[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species (ROS) possess important cell signalling properties. This contradicts traditional thought which associated ROS activity with cell death. Emerging evidence clearly demonstrates that ROS signalling acts as a key regulator in tumour cell survival and in the cellular processes required for tumour cells to successfully metastasise and proliferate. The discovery of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) family of enzymes in the last decade has unravelled much of the mystery surrounding how ROS are generated. Tumour cells are now known to express Nox enzymes which produce ROS required for cellular signalling. Activation of Nox enzymes occurs via interaction with proinflammatory cytokines and growth factors, all of which are released following surgical trauma. As our understanding of the signalling capabilities of ROS grows, the oncological implications of ROS activity are gradually being revealed. Nox-derived ROS are known to play a central role in each step of the metastatic cascade including invasion, adhesion, angiogenesis and proliferation. This article describes how surgery creates a ROS-rich environment, which facilitates redox signalling, and also examines the role played by Nox enzymes in this process. The authors then explore current knowledge of the oncological implications of surgery-induced redox signalling, and discuss current and future therapeutic strategies targeted at ROS and Nox enzymes in cancer patients.
Gut 12/2011; 62(3). DOI:10.1136/gutjnl-2011-300948 · 14.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumour stroma gene expression in biopsy specimens may obscure the expression of tumour parenchyma, hampering the predictive power of microarrays. We aimed to assess the utility of fluorescence-activated cell sorting (FACS) for generating cell populations for gene expression analysis and to compare the gene expression of FACS-purified tumour parenchyma to that of whole tumour biopsies. Single cell suspensions were generated from colorectal tumour biopsies and tumour parenchyma was separated using FACS. Fluorescence-activated cell sorting allowed reliable estimation and purification of cell populations, generating parenchymal purity above 90%. RNA from FACS-purified and corresponding whole tumour biopsies was hybridised to Affymetrix oligonucleotide microarrays. Whole tumour and parenchymal samples demonstrated differential gene expression, with 289 genes significantly overexpressed in the whole tumour, many of which were consistent with stromal gene expression (e.g., COL6A3, COL1A2, POSTN, TIMP2). Genes characteristic of colorectal carcinoma were overexpressed in the FACS-purified cells (e.g., HOX2D and RHOB). We found FACS to be a robust method for generating samples for gene expression analysis, allowing simultaneous assessment of parenchymal and stromal compartments. Gross stromal contamination may affect the interpretation of cancer gene expression microarray experiments, with implications for hypotheses generation and the stability of expression signatures used for predicting clinical outcomes.
British Journal of Cancer 06/2009; 100(9):1452-64. DOI:10.1038/sj.bjc.6604931 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Perioperative exposure to lipopolysaccharide (LPS) is associated with accelerated metastatic colorectal tumour growth. LPS directly affects cells through Toll-like receptor 4 (TLR-4) and the transcription factor NF-kappaB. The urokinase plasminogen activator (u-PA) system is intimately implicated in tumour cell extracellular matrix (ECM) interactions fundamental to tumour progression. Thus we sought to determine if LPS directly induces accelerated tumour cell ECM adhesion and invasion through activation of the u-PA system and to elucidate the cellular pathways involved. Human colorectal tumour cell lines were stimulated with LPS. u-PA concentration, u-PA activity, active u-PA, surface urokinase plasminogen activator receptor (u-PAR) and TLR-4 expression were assessed by ELISA, colorimetric assay, western blot analysis and flow cytometry respectively. In vitro tumour cell vitronectin adhesion and ECM invasion were analysed by vitronectin adhesion assay and ECM invasion chambers. u-PA and u-PAR function was inhibited with anti u-PA antibodies or the selective u-PA inhibitors amiloride or WXC-340, TLR-4 by TLR-4-blocking antibodies and NF-kappaB by the selective NF-kappaB inhibitor SN-50. LPS upregulates u-PA and u-PAR in a dose-dependent manner, enhancing in vitro tumour cell vitronectin adhesion and ECM invasion by >40% (P<0.01). These effects were ameliorated by u-PA and u-PAR inhibition. LPS activates NF-kappaB through TLR-4. TLR-4 and NF-kappaB inhibition ameliorated LPS-enhanced u-PA and u-PAR expression, tumour cell vitronectin adhesion and ECM invasion. LPS promotes tumour cell ECM adhesion and invasion through activation of the u-PA system in a TLR-4- and NF-kappaB-dependent manner.
British Journal of Cancer 05/2009; 100(10):1589-602. DOI:10.1038/sj.bjc.6604942 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Surgical trauma potentiates tumour growth. Endothelial progenitor cells (EPC), derived from bone-marrow, enhance tumour angiogenesis and growth. We hypothesized that laparotomy and laparoscopy differentially affect the mobilization of EPCs and hence tumour angiogenesis.Methods: C57BL/6 mice, bearing Lewis lung (3LL) flank tumours, were randomized to laparotomy, laparoscopy (CO2 pneumoperitoneum), and anaesthetic-only treatment groups (n = 12 per group). Bone marrow EPC were detected by flow cytometry using stem cell antigen (SCA-1) at 6, 24 and 48 h after treatment. Differentiated EPC were identified, at the same time points, in splenic homogenates by dual staining for lectin/UEA-1 and acetylated low density lipoprotein uptake. These findings were correlated with changes in tumour volume and microvessel density for each group. In addition, EPC uptake in tumours was evaluated using CM-Dil-labelling. This was further correlated with tumour growth and angiogenesis.Results: Laparotomy induced an 11-fold (P = <0.001) increase in bone marrow EPC, compared with anaesthetic-only control (table) at 24 h. Laparoscopy significantly (P = <0.001) attenuated EPC mobilization compared with laparotomy. This correlated with reduced tumour growth and angiogenesis. Following laparotomy, increased EPC were demonstrated lining tumour microvessels. This phenomenon was significantly (P = <0.001) attenuated in the laparoscopy group.Conclusions: Laparotomy is associated with significant mobilization of EPC for perioperative tumour angiogenesis and growth. Laparoscopy attenuates this effect. These findings identify a novel pathway by which open surgery promotes tumour angiogenesis.
British Journal of Surgery 01/2009; 89(S1):31 - 32. DOI:10.1046/j.1365-2168.89.s.1.24_4.x · 5.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Periprosthetic osteolysis precipitates aseptic loosening of components, increases the risk of periprosthetic fracture and, through massive bone loss, complicates revision surgery and ultimately is the primary cause for failure of joint arthroplasty. The anti-inflammatory properties of HMG-CoA reductase inhibitors belonging to the statin family are well recognized. We investigated a possible role for status in initiating the first stage of the osteolytic cycle, namely monocytic activation.
We used an in vitro model of the human monocyte/macrophage inflammatory response to poly-methylmethacrylate (PMMA) particles after pretreat-ing cells with cerivastatin, a potent member of the statin family. Cell activation based upon production of TNF-alpha and MCP-1 cytokines was analyzed and the intracellular Raf-MEK-ERK signal transduction pathway was evaluated using western blot analysis, to identify its role in cell activation and in any cerivastatin effects observed.
We found that pretreatment with cerivastatin significantly abrogates the production of inflammatory cytokines TNF-alpha and MCP-1 by human monocytes in response to polymethylmethacrylate particle activation. This inflammatory activation and attenuation appear to be mediated through the intracellular Raf-MEK-ERK pathway.
We propose that by intervening at the upstream activation stage, subsequent osteoclast activation and osteolysis can be suppressed. We believe that the anti-inflammatory properties of statins may potentially play a prophylactic role in the setting of aseptic loosening, and in so doing increase implant longevity.
[Show abstract][Hide abstract] ABSTRACT: The urokinase plasminogen activator (u-PA) is intimately associated with tumour invasion and metastases. Surgery facilitates accelerated metastatic tumour growth in murine models, a phenomenon related to elevated perioperative bacterial lipopolysaccaride (LPS) and inflammatory cytokine levels. The objectives of the study were to examine the role of u-PA in cytokine-enhanced tumour cell invasion in vitro and surgery-induced accelerated metastatic tumour growth in vivo and to assess the potential benefit of a novel selective u-PA inhibitor WXC-340 in this setting. CT-26 murine colorectal carcinoma cells were stimulated with LPS, tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Cell supernatant u-PA expression and activity were determined using a colorimetric assay and Western blot analysis, respectively. Baseline and cytokine-stimulated in vitro invasion were assessed using ECmatrix invasion chambers. Two established murine models of accelerated metastatic tumour growth were used to investigate the consequences of u-PA inhibition on postoperative metastatic tumour burden. The effect of u-PA inhibition in vitro and in vivo was examined using the novel selective u-PA inhibitor, WXC-340. Proinflammatory cytokine stimulation significantly enhanced in vitro u-PA expression, activity and extracellular matrix invasion by approximately 50% compared to controls (P<0.05). This was abrogated by WXC-340. In vivo WXC-340 almost completely ameliorated both LPS- and surgery-induced, metastatic tumour growth compared to controls (P>0.05). In conclusion, u-PA cascade is actively involved in cytokine-mediated enhanced tumour cell invasion and LPS and surgery-induced metastatic tumour growth. Perioperative u-PA inhibition with WXC-340 may represent a novel therapeutic paradigm.
British Journal of Cancer 02/2007; 96(2):262-8. DOI:10.1038/sj.bjc.6603550 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Post-natal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells migrate, differentiate and incorporate into the nacent endothelium and thereby contribute to physiological and pathological neurovascularisation, has stimulated much interest. Its contribution to neovascularisation of tumours, wound healing and revascularisation associated with ischaemia of skeletal and cardiac muscles is well established. We evaluated the responses of endothelial precursor cells in bone marrow to musculoskeletal trauma in mice. Bone marrow from six C57 Black 6 mice subjected to a standardised, closed fracture of the femur, was analysed for the combined expression of cell-surface markers stem cell antigen 1 (sca-1(+)) and stem cell factor receptor, CD117 (c-kit(+)) in order to identify the endothelial precursor cell population. Immunomagnetically-enriched sca-1(+) mononuclear cell (MNC(sca-1+)) populations were then cultured and examined for functional vascular endothelial differentiation. Bone marrow MNC(sca-1+,c-kit+) counts increased almost twofold within 48 hours of the event, compared with baseline levels, before decreasing by 72 hours. Sca-1(+) mononuclear cell populations in culture from samples of bone marrow at 48 hours bound together Ulex Europus-1, and incorporated fluorescent 1,1'-dioctadecyl- 3,3,3,'3'-tetramethylindocarbocyanine perchlorate-labelled acetylated low-density lipoprotein intracellularily, both characteristics of mature endothelium. Our findings suggest that a systemic provascular response of bone marrow is initiated by musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of neovascularisation and the healing of fractures.
The Bone & Joint Journal 02/2007; 89(1):116-20. DOI:10.1302/0301-620X.89B1.18222 · 3.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Postnatal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells (EPC) migrate, differentiate, and incorporate into the nacent endothelium contributing to physiological and pathological neovascularization, has stimulated much interest. Its contribution to tumor nonvascularization, wound healing, and revascularization associated with skeletal and cardiac muscles ischaemia is established. We evaluated the mobilization of EPCs in response to musculoskeletal trauma. Blood from patients (n = 15) following AO type 42a1 closed diaphyseal tibial fractures was analyzed for CD34 and AC133 cell surface marker expression. Immunomagnetically enriched CD34+ mononuclear cell (MNC(CD34+)) populations were cultured and examined for phenotypic and functional vascular endothelial differentiation. Circulating MNC(CD34+) levels increased sevenfold by day 3 postinjury. Circulating MNC(AC133+) increased 2.5-fold. Enriched MNC(CD34+) populations from day 3 samples in culture exhibited cell cluster formation with sprouting spindles. These cells bound UEA-1 and incorporated fluorescent DiI-Ac-LDL intracellularily. Our findings suggest a systemic provascular response is initiated in response to musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of fracture healing.
Journal of Orthopaedic Research 01/2007; 25(1):44-50. DOI:10.1002/jor.20228 · 2.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial progenitor cells (EPCs) derived from bone marrow incorporate into foci of neovascularization to propagate tumor growth. These cells are mobilized in response to surgical injury. Laparoscopic surgery may protect against the oncologic adverse effects of open surgical tumor excision, and this may be related to attenuated mobilization of EPCs.
For this study, 132 C57BL/6 mice were randomized to standardized laparotomy, laparoscopy, or control groups. The animals were killed at 6, 24, 48, and 72 h. Femur bone marrow and peripheral blood were harvested. Bone marrow EPCs were detected by flow cytometric dual staining for the stem cell antigen-1/cKit phenotype. Circulating EPCs were characterized in blood by vascular endothelial growth factor receptor 2 positive/macrophage activating complement-1 negative staining. Separately, 12 C57/bl6 mice bearing 3LL Lewis lung tumors 12 days after laparotomy or laparoscopy had their tumors excised and examined for endothelial cell expression (marker P1H12).
Laparoscopy decreased circulating EPCs and bone-marrow EPC levels, as compared with laparotomy, at all time points. Bone marrow EPC levels were 2.95% +/- 0.32% after laparotomy, as compared with 0.65 +/- 0.21 in the laparoscopy group (p < 0.05). The circulating EPC level in the laparotomy group was 35.2% +/- 6% of cells, as compared with 3.1% +/- 0.2% in the laparoscopy group (p < 0.05). In homogenized tumors, the percentage of P1H12 expression among laparoscopy-treated animals was 22.1% +/- 4.2%, as compared with 39% +/- 8% in the laparotomy group (p < 0.05).
Laparoscopy decreased EPC levels in both bone marrow and circulation, resulting in decreased tumor endothelial cell burden. This may represent a novel mechanism by which laparoscopy protects against the oncologic adverse effects of open surgical tumor excision.
[Show abstract][Hide abstract] ABSTRACT: The toll-like receptor (TLR) system constitutes a pylogenetically ancient, evolutionary conserved, archetypal pattern recognition system, which underpins pathogen recognition by and activation of the immune system. Toll-like receptor agonists have long been used as immunoadjuvants in anti cancer immunotherapy. However, TLRs are increasingly implicated in human disease pathogenesis and an expanding body of both clinical and experimental evidence suggests that the neoplastic process may subvert TLR signalling pathways to advance cancer progression. Recent discoveries in the TLR system open a multitude of potential therapeutic avenues. Extrapolation of such TLR system manipulations to a clinical oncological setting demands care to prevent potentially deleterious activation of TLR-mediated survival pathways. Thus, the TLR system is a double-edge sword, which needs to be carefully wielded in the setting of neoplastic disease.
British Journal of Cancer 09/2006; 95(3):247-52. DOI:10.1038/sj.bjc.6603275 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the recent past, several papers have pointed to the possibility that tumour removal generates a permissive environment in which tumour growth is potentiated. This phenomenon has been coined "perioperative tumour growth" and whilst it represents a departure in terms of our attitude to the surgical process, this concept was first hinted at by Paget(1) himself. Despite this, the time interval immediately before and after cancer surgery (i.e. the perioperative period) remains an underutilised interval during which chemotherapeutic regimens are rarely implemented. Herein, we present a summarised review of the literature that supports the concept that tumour removal may potentiate the growth of residual neoplastic disease. We also outline current knowledge regarding underlying mechanisms and in this manner highlight potential therapeutic entry points. Finally, we emphasise the urgent need for trials of agents that could protect patients against the harmful host-tumour interactions that may occur during the perioperative period.
[Show abstract][Hide abstract] ABSTRACT: Activated protein C (APC) is an endogenous anti-coagulant with anti-inflammatory properties. The purpose of the present study was to evaluate the effects of activated protein C in the setting of skeletal muscle ischaemia reperfusion injury (IRI). IRI was induced in rats by applying rubber bands above the levels of the greater trochanters bilaterally for a period of 2h followed by 12h reperfusion. Treatment groups received either equal volumes of normal saline or activated protein C prior to tourniquet release. Following 12h reperfusion, muscle function was assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Activated protein C significantly attenuated skeletal muscle reperfusion injury as shown by reduced myeloperoxidase content, wet to dry ratio and electrical properties of skeletal muscle. Further in vitro work was carried out on neutrophils isolated from healthy volunteers to determine the direct effect of APC on neutrophil function. The effects of APC on TNF-alpha stimulated neutrophils were examined by measuring CD18 expression as well as reactive oxygen species generation. The in vitro work demonstrated a reduction in CD18 expression and reactive oxygen species generation. We conclude that activated protein C may have a protective role in the setting of skeletal muscle ischaemia reperfusion injury and that this is in part mediated by a direct inhibitory effect on neutrophil activation.
Journal of Orthopaedic Research 12/2005; 23(6):1454-9. DOI:10.1016/j.orthres.2005.04.009.1100230631 · 2.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lidocaine has actions potentially of benefit during ischaemia-reperfusion. Neutrophils and endothelial cells have an important role in ischaemia-reperfusion injury.
Isolated human neutrophil CD11b and CD18, and human umbilical vein endothelial cell (HUVEC) ICAM-1 expression and supernatant IL-1beta concentrations in response to hypoxia-reoxygenation were studied in the presence or absence of different concentrations of lidocaine (0.005, 0.05 and 0.5 mg mL(-1)). Adhesion molecule expression was quantified by flow cytometry and IL- 1beta concentrations by ELISA. Differences were assessed with analysis of variance and Student-Newman-Keuls as appropriate. Data are presented as mean+/-SD.
Exposure to hypoxia-reoxygenation increased neutrophil CD11b (94.33+/-40.65 vs. 34.32+/-6.83 mean channel fluorescence (MCF), P = 0.02), CD18 (109.84+/-35.44 vs. 59.05+/-6.71 MCF, P = 0.03) and endothelial ICAM-1 (146.62+/-16.78 vs. 47.29+/-9.85 MCF, P < 0.001) expression compared to normoxia. Neutrophil CD18 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL(-1)) treated cells compared to control (71.07+/-10.14 vs. 109.84+/-35.44 MCF, P = 0.03). Endothelial ICAM-1 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL(-1)) treated cells compared to control (133.25+/-16.05 vs. 146.62+/-16.78 MCF, P = 0.03). Hypoxia-reoxygenation increased HUVEC supernatant IL-1beta concentrations compared to normoxia (3.41+/-0.36 vs. 2.65+/-0.21 pg mL(-1), P = 0.02). Endothelial supernatant IL-1beta concentrations in lidocaine-treated HUVECs were similar to controls.
Lidocaine at clinically relevant concentrations decreased neutrophil CD18 and endothelial ICAM-1 expression but not endothelial IL-1beta concentrations.
European Journal of Anaesthesiology 12/2004; 21(12):967-72. DOI:10.1097/00003643-200412000-00007 · 2.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Changes in neutrophil and endothelial adhesion molecule expression occur during perioperative ischaemia and reperfusion (I/R) injury. We investigated the effects of lidocaine on neutrophil-independent changes in neutrophil and endothelial adhesion molecule expression associated with tourniquet-induced I/R.
Plasma was obtained from venous blood samples (tourniquet arm) taken before (baseline), during, 15 min, 2 and 24 h following tourniquet release in seven patients undergoing elective upper limb surgery with tourniquet application. Isolated neutrophils from healthy volunteers (n = 7) were pretreated in the presence or absence of lidocaine (0.005, 0.05 and 0.5 mg mL(-1) for 1 h, and then incubated with I/R plasma for 2 h. Human umbilical vein endothelial cells (HUVECs) were pretreated in the presence or absence of lidocaine (0.005, 0.05 and 0.5 mg mL(-1)) for 1 h, and then incubated with the plasma for 4 h. Adhesion molecule expression was estimated using flow cytometry. Data were analysed using ANOVA and post hoc Student-Newman-Keuls tests.
I/R plasma (withdrawn 15 min following tourniquet release) increased isolated neutrophil CD11b (P = 0.03), CD18 (P = 0.01) and endothelial intercellular adhesion molecule-1 (ICAM-1) (P = 0.008) expression compared to baseline. CD11b, CD18 and ICAM-1 expression on lidocaine (0.005 mg mL(-1)) treated neutrophils was similar to control. CD11b (P < 0.001), CD18 (P = 0.03) and ICAM-1 (P = 0.002) expression on lidocaine (0.05 mg mL(-1)) treated neutrophils and HUVECs was less than that on controls.
Increased in vitro neutrophil and endothelial cell adhesion molecule expression on exposure to plasma obtained during the early reperfusion phase is diminished by lidocaine at greater than clinically relevant plasma concentrations.
European Journal of Anaesthesiology 11/2004; 21(11):892-7. DOI:10.1017/S0265021504000249 · 2.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial progenitor cells (EPCs) are derived from the bone marrow and incorporate into the foci of tumor neovascularization to increase tumor growth. We hypothesized that surgery induces the mobilization of EPCs.
C57BL/6 mice were assigned randomly to standardized laparotomy or anesthesia-only treatment groups (n=102 mice). Animals were killed at 6, 24, 48, and 72 hours. Bone marrow EPCs were detected by blood flow cytometric dual staining for stem cell antigen-1/cKit. Circulating EPCs were characterized in blood by vascular endothelial growth factor receptor 2(+)/macrophage activating complement-1(-) staining. EPCs were detected in splenic homogenates by dual staining for lectin and acetylated low-density lipoprotein uptake. Plasma vascular endothelial growth factor was determined by enzyme-linked immunosorbent assay.
Surgery induced increases in bone marrow and splenic EPC levels (0.2% +/- 0.01% vs 2.9% +/- 0.3%) at 24 hours and in circulating EPC levels (2.5% +/- 0.01% vs 35.2% +/- 6%) at 48 hours compared with control subjects (P <.001). Surgical injury also caused an increase in vascular endothelial growth factor release (81 +/- 8 vs 14 +/- 2 pg; P>.02).
EPCs were mobilized by surgical injury, which may have implications for residual and metastatic tumor growth during the perioperative period.
Surgery 06/2004; 135(6):657-61. DOI:10.1016/j.surg.2003.10.012 · 3.38 Impact Factor