLR4) expression]. Results: PICU patients had significantly decreased protein c levels and LPS responses compared with adult and pediatric controls for all parameters. APC reduced LPS-induced neutrophil PICU TLR4 and adult ROI (p < 0.05). PICU non-survivors had increased LPS induced neutrophil and monocyte ROI production vs. survivors which was significantly reduced by APC. Conclusion: PICU patients demonstrate significantly reduced endotoxin reactivity which may predispose them to sepsis and alter effective antibacterial responses. APC reduces LPS-induced ROI production in adults and may have a role in treating severely compromised PICU patients especially given that newer APC forms are associated with decreased bleeding risk and enhanced anti-inflammatory effects.

Perinatal Asphyxia and Erythropoietin and VEGF: Serial Serum and Cerebrospinal Fluid Responses.

BACKGROUND: Infants with neonatal encephalopathy (NE) of hypoxic-ischaemic origin are at risk of oxidative and ischaemia-reperfusion injury, which may induce abnormal inflammatory responses involving excessive cytokine production and release in serum and cerebrospinal fluid (CSF). Systemic inflammation is found in infants with NE, and we therefore were interested in cytokines associated with hypoxia, including vascular endothelial growth factor (VEGF) and erythropoietin (Epo). OBJECTIVE: To investigate the relationship between Epo, VEGF levels, brain injury and outcome in a group of term infants exposed to perinatal asphyxia (PA) compared to controls. METHODS: Serum and CSF biomarkers associated with hypoxia (VEGF, Epo) were serially measured using multiplex immunoassays over days 1-4 in term infants exposed to PA including infants with NE and controls. Results were compared to severity of encephalopathy, MR brain imaging and mortality. RESULTS: Ninety-four infants had 247 serum samples collected (n = 12 controls, 82 exposed to PA with 34 CSF samples), and 4 infants died. Controls had significantly lower serum Epo levels on days 1 and 2 compared to those exposed to PA (p = 0.02). Grade II/III NE was significantly associated with elevated day 2 Epo and decreased day 1 VEGF (p < 0.05; day 2 Epo AUC = 0.74, cut-off 10.05 IU/ml). Elevated serum Epo was associated with severely abnormal MRI. Mortality was associated with elevated day 3 Epo and decreased day 1 VEGF. CSF levels were all after hypothermia and were not significantly associated with outcome. CONCLUSION: Serum Epo and VEGF may be markers of severity of hypoxia-ischaemia and brain injury as they are closely related to hypoxic exposure.

Endosomal gene expression: a new indicator for prostate cancer patient prognosis?

Prostate cancer continues to be a major cause of morbidity and mortality in men, but a method for accurate prognosis in these patients is yet to be developed. The recent discovery of altered endosomal biogenesis in prostate cancer has identified a fundamental change in the cell biology of this cancer, which holds great promise for the identification of novel biomarkers that can predict disease outcomes. Here we have identified significantly altered expression of endosomal genes in prostate cancer compared to non-malignant tissue in mRNA microarrays and confirmed these findings by qRT-PCR on fresh-frozen tissue. Importantly, we identified endosomal gene expression patterns that were predictive of patient outcomes. Two endosomal tri-gene signatures were identified from a previously published microarray cohort and had a significant capacity to stratify patient outcomes. The expression of APPL1, RAB5A, EEA1, PDCD6IP, NOX4 and SORT1 were altered in malignant patient tissue, when compared to indolent and normal prostate tissue. These findings support the initiation of a case-control study using larger cohorts of prostate tissue, with documented patient outcomes, to determine if different combinations of these new biomarkers can accurately predict disease status and clinical progression in prostate cancer patients.

Critical involvement of stress-activated mitogen-activated protein kinases in the regulation of intracellular adhesion molecule-1 in serosal fibroblasts isolated from patients with Crohn's disease.

BACKGROUND: Stricture formation in Crohn's disease occurs as a result of persistent fibroblast activation. Chronic inflammation seen in patients with Crohn's disease leads to enhanced adhesion molecule expression in fibroblasts. Stress-activated mitogen-activated protein kinases are critical signaling pathways that control expression of intracellular adhesion molecule-1 (ICAM-1) in inflammation. The purpose of this study was to investigate the involvement of stress-activated mitogen-activated protein kinases in the regulation of ICAM-1 expression by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in serosal fibroblasts isolated from patients with Crohn's disease. STUDY DESIGN: Fibroblasts were isolated from serosal biopsies of strictures in patients with Crohn's disease and normal colon in patients with colorectal carcinoma. Cell surface and whole cell ICAM-1 expression were evaluated by flow cytometry and Western blot analysis, respectively. Cells were stimulated with TNF-alpha and IL-1beta. To determine the mitogen-activated protein kinase signaling pathway required for ICAM-1 induction, cells were pretreated with inhibitors to Jun N-terminal kinase, p38 kinase, and p42/44 kinase. RESULTS: Baseline ICAM-1 expression was higher (p < 0.001) in fibroblasts isolated from strictures in patients with Crohn's disease (3.2 +/- 0.3) as compared with nonstrictured Crohn's fibroblasts (2.1 +/- 0.3) and control fibroblasts (1.6 +/- 0.1). TNF-alpha and IL-1beta increased ICAM-1 expression in both control and Crohn's disease. Pretreatment of fibroblasts with the Jun N-terminal kinase inhibitor dimethylaminopurine abolished TNF-alpha- and IL-1beta-stimulated ICAM-1 expression. CONCLUSIONS: Serosal fibroblasts isolated from strictures of patients with Crohn's disease demonstrate enhanced expression of ICAM-1. TNF-alpha and IL-1beta upregulate ICAM-1 expression in serosal fibroblasts through a Jun N-terminal kinase signaling pathway. Specific inhibition of inflammatory signaling pathways could provide novel therapeutic targets for treatment of Crohn's disease.

The role of transcription factors in prostate cancer and potential for future RNA interference therapy.

INTRODUCTION: Prostate cancer is a leading cause of cancer-related death in men and current treatments offer only a modest survival benefit in advanced stages of the disease. RNA interference (RNAi) is a therapeutic option that has received great attention in recent years with the potential to treat a variety of disorders, including prostate cancer. Transcription factors are cellular proteins that can up-regulate or down-regulate the transcription of genes and offer promising therapeutic targets. AREAS COVERED: This review will focus on transcription factors that have been identified as key molecules in drug resistance, disease progression and metastases in prostate cancer, which may offer potential as therapeutic targets for RNAi in the future. EXPERT OPINION: By identifying therapeutically viable transcription factor targets in prostate cancer, it is hoped that treatment strategies using RNAi will augment the effect of current chemotherapy regimens, slow disease progression and reduce metastases in prostate cancer, resulting in disease regression.

Preoperative neutrophil response as a predictive marker of clinical outcome following open heart surgery and the impact of leukocyte filtration.

OBJECTIVES: Open heart surgery is associated with a massive systemic inflammatory response. Neutrophils, are the main mediator of this response. We hypothesised that the degree of neutrophil activation and inflammatory response to open heart surgery varies individually and correlates with clinical outcome. The aim of this study was to determine if individual clinical outcome can be predicted preoperatively through assessment of in-vitro stimulated neutrophil responses. Following that, the effects of neutrophil depletion through leukocyte filters are examined. METHODS: Neutrophil responses were assessed preoperatively (n=40) through change in neutrophil adhesion molecule [CD11b, CD62L and P Selectin Glycoprotein-1 (PSGL-1)] expression before and after in-vitro stimulation with Phorbol 12-myristate 13-acetate, PMA (1 ng/ml), lipopolysaccharide, LPS (1 µg/ml) and N-Formyl-Met-Leu-Phe, fMLP (1 ng/ml). Stimulated neutrophil responses were then correlated with postoperative clinical outcome. Patients were then randomised to leukocyte filtration (n=20) and a control group (n=20) and the effect of leukocyte filtration on neutrophil response and clinical outcome were investigated. RESULTS: An individual variation in in-vitro stimulated neutrophil responses was demonstrated. Significant correlations were shown between neutrophil responses and maximum serum creatinine change, CKMB-fraction, adrenaline requirement, noradrenaline requirement, duration of adrenaline required and time to extubation. White cell count and percentage neutrophils were lower in the LD group (P=0.05). CD11b expression (P=0.005) and PSGL-1 expression (P=0.043) across leukocyte filters were also increased. However, no significant difference was detected in clinical outcome between the LD and control groups. CONCLUSION: Preoperative neutrophil responses to in-vitro stimuli can predict clinical outcome following open heart surgery. However, leukocyte filtration did not offer significant benefit in clinical outcome in our study.

The HIF-1alpha C1772T polymorphism may be associated with susceptibility to clinically localised prostate cancer but not with elevated expression of hypoxic biomarkers.

We investigated the role of the C1772T polymorphisms in exon 12 of the Hypoxia-inducible factor-1 alpha (HIF-1alpha) gene C1772T genotype in prostate cancer (PCa) and amplification of the hypoxic response. We identified the heterozygous germline CT genotype as an increased risk factor for clinically localised prostate cancer (Odds ratio = 6.2; p < 0.0001). While immunostaining intensity for HIF-1alpha and VEGF was significantly enhanced in 75% of PCa specimens when compared to matched benign specimens (p < 0.0001), the CT genotype did not modulate the kinetics of HIF-1alpha protein expression in hypoxia in vitro, and was not associated with enhanced expression of hypoxic biomarkers. This study provides the first evidence of an increased risk for clinically localised prostate cancer in men carrying the C1772T HIF-1alpha gene polymorphism. Although our results did not suggest an association between expression of hypoxic biomarkers and genotype status, the correlation may merit further investigation.

A retrospective study comparing clinical outcomes after obturation with Resilon/Epiphany or Gutta-Percha/Kerr sealer.

The purpose of this retrospective study was to evaluate the treatment outcome of root canal systems obturated with gutta-percha and Kerr Pulp Canal Sealer compared with Resilon and Epiphany sealer. One hundred three teeth treated in a private endodontic practice were included in the study. Clinical outcomes (healed versus nonhealed) were assessed by using the Periapical Index determination and clinical evaluation at recall appointments. The magnitude of the association between obturation materials used and outcome measured was evaluated with univariate and multivariate logistic regression analysis. Univariate analysis indicated that pulpal vitality, presence of a preoperative lesion, and length of recall times were statistically significant in predicting the outcome. Logistic regression analysis showed that age, tooth position, and length of recall times were statistically significant in predicting the outcome. Root canal systems obturated with gutta-percha and Kerr Pulp Canal Sealer or Resilon and Epiphany sealer had statistically indistinguishable differences in clinical outcome.

Alterations in the expression of inhibitors of apoptosis during differentiation of prostate epithelial cells.

OBJECTIVE: To investigate alterations in the apoptotic phenotype, specifically the inhibitors of apoptosis (IAP) family, in prostate epithelial cells after differentiation from an apoptotic-resistant basal cell to an apoptotic-susceptible secretory cell. MATERIALS AND METHODS: Cells of the immortalized human prostate epithelial line HPr-1AR were cultured with and with no 5alpha-dihydrotestosterone (DHT) to drive differentiation. Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine changes in differentiation markers such as cytokeratins (CK) 14 and 18, and in XIAP, cIAP-1 and cIAP-2. Flow cytometry was used to assess viability and apoptosis, by propidium iodide DNA staining of the cells during differentiation. RESULTS: Morphological changes and the increased CK-18 and decreased CK-14 expression confirmed differentiation of cells towards a secretory phenotype. Real-time PCR and Western blotting confirmed the expression of the IAPs in the HPr-1AR cells. There was a time-dependent decrease in the mRNA expression of XIAP, cIAP-1 and cIAP-2 after treatment with DHT. Differentiation also resulted in decreased cIAP-1 and XIAP protein expression, but cIAP-2 remained unchanged. Spontaneous apoptosis was significantly increased during cellular differentiation. CONCLUSION: We show for the first time that differentiation of HPr-1AR prostate epithelial cells results in the development of a transient end-stage cell that might be explained by the loss of the IAP family of proteins.