D. Bouchier-Hayes

Angiogenic and cell survival functions of Vascular Endothelial Growth Factor (VEGF)

Vascular endothelial growth factor (VEGF) was originally identified as an endothelial cell specific growth factor stimulating angiogenesis and vascular permeability. Some family members, VEGF C and D, are specifically involved in lymphangiogenesis. It now appears that VEGF also has autocrine functions acting as a survival factor for tumour cells protecting them from stresses such as hypoxia, chemotherapy and radiotherapy. The mechanisms of action of VEGF are still being investigated with emerging insights into overlapping pathways and cross‐talk between other receptors such as the neuropilins which were not previously associated with angiogenesis. VEGF plays an important role in embryonic development and angiogenesis during wound healing and menstrual cycle in the healthy adult. VEGF is also important in a number of both malignant and non‐malignant pathologies. As it plays a limited role in normal human physiology, VEGF is an attractive therapeutic target in diseases where VEGF plays a key role. It was originally thought that in pathological conditions such as cancer, VEGF functioned solely as an angiogenic factor, stimulating new vessel formation and increasing vascular permeability. It has since emerged it plays a multifunctional role where it can also have autocrine pro‐survival effects and contribute to tumour cell chemoresistance. In this review we discuss the established role of VEGF in angiogenesis and the underlying mechanisms. We discuss its role as a survival factor and mechanisms whereby angiogenesis inhibition improves efficacy of chemotherapy regimes. Finally, we discuss the therapeutic implications of targeting angiogenesis and VEGF receptors, particularly in cancer therapy.

Tumour hypoxia, chemotherapeutic resistance and hypoxia-related therapies

Tissue hypoxia occurs where there is an imbalance between oxygen supply and consumption. Hypoxia occurs in solid tumours as a result of an inadequate supply of oxygen, due to exponential cellular proliferation and an inefficient vascular supply. It is an adverse prognostic indicator in cancer as it is associated with tumour progression and resistance to therapy. The expression of several genes controlling tumour cell survival are regulated by hypoxia, e.g., growth factors governing the formation of new blood vessels, and hypoxia-responsive transcription factors modulating the expression of genes, which promote tumour cell survival. This review outlines some of the pathways by which tumour hypoxia leads to chemotherapeutic resistance, directly due to lack of oxygen availability, and indirectly due to alterations in the proteome/genome, angiogenesis and pH changes. Some innovative therapies are also detailed which may potentially minimise or eliminate these problems associated with targeting solid tumours.

Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion.

Endotoxin/lipopolysaccharide (LPS), a cell wall component of Gram‐negative bacteria, is a potent inflammatory stimulus. We previously reported that LPS increased the growth of experimental metastases in a murine tumor model. Here, we examined the effect of LPS exposure on key determinants of metastasis—angiogenesis, tumor cell invasion, vascular permeability, nitric oxide synthase (NOS) and matrix metalloproteinase 2 (MMP2) expression. BALB/c mice bearing 4T1 lung metastases were given an intraperitoneal (i.p.) injection of 10 μg LPS or saline. LPS exposure resulted in increased lung weight and incidence of pleural lesions. LPS increased angiogenesis both in vivo and in vitro. Vascular permeability in lung tissue was increased 18 hr after LPS injection. LPS increased inducible nitric oxide synthase (iNOS) and MMP2 expression in lung tumor nodules. 4T1 cells transfected with green fluorescent protein (4T1‐GFP) were injected via lateral tail vein. LPS exposure resulted in increased numbers of 4T1‐GFP cells in mouse lung tissue compared to saline controls, an effect blocked by the competitive NOS inhibitor, NG methyl‐L‐arginine (NMA). LPS‐induced growth and metastasis of 4T1 experimental lung metastases is associated with increased angiogenesis, vascular permeability and tumor cell invasion/migration with iNOS expression implicated in LPS‐induced metastasis.

Immunonutrition: the role of taurine.

Taurine is a sulfonated beta amino acid derived from methionine and cysteine metabolism. It is present in high concentrations in most tissues and in particular in proinflammatory cells such as polymorphonuclear phagocytes. Initial investigation into the multifaceted properties of this non-toxic physiologic amino acid revealed a link between retinal dysfunction and dietary deficiency. Since then a role for this amino acid has been found in membrane stabilization, bile salt formation, antioxidation, calcium homeostasis, growth modulation, and osmoregulation. Our own group has demonstrated a key role for taurine in modulation of apoptosis in a variety of cell types. This review summarizes our current knowledge of taurine in nutrition, host proinflammatory cell homeostasis, therapeutic applications, and its potential immunoregulatory properties. It is our belief that taurine, similar to arginine and glutamine, is now more than worthy of critical clinical analysis.

Excisional surgery for cancer cure: therapy at a cost

Excisional surgery is one of the primary treatment modalities for cancer. Minimal residual disease (MRD) is the occult neoplastic disease that remains in situ after curative surgery. There is increasing evidence that tumour removal alters the growth of MRD, leading to perioperative tumour growth. Because neoplasia is a systemic disease, this phenomenon may be relevant to all patients undergoing surgery for cancer. In this review we discuss the published work that addresses the effects of tumour removal on subsequent tumour growth and the mechanisms by which tumour excision may alter residual tumour growth. In addition, we describe therapeutic approaches that may protect patients against any oncologically adverse effects of tumour removal. On the basis of the evidence presented, we propose a novel therapeutic paradigm; that the postoperative period represents a window of opportunity during which the patient may be further protected against the oncological effects of tumour removal.

Regulation of macrophage production of vascular endothelial growth factor (VEGF) by hypoxia and transforming growth factor β-1

AbstractBackground: Breast tumors contain high numbers of infiltrating macrophages. The role and function of these cells within the tumor remain unclear, but a number of studies have found an association between poor prognosis and macrophage content in human breast cancer. Both hypoxia and TGFβ-1 have been shown to regulate VEGF in other cell types. We hypothesized that breast tumor-associated macrophages produce VEGF and that macrophage production of this factor is regulated by both hypoxia and TGFβ-1. Methods: Paraffin-embedded breast tumor sections were stained immunohistochemically with anti-VEGF, anti-CD68, and anti-cytokeratin. Monocytes were matured for 3 days in 20% autologous plasma and activated with 1000 U/mL interferon-γ for 24 hours. Supernatants were assayed for VEGF protein by ELISA. Total RNA was isolated from cells and reverse transcribed to cDNA, which was used as a template in PCR reactions for VEGF and β-actin. Results: Both tumor cells and tumor macrophages produce VEGF in human breast tumors. Hypoxia increases VEGF protein and mRNA levels in monocyte-derived macrophages, whereas TGFβ-1 increases VEGF protein but not mRNA under hypoxic growth conditions. Conclusions: Breast tumor-associated macrophages may contribute to the angiogenic activity of human breast tumors by producing VEGF. Macrophage production of VEGF is upregulated by hypoxia and TGFβ-1, both of which occur in the tumor environment. Macrophage production of VEGF is regulated at both the mRNA and protein levels.

Vascular endothelial growth factor (VEGF) upregulates BCL-2 and inhibits apoptosis in human and murine mammary adenocarcinoma cells

Tumour progression is regulated by the balance of proliferation and apoptosis in the tumour cell population. To date, the role of vascular endothelial growth factor (VEGF) in tumour growth has been attributed to the induction of angiogenesis. VEGF has been shown to be a survival factor for endothelial cells, preventing apoptosis by inducing Bcl-2 expression. In both murine (4T1) and human (MDA-MB-231) metastatic mammary carcinoma cell lines, we found that VEGF upregulated Bcl-2 expression and anti-VEGF antibodies reduced Bcl-2 expression. These alterations in Bcl-2 expression were reflected by the levels of tumour cell apoptosis. VEGF resulted in reduced tumour cell apoptosis, whereas its inhibition with anti-VEGF neutralizing antibodies induced apoptosis directly in tumour cells. Therefore, in addition to its role in angiogenesis and vessel permeability, VEGF acts as a survival factor for tumour cells, inducing Bcl-2 expression and inhibiting tumour cell apoptosis.

Taurine prevents high-glucose-induced human vascular endothelial cell apoptosis

Elevated blood glucose in uncontrolled diabetes is causally correlated with diabetic microangiopathy. Hyperglycemia-triggered accelerated endothelial cell apoptosis is a critical event in the process of diabetes-associated microvascular disease. The conditionally semiessential amino acid taurine has been previously shown to protect against human endothelial cell apoptosis. Therefore, this study was designed to investigate the role of taurine in the prevention of high-glucose-mediated cell apoptosis in human umbilical vein endothelial cells (HUVEC) and the mechanisms involved. Exposure of HUVEC to 30 mM glucose for 48 h (short-term) and 14 days (long-term) resulted in a significant increase in apoptosis, compared with normal glucose (5.5 mM; P < 0.05). High-glucose-induced DNA fragmentation preferentially occurred in the S phase cells. Mannitol (as osmotic control) at 30 mM failed to induce HUVEC apoptosis. Taurine prevented high-glucose-induced HUVEC apoptosis, which correlates with taurine attenuation of high-glucose-mediated increased intracellular reactive oxygen species (ROS) formation and elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) level. Antioxidants, DMSO, N-acetyl cysteine, and glutathione, only partly attenuated high-glucose-induced HUVEC apoptosis. Glucose at 30 mM did not cause HUVEC necrosis. However, both glucose and mannitol at 60 mM caused HUVEC necrosis as represented by increased lactate dehydrogenase release and cell lysis. Taurine failed to prevent hyperosmolarity-induced cell necrosis. These results demonstrate that taurine attenuates hyperglycemia-induced HUVEC apoptosis through ROS inhibition and [Ca(2+)](i) stabilization and suggest that taurine may exert a beneficial effect in preventing diabetes-associated microangiopathy.Elevated blood glucose in uncontrolled diabetes is causally correlated with diabetic microangiopathy. Hyperglycemia-triggered accelerated endothelial cell apoptosis is a critical event in the process of diabetes-associated microvascular disease. The conditionally semiessential amino acid taurine has been previously shown to protect against human endothelial cell apoptosis. Therefore, this study was designed to investigate the role of taurine in the prevention of high-glucose-mediated cell apoptosis in human umbilical vein endothelial cells (HUVEC) and the mechanisms involved. Exposure of HUVEC to 30 mM glucose for 48 h (short-term) and 14 days (long-term) resulted in a significant increase in apoptosis, compared with normal glucose (5.5 mM; P < 0.05). High-glucose-induced DNA fragmentation preferentially occurred in the S phase cells. Mannitol (as osmotic control) at 30 mM failed to induce HUVEC apoptosis. Taurine prevented high-glucose-induced HUVEC apoptosis, which correlates with taurine attenuation of high-glucose-mediated increased intracellular reactive oxygen species (ROS) formation and elevated intracellular Ca2+ concentration ([Ca2+]i) level. Antioxidants, DMSO, N-acetyl cysteine, and glutathione, only partly attenuated high-glucose-induced HUVEC apoptosis. Glucose at 30 mM did not cause HUVEC necrosis. However, both glucose and mannitol at 60 mM caused HUVEC necrosis as represented by increased lactate dehydrogenase release and cell lysis. Taurine failed to prevent hyperosmolarity-induced cell necrosis. These results demonstrate that taurine attenuates hyperglycemia-induced HUVEC apoptosis through ROS inhibition and [Ca2+]istabilization and suggest that taurine may exert a beneficial effect in preventing diabetes-associated microangiopathy.

Endotoxin/Lipopolysaccharide Activates NF-κB and Enhances Tumor Cell Adhesion and Invasion Through a β1 Integrin-Dependent Mechanism

β1 integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a β1 integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the β1 subunit, whereas various low levels of α1, α2, α4, and α6 expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated β1 integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-β1 blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-κB activation in both SW480 and SW620 cells. Inhibition of the NF-κB pathway significantly attenuated LPS-induced up-regulation of β1 integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-κB, up-regulate β1 integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is β1 subunit-dependent.

The role of endotoxin/lipopolysaccharide in surgically induced tumour growth in a murine model of metastatic disease.

SummarySurgical removal of a primary tumour is often followed by rapid growth of previously dormant metastases. Endotoxin or lipopolysaccharide, a cell wall constituent of Gram-negative bacteria, is ubiquitously present in air and may be introduced during surgery. BALB/c mice received a tail vein injection of 105 4T1 mouse mammary carcinoma cells. Two weeks later, animals were subjected to surgical trauma or an intraperitoneal injection of endotoxin (10 μg per animal). Five days later, animals which underwent open surgery, laparoscopy with air sufflation or received an endotoxin injection displayed increased lung metastasis compared to anaesthetic controls. These increases in metastatic tumour growth were reflected in increased tumour cell proliferation and decreased apoptosis within lung metastases. Circulating levels of the angiogenic cytokine, vascular endothelial growth factor (VEGF), were also elevated in these groups and correlated with increased plasma levels of endotoxin. Endotoxin treatment for 18 h (>10 ng ml–1) directly up-regulated VEGF production by the 4T1 tumour cells in vitro. Metastatic tumour growth in mice undergoing carbon dioxide laparoscopy, where air is excluded, was similar to anaesthetic controls. These data indicate that endotoxin introduced during surgery is associated with the enhanced growth of metastases following surgical trauma, by altering the critical balances governing cellular growth and angiogenesis.