Simon A. Fox

Secreted frizzled related proteins: Implications in cancers

The Wnt (wingless-type) signaling pathway plays an important role in embryonic development, tissue homeostasis, and tumor progression becaluse of its effect on cell proliferation, migration, and differentiation. Secreted frizzled-related proteins (SFRPs) are extracellular inhibitors of Wnt signaling that act by binding directly to Wnt ligands or to Frizzled receptors. In recent years, aberrant expression of SFRPs has been reported to be associated with numerous cancers. As gene expression of SFRP members is often lost through promoter hypermethylation, inhibition of methylation through the use of epigenetic modifying agents could renew the expression of SFRP members and further antagonize deleterious Wnt signaling. Several reports have described epigenetic silencing of these Wnt signaling antagonists in various human cancers, suggesting their possible role as tumor suppressors. SFRP family members thus come across as potential tools in combating Wnt-driven tumorigenesis. However, little is known about SFRP family members and their role in different cancers. This review comprehensively covers all the available information on the role of SFRP molecules in various human cancers.

Synthesis and antimalarial evaluation of novel isocryptolepine derivatives

A series of mono- and di-substituted analogues of isocryptolepine have been synthesized and evaluated for in vitro antimalarial activity against chloroquine sensitive (3D7) and resistant (W2mef) Plasmodium falciparum and for cytotoxicity (3T3 cells). Di-halogenated compounds were the most potent derivatives and 8-bromo-2-chloroisocryptolepine displayed the highest selectivity index (106; the ratio of cytotoxicity (IC(50)=9005 nM) to antimalarial activity (IC(50)=85 nM)). Our evaluation of novel isocryptolepine compounds has demonstrated that di-halogenated derivatives are promising antimalarial lead compounds.

Chemotherapy broadens the range of tumor antigens seen by cytotoxic CD8+ T cells in vivo

Cytotoxic chemotherapies may expose the immune system to high levels of tumor antigens and expand the CD8+ T-cell response to include weak or subdominant antigens. Here, we evaluated the in vivo CTL response to tumor antigens using a murine mesothelioma tumor cell line transfected with a neotumor antigen, ovalbumin, that contains a known hierarchy of epitopes for MHC class I molecules. We show that as tumors progress, effector CTLs are generated in vivo that focus on the dominant epitope SIINFEKL, although a weak response was seen to one (KVVRFDKL) subdominant epitope. These CTLs did not prevent tumor growth. Cisplatin treatment slowed tumor growth, slightly improved in vivo SIINFEKL presentation to T cells and reduced SIINFEKL-CTL activity. However, the CTL response to KVVRFDKL was amplified, and a response to another subdominant epitope, NAIVFKGL, was revealed. Similarly, gemcitabine cured most mice, slightly enhanced SIINFEKL presentation, reduced SIINFEKL-CTL activity yet drove a significant CTL response to NAIVFKGL, but not KVVRFDKL. These NAIVFKGL-specific CTLs secreted IFNγ and proliferated in response to in vitro NAIVFKGL stimulation. IL-2 treatment during chemotherapy refocused the response to SIINFEKL and simultaneously degraded the cisplatin-driven subdominant CTL response. These data show that chemotherapy reveals weaker tumor antigens to the immune system, a response that could be rationally targeted. Furthermore, while integrating IL-2 into the chemotherapy regimen interfered with the hierarchy of the response, IL-2 or other strategies that support CTL activity could be considered upon completion of chemotherapy.

WNT signaling in malignant mesothelioma.

Neoplastic transformation of mesothelium is commonly associated with exposure to asbestos and gives rise to malignant mesothelioma, an aggressive disease that has proved particularly refractory to conventional anti-cancer therapies. The Wnt signaling pathways play key roles in fundamental processes, which include both development and homeostasis. The importance of these pathways in tumorigenesis is emphasized by the many cancers which show aberrations in Wnt signaling. In this review we examine the current evidence for activation of Wnt signaling and the abnormal expression of specific molecules in malignant mesothelioma.

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.

Inclusion body myositis : investigation of the mumps virus hypothesis by polymerase chain reaction

Inclusion body myositis (IBM) is a distinctive form of chronic inflammatory myopathy characterized pathologically by the finding of rimmed vacuoles and 15–18nm microtubular filamentous inclusions in muscle fiber nuclei and cytoplasm. The observation that these filaments resembled nucleocapsids of the paramyxovirus group and showed immunoreactivity with mumps virus (MV) antibodies has led to a long‐standing postulate that IBM may be a “slow” mumps infection. We searched for the presence of MV RNA in 34 muscle biopsies (17 frozen and 17 paraffin‐embedded) from 18 patients with IBM and 43 control biopsies (mainly from patients with other forms of inflammatory myopathy) using a polymerase chain reaction (PCR). The MV PCR was shown to be sensitive and specific for MV strains (including J‐L) and the integrity of muscle RNA extracts was confirmed by PCR detection of constitutive Ableson tyrosine kinase mRNA. MV RNA was not found in any biopsy from the IBM group nor any of the control cases. Our results therefore do not support the mumps hypothesis for IBM.

Mechanisms of cisplatin-induced cell death in malignant mesothelioma cells: Role of inhibitor of apoptosis proteins (IAPs) and caspases

Malignant mesothelioma (MM) is an aggressive and highly chemoresistant tumour. Although cisplatin is used in frontline therapy of this disease treatment remains palliative at best. The biochemical pathways activated by cisplatin and the mechanisms of resistance in mesothelioma cells are poorly understood. Overexpression of inhibitor of apoptosis proteins (IAPs) has been described in clinical mesothelioma tumours and proposed as therapeutic targets. In this study, we examined cisplatin-induced cell death pathways and IAPs in three mesothelioma-derived cell lines. Cisplatin induced cell death in mesothelioma cell lines was characterised by biochemical mechanisms classically associated with apoptosis including: mitochondrial depolarisation, phosphatidylserine translocation and caspase activation. Surprisingly mRNA expression of IAPs in mesothelioma was not upregulated relative to primary mesothelial cells except for survivin which was higher in the most resistant cell line. In contrast, protein expression of both XIAP and survivin was upregulated in all mesothelioma cells, consistent with post-translational regulation. Knockdown of either XIAP or survivin by RNAi did not affect the sensitivity to cisplatin in any of the cell lines. Survivin RNAi did, however, inhibit proliferation in the highest expressing cell line, ONE58. The pan-caspase inhibitor z-VAD and the more selective caspase 3/7 inhibitor z-DEVD had no effect upon the sensitivity of any of the cell lines to cisplatin indicating that caspase-independent pathways predominate. The findings of the present study provide insights into cisplatin-induced mechanisms in mesothelioma cells and show that alternative pathways are operating which may provide new options for targeting this extremely resistant tumour.

Therapeutic approach to target mesothelioma cancer cells using the Wnt antagonist, secreted frizzled-related protein 4: Metabolic state of cancer cells.

Malignant mesothelioma (MM) is an aggressive cancer, characterized by rapid progression, along with late metastasis and poor patient prognosis. It is resistant to many forms of standard anti-cancer treatment. In this study, we determined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt pathway inhibitor, on cancer cell proliferation and metabolism using the JU77 mesothelioma cell line. Treatment with sFRP4 (250 pg/ml) resulted in a significant reduction of cell proliferation. The addition of the Wnt activator Wnt3a (250 pg/ml) or sFRP4 had no significant effect on ATP production and glucose utilisation in JU77 cells at both the 24 and 48 h time points examined. We also examined their effect on Akt and Glycogen synthase kinase-3 beta (GSK3β) phosphorylation, which are both important components of Wnt signalling and glucose metabolism. We found that protein phosphorylation of Akt and GSK3β varied over the 24h and 48 h time points, with constitutive phosphorylation of Akt at serine 473 (pAkt) decreasing to its most significant level when treated with Wnt3a+sFRP4 at the 24h time point. A significant reduction in the level of Cytochrome c oxidase was observed at the 48 h time point, when sFRP4 and Wnt3a were added in combination. We conclude that sFRP4 may function, in part, to reduce/alter cancer cell metabolism, which may lead to sensitisation of cancer cells to chemotherapeutics, or even cell death.

Regulated chemokine gene expression in mouse mesothelioma and mesothelial cells: TNF-α upregulates both CC and CXC chemokine genes.

Many cancers express an array of chemokines which have the capacity to modulate the nature and function of intratumoural leukocyte infiltrates. In malignant mesothelioma (MM) neither the chemokine signalling networks nor their regulation have been investigated despite the prominence of leucocytic infiltrates in both clinical and experimental tumours. In this study, we examined constitutive and cytokine-regulated expression of CC and CXC chemokine genes in mesothelioma and mesothelial cell cultures derived from two different mouse strains (BALB/C and CBA/CaH). In mouse MM and mesothelial cells MCP-1/JE, GRO-α/KC and RANTES were expressed whereas MIP-1α and MIP-2 were infrequently expressed. Comparison of basal chemokine expression showed that GRO-α/KC mRNA was overexpressed in the malignant cells whereas MCP-1 gene expression and release was downregulated. Treatment of mesothelioma cells with IL-4, IFN-γ or TNF-α revealed that chemokine genes could be more responsive to cytokines in the malignant compared to their mesothelial cells. TNF-α was consistently the most potent positive regulator of both CC and CXC chemokine expression and MCP-1 release. The present study for the first time provides a mechanistic insight into the differential regulation of chemokine expression in malignant mesothelioma cells and has implications for mesothelial chemokine signalling in mouse models.

Enterovirus hypothesis for motor neurone disease

EDITOR, — A longstanding hypothesis based on indirect and epidemiological evidence proposes that motor neurone disease is a late consequence of subclinical infection with poliovirus. C J Woodall and colleagues provide supportive evidence for this “enterovirus hypothesis,” reporting the finding of conserved enterovirus sequences in the spinal cords of nine of 13 patients with motor neurone disease when they used conventional polymerase chain reaction and hybridisation; they conclude that these sequences were related to Coxsackie B virus rather than poliovirus.1 The enterovirus hypothesis has also been proposed for idiopathic inflammatory myopathy, and Behan and Behan, from the same group as Woodall and colleagues, have reported finding enterovirus in 56% …