Deirdre Toomey

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.

Activation-induced cell death: the controversial role of Fas and Fas ligand in immune privilege and tumour counterattack.

Activation‐induced cell death (AICD) is the process by which cells undergo apoptosis in a controlled manner through the interaction of a death factor and its receptor. Programmed cell death can be induced by a number of physiological and pathological factors including Fas (CD95)–Fas ligand (FasL/CD95L) interaction, tumour necrosis factor (TNF), ceramide, and reactive oxygen species (ROS). Fas is a 48‐kDa type I transmembrane protein that belongs to the TNF/nerve growth factor receptor superfamily. FasL is a 40‐kDa type II transmembrane protein that belongs to the TNF superfamily. The interaction of Fas with FasL results in a series of signal transductions which initiate apoptosis. The induction of apoptosis in this manner is termed AICD. Activation‐induced cell death and Fas–FasL interactions have been shown to play significant roles in immune system homeostasis. In this review the involvement of Fas and Fas ligand in cell death, with particular reference to the T cell, and the mechanism(s) by which they induce cell death is described. The role of AICD in immune system homeostasis and the controversy surrounding the role of FasL in immune privilege, inflammation, and so‐called tumour counterattack is also discussed.

TGFβ-1 regulation of VEGF production by breast cancer cells

AbstractBackground: Angiogenesis is essential for tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor identified to date. TGFβ-1 acts as an indirect angiogenic agent. Methods: VEGF and TGFβ-1 were measured in the serum of breast cancer patients and agematched controls and in tumor tissue of cancer patients by ELISA. VEGF protein and mRNA expression by breast tumor cell lines were examined, and the effect of TGFβ-1 on VEGF production in these cells was assessed. Results: VEGF levels were significantly higher (P=.03) in the serum of patients with breast cancer compared to age-matched controls. A positive correlation was found between serum (r=0.539) and tumor tissue (r=0.688) levels of VEGF and TGFβ-1. Metastatic MDA-MB-231 breast cancer cells produce more VEGF than do the primary BT474 cells. TGFβ-1 significantly (P<.05) increased production of VEGF. Conclusions: Breast cancer cells constitutively produce VEGF protein and mRNA. There is a relationship between VEGF and TGFβ-1 levels in breast cancer patients, and TGFβ-1 regulates VEGF expression by breast cancer cells.

Mechanisms mediating cancer cachexia.

Background. Cancer cachexia encompasses a wide range of metabolic, hormonal, and cytokine‐related abnormalities that result in a wasting syndrome possibly accounting for up to 30% of cancer‐related deaths.

Taurine attenuates CD3/interleukin‐2‐induced T cell apoptosis in an in vitro model of activation‐induced cell death (AICD)

Interleukin (IL)‐2 immunotherapy is used for the treatment of metastatic melanoma and renal cell carcinoma and mediates its effects through the clonal expansion of lymphocytes. Although IL‐2 remains the most effective form of therapy for these cancers, response rates are poor and dose escalation is hampered by side effects, which include vascular leak and lymphopenia. The mechanism underlying T cell loss is currently unidentified but could be the induction of activation‐induced cell death (AICD) mediated by FasL. Our previous studies have shown that the amino acid taurine can attenuate apoptosis induced by a number of factors in different cell types. Here, we induced T cell AICD via CD3 and IL‐2 stimulation and investigated the effect of taurine on lymphocyte apoptosis. Anti‐CD3‐activated Jurkat T cells treated with IL‐2 significantly increased FasL expression, which was associated with increased apoptosis. Treatment with taurine prior to stimulation down‐regulated FasL protein expression and partially inhibited apoptosis. Inhibition of FasL‐signalling resulted in an identical reduction in apoptosis. As the kinetics of AICD are completely different in circulating T cells, we repeated these experiments in such cells to confirm our finding. Stimulation of CD4+ circulating T cells induced apoptosis in sensitized, but not freshly isolated T cells, which was abrogated partially by taurine. In Jurkat cells it was determined that taurine‐mediated down‐regulation of FasL protein expression was associated with decreased FasL mRNA expression and reduced NFκB activation. These results reveal one possible mechanism underlying the lymphopenia observed with IL‐2 immunotherapy, involving increased FasL expression leading to apoptosis. Taurine may be of use in reversing the lymphopenia associated with IL‐2, thereby augmenting its immunotherapeutic potential.

Infiltrating immune cells, but not tumour cells, express FasL in non-small cell lung cancer: No association with prognosis identified in 3-year follow-up.

Non‐small cell lung cancer (NSCLC) remains a difficult disease to treat and independent prognostic markers other than tumour stage and histology have not emerged. The immune cell content of solid tumours has been associated with tumour regression and at times, tumour progression. The involvement of immune cells in prognosis of NSCLC is poorly described. Poor immune responses within solid tumours have been linked with tumour production of immunosuppressive cytokines. Tumour expression of FasL is thought to disarm responses through the transduction of a death signal in Fas‐expressing T cells. The existence of the ‘tumour counterattack’ in vivo has been questioned. We undertook to measure T cell and macrophage infiltration of the tumour bed in NSCLC and report the association between immune cell content and prognosis in a limited, 3‐year analysis of survival (n = 113). In addition we investigated FasL expression (n = 45). T cells and macrophages were found to frequently infiltrate lung tumours, albeit in small numbers. Generally there were more T cells infiltrating than macrophages. T cell and macrophage numbers were not associated with prognosis. Lung tumours were found not to express FasL, although occasional immune cells surrounding tumour cells were strongly positive. FasL expression was not associated with prognosis in this series. Thus, immune cells infiltrating NSCLC are not capable of suppressing tumour growth, nor are they associated with tumour progression. We report that lung tumours do not express the FasL, and that although some immune cells are FasL positive, this is not a reflection of general immune cell activation.

Phenotyping of immune cell infiltrates in breast and colorectal tumours.

White cell infiltration of solid tumors is an important prognostic indicator in malignant disease. Although macrophage infiltration is associated with good outcome in colorectal cancer, a high macrophage content is associated with poor prognosis in breast cancer. Suppressor macrophages prevent T cell activation in normal tissues such as mucosal linings exposed to continuous antigenic challenge. Interleukin 10 (IL-10), an immunosuppressive cytokine, inhibits macrophage co-stimulation of T cells. Suppressor macrophage numbers, T cell numbers and T cell activation status were assessed in cell suspensions obtained from fresh specimens of breast and colorectal tumours and matched normal tissues. IL-10 production by both malignant and matched normal tissue was also assessed. This study identified elevated numbers of suppressor macrophages in breast tumors compared to matched normal breast tissue. Colorectal tumors did not contain significant numbers of these cells. Although T cell numbers are increased in breast tumors, these cells do not appear to be fully activated, as assessed by major histocompatibility complex class II and Interleukin 2 receptor expression. In contrast, T cells in colorectal tumors exhibit greater expression levels of these markers. Breast tumors produce significantly higher levels of IL-10 than normal breast tissue whereas IL-10 levels in colorectal tumors are similar to normal colon tissue. Our findings of high suppressor macrophage numbers, high levels of IL-10 and poorly activated T cells in breast tumors compared to low suppressor macrophage numbers, low IL-10 and fully activated T cells in colorectal tumors may explain why high macrophage content is associated with poor prognosis in breast cancer and good prognosis in colorectal malignancy.

Taurine attenuates calcium-dependent, Fas-mediated neutrophil apoptosis

The pathway involved in Fas-mediated neutrophil apoptosis remains to be fully elucidated. We examined whether this pathway involved either oxygen-dependent or calcium-dependent mechanisms. We also investigated whether taurine, a powerful antioxidant and regulator of intracellular calcium fluxes, could inhibit Fas-mediated neutrophil apoptosis. Neutrophils were stimulated with Fas monoclonal antibody in the presence or absence of taurine. Fas receptor ligation resulted in significant neutrophil apoptosis at 18 h. Engagement of the Fas receptor rapidly resulted in a significant decrease in intracellular calcium. Apoptosis was inhibited and intracellular calcium levels were maintained in the presence of calcium ionophore A23187 or taurine. Fas ligation did not result in an increase in intracellular reactive oxygen species. We have demonstrated that Fas-mediated neutrophil apoptosis occurs after a decrease in intracellular calcium and is reactive oxygen intermediate independent. Furthermore, the amino acid taurine attenuates this pathway of neutrophil apoptosis by calcium regulation. This newly identified role of taurine in the inhibition of Fas-mediated neutrophil apoptosis may have significant implications for future manipulation of host pro-inflammatory cell function.

Effect of neoadjuvant chemoradiotherapy on angiogenesis in oesophageal cancer

Vascular endothelial growth factor (VEGF) levels are raised in the serum of patients with oesophageal carcinoma. The aim of this study was to evaluate the tumour microvasculature and the role of tumour‐associated macrophages in VEGF production after neoadjuvant chemoradiotherapy and surgery for oesophageal cancer.

N-acetylcysteine attenuates lung injury in a rodent model of fracture

BACKGROUND Neutrophil-mediated lung injury is a cause of significant morbidity and mortality in patients with multiple injuries. We have shown previously that fracture hematoma can activate neutrophils and is thus a putative mediator of the systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS) and multiple organ failure (MOF) in those patients with severe skeletal trauma. Our aim was to establish a rodent model of fracture which caused lung injury and subsequently to administer a drug following fracture to attenuate the lung injury. The drug we chose was N-acetylcysteine, a potent antioxidant. ANIMALS AND METHODS Adult Sprague-Dawley rats were assigned to 4 groups: (1) general anesthetic only, (2) general anesthetic with bilateral femur fractures and nailing, (3) general anesthetic and N-acetylcysteine, (4) general anesthetic with bilateral femur fractures and nailing and N-acetylcysteine after the injury (n = 6 in each group). The dose of N-acetylcysteine was 0.5 mg/kg which was given intraperitoneally after injury to the treated groups. The rats were killed 24 hours after injury and some parameters of lung injury were evaluated--i.e., bronchoalveolar lavage (BAL), lung tissue myeloperoxidase levels (MPO) and wet/dry ratios of lung tissue. The results were analyzed, using one-way analysis of variance. RESULTS Bilateral femur fracture produced a significant lung injury, measured by increases in MPO (25-43 microg/g tissue) and BAL protein (460-605 microg/mL). This effect was attenuated by treatment with N-acetylcysteine (MPO 43-9 microg/mL, BAL protein 605-198 microg/mL). INTERPRETATION N-acetyl cysteine, if given after skeletal trauma, is of potential therapeutic benefit, in preventing SIRS, ARDS and MOF.