Duramycin-induced calcium release in cancer cells

Introduction: Duramycin through binding with phosphatidylethanolamine (PE) has shown potential to be an effective anti-tumour agent. However its mode of action in relation to tumour cells is not fully understood. Methods: PE expression on the surface of a panel of cancer cell lines was analysed using duramycin and subsequent antibody labelling then analysed by flow cytometry. Cell viability was also assessed via flow cytometry using annexin V and propidium iodide (PI). Calcium ion (Ca²⁺) release by tumour cells in response to duramycin was determined by spectrofluorometry following incubation with Fluo-3, AM. Confocal microscopy was performed on the cancer cell line AsPC-1 to assess real time cell response to duramycin treatment. Results: Duramycin was able to detect cell surface PE expression on all 15 cancer cell lines screened, which was shown to be duramycin concentration dependent. However higher concentrations induced necrotic cell death. Duramycin induced calcium ion (Ca²⁺) release from the cancer cell lines also in a concentration and time dependent manner. Confocal microscopy showed an influx of PI into the cells over time and induced morphological changes. Conclusion: Duramycin induces Ca²⁺ release from cancer cell lines in a time and concentration dependent relationship

Targeted Therapy in the Management of Elderly Patients with Pancreatic Metastases from Renal Cell Carcinoma

Background: The pancreas is an uncommon but recognizable site for metastases from renal cell carcinoma (RCC). Isolated pancreatic RCC metastases are still rarer and often present years after initial nephrectomy. Surgical resection has been the treatment of choice because of superior patient survival compared with traditional immunotherapy. In recent years, the advent of targeted therapy has transformed the outcomes of patients with metastatic RCC although little evidence is available on its effectiveness on this subset of patients. We report our experience of 6 patients with pancreatic RCC metastases. Patients and Methods: Between 2007 and 2012, 6 patients (2 men, 4 women; median age 78 years) were diagnosed to have pancreatic RCC metastases at our institute. The clinical features, treatment and outcomes were examined. Results: All 6 patients had a primary RCC of clear cell type. The median interval between initial curative nephrectomy and re-presentation with pancreatic metastases was 12.5 years. Four patients were asymptomatic at the time of diagnosis, one presented with obstructive jaundice and another with acute gastrointestinal bleed. Four patients had extra-pancreatic disease. All were deemed unsuitable or unfit for surgical metastasectomy. Five patients had a Memorial Sloan-Kettering Cancer Center (MSKCC) score of 1 (moderate risk) and the other patient had a score of 0 (good risk). Two patients were commenced on Sunitinib, one received Pazopanib and one received Temsirolimus. Two patients did not undergo further treatment. Of the 4 patients who underwent targeted therapy, the median follow up was 33 months with a median progression free survival of 16 months. One achieved complete response but recurred soon after treatment was stopped. Targetted therapy was recommenced and the disease remained stable. A second patient had long period of stable disease before disease progression. A third achieved partial response since started on targeted therapy and a fourth had disease progression despite treatment. Of the four patients who underwent systemic therapy, three are still alive at the time of this report. Conclusion: Pancreatic metastasis from RCC is a unique subgroup of disease which runs an indolent course, and a higher incidence in an elderly population. Our results demonstrate that targeted therapy can be efficacious in some patients where surgical resection is not suitable or possible.published_or_final_versio

Duramycin-porphyrin conjugates for targeting of tumour cells using photodynamic therapy

Duramycin, through binding with phosphatidylethanolamine (PE), has been shown to be a selective molecular probe for the targeting and imaging of cancer cells. Photodynamic therapy aims to bring about specific cytotoxic damage to tumours through delivery of a photosensitising agent and light irradiation. Conjugation to biological molecules that specifically target cancer has been shown to increase photosensitiser (PS) selectivity and decrease damage to surrounding normal tissue. The aim of this study was to target tumour cells with a PE-specific PS therefore duramycin was conjugated to a porphyrin based PS which was achieved via direct reaction with the ε-amino group on the lysine residue near duramycin's N-terminal. The compound was subsequently purified using RP-HPLC and confirmed using mass spectrometry. Binding of the conjugate to ovarian and pancreatic cancer cell lines was assessed by flow cytometry. Light irradiation with a light fluence of 7.5 J/cm2 was delivered to conjugate treated cancer cells and cell proliferation analysed by MTT assay. The conjugate detected PE on all 4 cancer cell lines in a concentration dependent manner and conjugate plus irradiation effectively reduced cell proliferation at concentrations ≥0.5 μM, dependent on cancer cell line. Reduction in cell proliferation by the irradiated conjugate was enhanced over unconjugated duramycin in A2780, AsPC-1 and SK-OV-3 (p b 0.05). In this study we have shown that a duramycin-porphyrin conjugate retained good binding affinity for its target and, following irradiation, reduced cell proliferation of pancreatic and ovarian cancer cell lines

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses

BACKGROUND: Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis. METHODS: In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines. RESULTS: BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th(1)-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th(1)-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement. CONCLUSION: These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility

Tissue factor-bearing microparticles and inflammation: a potential mechanism for the development of venous thromboembolism in cancer

© 2017 International Society on Thrombosis and Haemostasis Summary: Cancer is associated with an increased risk of venous thromboembolism (VTE); the exact mechanisms for the induction of VTE remain to be fully elucidated, but it is widely acknowledged that tissue factor (TF)-bearing microparticles (TF-MPs) may play a significant role. However, TF-MPs have yet to be accepted as a genuine biomarker for cancer-associated VTE, as the presence of elevated TF-MP levels is not always accompanied by thrombosis; interestingly, in certain cases, particularly in pancreatic cancer, VTE seems to be more likely in the context of acute inflammation. Although several potential mechanisms for the development of VTE in cancer have been postulated, this review explores the homeostatic disruption of TF-MPs, as the main reservoir of bloodborne TF, in the context of cancer and inflammation, and considers the abrogated responses of the activated endothelium and mononuclear phagocyte system in mediating this disruption

Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression

BackgroundDespite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear.MethodsIn this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson’s correlation coefficients.ResultsTF release as microvesicles peaked between 30–60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p

Circulating biomarkers during treatment in patients with advanced biliary tract cancer receiving cediranib in the UK ABC-03 trial

BACKGROUND: Advanced biliary tract cancer (ABC) has a poor prognosis. Cediranib, in addition to cisplatin/gemcitabine [CisGem], improved the response rate, but did not improve the progression-free survival (PFS) in the ABC-03 study. Minimally invasive biomarkers predictive of cediranib benefit may improve patient outcomes. METHODS: Changes in 15 circulating plasma angiogenesis or inflammatory-related proteins and cytokeratin-18 (CK18), measured at baseline and during therapy until disease progression, were correlated with overall survival (OS) using time-varying covariate Cox models (TVC). RESULTS: Samples were available from n=117/124 (94%) patients. Circulating Ang1&2, FGFb, PDGFbb, VEGFC, VEGFR1 and CK18 decreased as a result of the therapy, independent of treatment with cediranib. Circulating VEGFR2 and Tie2 were preferentially reduced by cediranib. Patients with increasing levels of VEGFA at any time had a worse PFS and OS; this detrimental effect was attenuated in patients receiving cediranib. TVC analysis revealed CK18 and VEGFR2 increases correlated with poorer OS in all patients (P< 0.001 and P=0.02, respectively). CONCLUSIONS: Rising circulating VEGFA levels in patients with ABC, treated with CisGem, are associated with worse PFS and OS, not seen in patients receiving cediranib. Rising levels of markers of tumour burden (CK18) and potential resistance (VEGFR2) are associated with worse outcomes and warrant validation

Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

Tissue factor (TF) signalling has been associated with alterations in Akt activity influencing cellular survival and proliferation. TF is also shown to induce signalling through activation of the protease activated receptor (PAR)2. Seven cell lines were exposed to recombinant-TF (rec-TF), or activated using a PAR2-agonist peptide and the phosphorylation state of PTEN, and the activities of PTEN and Akt measured. Furthermore, by measuring the association of PTEN with MAGI proteins a mechanism for the induction of signalling by TF was proposed. Short term treatment of cells resulted in de-phosphorylation of PTEN, increased lipid-phosphatase activity and reduced Akt kinase activity in most of the cell lines examined. In contrast, continuous exposure to rec-TF up to 14 days, resulted in lower PTEN antigen levels, enhanced Akt activity and increased rate of cell proliferation. To explore the mechanism of activation of PTEN by TF, the association of &quot;membrane-associated guanylate kinase-with inverted configuration&quot; (MAGI)1–3 proteins with PTEN was assessed using the proximity ligation assay and by co-immunoprecipitation. The interaction of PTEN with all three MAGI proteins was transiently reduced following PAR2 activation and explains the changes in PTEN activity. Our data is first to show that PAR2 activation directly, or through exposure of cells to TF releases PTEN from MAGI proteins and is concurrent with increases in PTEN phosphatase activity. However, prolonged exposure to TF results in the reduction in PTEN antigen with concurrent increase in Akt activity which may explain the aberrant cell survival, proliferation and invasion associated with TF during chronic diseases

Isolated tumour microparticles induce endothelial microparticle release in vitro

© 2019 Wolters Kluwer Health, Inc. All rights reserved. Cancer induces a hypercoagulable state, resulting in an increased risk of venous thromboembolism. One of the mechanisms driving this is tissue factor (TF) production by the tumour, released in small lipid bound microparticles. We have previously demonstrated that tumour cell line media-induced procoagulant changes in HUVEC. The aim of this study was to investigate the effect of tumour microparticles and recombinant human TF (rhTF) on the endothelium. Procoagulant microparticles from the PANC-1 cell line were harvested by ultrafiltration. HUVEC were then incubated with these procoagulant microparticles or rhTF. Flow cytometry was used to investigate the effect of endothelial cell surface protein expression and microparticle release. Microparticles but not soluble TF was responsible for the procoagulant activity of cell-free tumour media. We also demonstrated an increase in endothelial microparticle release with exposure to tumour microparticles, with a positive linear relationship observed (R2 = 0.6630 P ≤ 0.0001). rhTF did not induce any of the changes observed with microparticles. Here we demonstrate that procoagulant activity of tumour cell line media is dependent on microparticles, and that exposure of endothelial cells to these microparticles results in an increase in microparticle release from HUVEC. This suggests a mechanism of transfer of procoagulant potential from the cancer to the remote endothelium