Chronic immune activation and inflammation as the cause of malignancy

Several chronic infections known to be associated with malignancy have established oncogenic properties. However the existence of chronic inflammatory conditions that do not have an established infective cause and are associated with the development of tumours strongly suggests that the inflammatory process itself provides the prerequisite environment for the development of malignancy. This environment includes upregulation of mediators of the inflammatory response such as cyclo-oxygenase (COX)-2 leading to the production of inflammatory cytokines and prostaglandins which themselves may suppress cell mediated immune responses and promote angiogenesis. These factors may also impact on cell growth and survival signalling pathways resulting in induction of cell proliferation and inhibition of apoptosis. Furthermore, chronic inflammation may lead to the production of reactive oxygen species and metabolites such as malondialdehyde within the affected cells that may in turn induce DNA damage and mutations and, as a result, be carcinogenic. Here it is proposed that the conditions provided by a chronic inflammatory environment are so essential for the progression of the neoplastic process that therapeutic intervention aimed at inhibiting inflammation, reducing angiogenesis and stimulating cell mediated immune responses may have a major role in reducing the incidence of common cancers. © 2001 Cancer Research Campaign http://www.bjcancer.com.

Lipoxygenase metabolism : roles in tumor progression and survival

The metabolism of arachidonic acid through lipoxygenase pathways leads to the generation of various biologically active eicosanoids. The expression of these enzymes vary throughout the progression of various cancers, and thereby they have been shown to regulate aspects of tumor development. Substantial evidence supports a functional role for lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Pharmacologic and natural inhibitors of lipoxygenases have been shown to suppress carcinogenesis and tumor growth in a number of experimental models. Signaling of hydro[peroxy]fatty acids following arachidonic or linoleic acid metabolism potentially effect diverse biological phenomenon regulating processes such as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, the effects of distinct LOX isoforms differ considerably with respect to their effects on both the individual mechanisms described and the tumor being examined. 5-LOX and platelet type 12-LOX are generally considered pro-carcinogenic, with the role of 15-LOX-1 remaining controversial, while 15-LOX-2 suppresses carcinogenesis. In this review, we focus on the molecular mechanisms regulated by LOX metabolism in some of the major cancers. We discuss the effects of LOXs on tumor cell proliferation, their roles in cell cycle control and cell death induction, effects on angiogenesis, migration and the immune response, as well as the signal transduction pathways involved in these processes. Understanding the molecular mechanisms underlying the anti-tumor effect of specific, or general, LOX inhibitors may lead to the design of biologically and pharmacologically targeted therapeutic strategies inhibiting LOX isoforms and/or their biologically active metabolites, that may ultimately prove useful in the treatment of cancer, either alone or in combination with conventional therapies. © 2007 Springer Science+Business Media, LLC.

A stimulatory effect of Ca3ZrSi2O9 bioceramics on cementogenic/osteogenic differentiation of periodontal ligament cells

The regeneration of periodontal tissues to cure periodontitis remains a medical challenge. Therefore, it is of great importance to develop a novel biomaterial that could induce cementogenesis and osteogenesis in periodontal tissue engineering. Calcium silicate (Ca–Si) based ceramics have been found to be potential bioactive materials due to their osteostimulatory effect. Recently, it is reported that zirconium modified calcium-silicate-based (Ca3ZrSi2O9) ceramics stimulate cell proliferation and osteogenic differentiation of osteoblasts. However, it is unknown whether Ca3ZrSi2O9 ceramics possess specific cementogenic stimulation for human periodontal ligament cells (hPDLCs) in periodontal tissue regeneration in vitro. The purpose of this study was to investigate whether Ca3ZrSi2O9 ceramic disks and their ionic extracts could stimulate cell growth and cementogenic/osteogenic differentiation of hPDLCs; the possible molecular mechanism involved in this process was also explored by investigating the Wnt/β-catenin signalling pathway of hPDLCs. Our results showed that Ca3ZrSi2O9 ceramic disks supported cell adhesion, proliferation and significantly up-regulated relative alkaline phosphatase (ALP) activity, cementogenic/osteogenic gene expression (CEMP1, CAP, ALP and OPN) and Wnt/β-catenin signalling pathway-related genes (AXIN2 and CTNNB) for hPDLCs, compared to that of β-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic extracts from Ca3ZrSi2O9 powders also significantly enhanced relative ALP activity, cementogenic/osteogenic and Wnt/β-catenin-related gene expression of hPDLCs. The present results demonstrate that Ca3ZrSi2O9 ceramics are capable of stimulating cementogenic/osteogenic differentiation of hPDLCs possibly via activation of the Wnt/β-catenin signalling pathway, suggesting that Ca3ZrSi2O9 ceramics have the potential to be used for periodontal tissue regeneration.

Correlating flow induced shear stress and chondrocytes activity in micro-porous scaffold using computational fluid dynamics and rapid prototyping

Flow induced shear stress plays an important role in regulating cell growth and distribution in scaffolds. This study sought to correlate wall shear stress and chondrocytes activity for engineering design of micro-porous osteochondral grafts based on the hypothesis that it is possible to capture and discriminate between the transmitted force and cell response at the inner irregularities. Unlike common tissue engineering therapies with perfusion bioreactors in which flow-mediated stress is the controlling parameter, this work assigned the associated stress as a function of porosity to influence in vitro proliferation of chondrocytes. D-optimality criterion was used to accommodate three pore characteristics for appraisal in a mixed level fractional design of experiment (DOE); namely, pore size (4 levels), distribution pattern (2 levels) and density (3 levels). Micro-porous scaffolds (n=12) were fabricated according to the DOE using rapid prototyping of an acrylic-based bio-photopolymer. Computational fluid dynamics (CFD) models were created correspondingly and used on an idealized boundary condition with a Newtonian fluid domain to simulate the dynamic microenvironment inside the pores. In vitro condition was reproduced for the 3D printed constructs seeded by high pellet densities of human chondrocytes and cultured for 72 hours. The results showed that cell proliferation was significantly different in the constructs (p<0.05). Inlet fluid velocity of 3×10-2mms-1 and average shear stress of 5.65×10-2 Pa corresponded with increased cell proliferation for scaffolds with smaller pores in hexagonal pattern and lower densities. Although the analytical solution of a Poiseuille flow inside the pores was found insufficient for the description of the flow profile probably due to the outside flow induced turbulence, it showed that the shear stress would increase with cell growth and decrease with pore size. This correlation demonstrated the basis for determining the relation between the induced stress and chondrocyte activity to optimize microfabrication of engineered cartilaginous constructs.

Escape from apoptosis after prolonged serum deprivation is associated with the regulation of the mitochondrial death pathway by Bcl-x(l)

Bcl-x(l) and Bax play important roles in the regulation of apoptosis. This study investigated the involvement of the mitochondrial death pathway and the role of Bcl-x(l) and Bax in the escape from apoptosis after prolonged serum deprivation in Madin-Darby canine kidney (MDCK) cells. Low level apoptosis and basal activity of the mitochondrial death pathway were detectable in normal cell growth. In serum deprivation, mitosis was partially suppressed, and the mitochondrial activity was stimulated. The level of apoptosis continuously rose over 48 h. This rise was concomitant with the increasing presence of cytochrome c in cytosol. However, both apoptosis and cytosolic cytochrome c fell dramatically at 72 h. Elevation of whole cell Bcl-x(l) and redistribution of Bcl-x(l) protein from cytosol to the membrane at 48 h and 72 h was observed. Redistribution of Bax protein from the membrane to cytosol occurred at 24 h, and remained steady to 72 h. Bax/Bcl-x(l) coimmunoprecipitation by anti-Bax antibody showed reduced Bax/Bcl-x(l) interaction at the membrane at 72 h, but not at 24 or 48 h. These results suggest that apoptosis upon serum withdrawal results from the leakage of cytochrome c to cytosol. Amelioration of the leakage of cytochrome c and apoptosis requires not only the increase of Bcl-x(l)/Bax ratio, but also the release of Bcl-x(l) from Bax at the membrane.

The role of the arachidonic acid pathway in NSCLC development and progression : novel approaches for therapeutic intervention

Arachidonic acid metabolism through cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P-450 epoxygenase (EPOX) pathways is responsible for the formation of biologically active eicosanoids, including prostanoids, leukotrienes, hydroxyeicosatetraenoic acid, epoxyeicosatrienoic acid and hydroperoxyeicosatetraenoic acids. Altered eicosanoid expression levels are commonly observed during tumour development and progression of a range of malignancies, including non-small cell lung cancer (NSCLC). Arachidonic acid-derived eicosanoids affect a range of biological phenomena to modulate tumour processes such as cell growth, survival, angiogenesis, cell adhesion, invasion and migration and metastatic potential. Numerous studies have demonstrated that eicosanoids modulate NSCLC development and progression, while targeting these pathways has generally been shown to inhibit tumour growth/progression. Modulation of these arachidonic acid-derived pathways for the prevention and/or treatment of NSCLC has been the subject of significant interest over the past number of years, with a number of clinical trials examining the potential of COX and LOX inhibitors in combination with traditional and novel molecular approaches. However, results from these trials have been largely disappointing. Furthermore, enthusiasm for the use of selective COX-2 inhibitors for cancer prevention/treatment waned, due to their association with adverse cardiovascular events in chemoprevention trials. While COX and LOX targeting may both retain promise for NSCLC prevention and/or treatment, there is an urgent need to understand the downstream signalling mechanisms through which these and other arachidonic acid-derived signalling pathways mediate their effects on tumourigenesis. This will allow for development of safer and potentially more effective strategies for NSCLC prevention and/or treatment. Chemoprevention studies with PGI2 analogues have demonstrated considerable promise, while binding to/signalling through PGE2 receptors have also been the subject of interest for NSCLC treatment. In this chapter, the role of the eicosanoid signalling pathways in non-small cell lung cancer will be discussed. In particular, the effect of the eicosanoids on tumour cell proliferation, their roles in induction of cell death, effects on angiogenesis, migration, invasion and their regulation of the immune response will be assessed, with signal transduction pathways involved in these processes also discussed. Finally, novel approaches targeting these arachidonic acid-derived eicosanoids (using pharmacological or natural agents) for chemoprevention and/or treatment of NSCLC will be outlined. Elucidating the molecular mechanisms underlying the effects of specific or general arachidonic acid pathway modulators may lead to the design of biologically and pharmacologically targeted therapeutic strategies for NSCLC prevention/treatment, which may be used alone or in combination with conventional therapies.

Strategies for co-targeting the PI3K/AKT/mTOR pathway in NSCLC

The PI3K/AKT/mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancer due to mutation, amplification, deletion, methylation and post-translational modifications. We and others have shown that activation of this pathway in non-small cell lung cancer (NSCLC) leads to a more aggressive disease which correlates to poor prognosis for patients. A multitude of selective inhibitors are in development which target key regulators in this pathway, however the success of PI3K targeted inhibition has been hampered by a high rate of innate and acquired resistance. Response to PI3K inhibition may be improved by co-targeting potential mediators of resistance, such as related cell surface receptors or other intracellular signaling pathways which cross-talk with the PI3K pathway. Inhibition of the PI3K pathway may also overcome radioresistance, chemoresistance and immune evasion in NSCLC. The identification of appropriate patient cohorts who will benefit from PI3K co-targeted inhibition strategies will be key to the success of these inhibitors.

Temporal and functional changes in glycosaminoglycan expression during osteogenesis

Heparan sulfate proteoglycans (HSPGs) are complex and labile macromolecular moieties on the surfaces of cells that control the activities of a range of extracellular proteins, particularly those driving growth and regeneration. Here, we examine the biosynthesis of heparan sulfate (HS) sugars produced by cultured MC3T3-E1 mouse calvarial pre-osteoblast cells in order to explore the idea that changes in HS activity in turn drive phenotypic development during osteogenesis. Cells grown for 5 days under proliferating conditions were compared to cells grown for 20 days under mineralizing conditions with respect to their phenotype, the forms of HS core protein produced, and their HS sulfotransferase biosynthetic enzyme levels. RQ-PCR data was supported by the results from the purification of day 5 and day 20 HS forms by anionic exchange chromatography. The data show that cells in active growth phases produce more complex forms of sugar than cells that have become relatively quiescent during active mineralization, and that these in turn can differentially influence rates of cell growth when added exogenously back to preosteoblasts.

An adipoinductive role of inflammation in adipose tissue engineering : key factors in the early development of engineered soft tissues

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1β are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.

Modulation of breast cancer progression and differentiation by the gp30/neregulin

In the last decade we have come to understand that the growth of cancer cells in general and of breast cancer in particular depends, in many cases, upon growth factors that will bind to and activate their receptors. One of these growth factor receptors is the erbB-2 protein which plays an important role in the prognosis of breast cancer and is overexpressed in nearly 30% of human breast cancer patients. While evidence accumulates to support the relationship between erbB-2 overexpression and poor overall survival in breast cancer, understanding of the biological consequence(s) of erbB-2 overexpression remains elusive. Our recent discovery of the gp30 has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through the erbB-2 receptor. These endpoints of growth, invasiveness, and differentiation have clear implications for the emergence, maintenance and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in breast cancer. We have shown that gp30 induces a biphasic growth effect on cells with erbB-2 over-expression. We have recently determined the protein sequence of gp30 and cloned its full length cDNA sequence. We have also cloned two additional forms to the ligand, that are believed to be different isoforms. We are currently expressing the different forms in order to determine their biological effects. To elucidate the cellular mechanisms underlying cell growth inhibition by gp30, we tested the effect of this ligand on cell growth and differentiation of the human breast cancer cells which overexpress erbB-2 and cells which express low levels of this protooncogene. High concentrations of ligand induced differentiation of cells overexpressing erbB-2, as measured by inhibition of cell growth, and increased synthesis of milk components, and modulation of E-cadherin and up- regulation of c-jun and c-fos. These findings indicate that ligand-induced growth inhibition in cells overexpressing erbB-2 is associated with an apparent induction of differentiation. The availability of gp30 derived synthetic peptides and its full cDNAs provides tools necessary to acquire a better understanding of the mechanism of action of the this ligands and the erbB-2 receptor in breast cancer.

miR-126 in human cancers : clinical roles and current perspectives

miR-126 has been implicated in the processes of inflammation and angiogenesis. Through these processes, miR-126 is implicated in cancer biology, but its role there has not been well reviewed. The aim of this review is to examine the molecular mechanisms and clinicopathological significance of miR-126 in human cancers. miR-126 was shown to have roles in cancers of the gastrointestinal tract, genital tracts, breast, thyroid, lung and some other cancers. Its expression was suppressed in most of the cancers studied. The molecular mechanisms that are known to cause aberrant expression of miR-126 include alterations in gene sequence, epigenetic modification and alteration of dicer abundance. miR-126 can inhibit progression of some cancers via negative control of proliferation, migration, invasion, and cell survival. In some instances, however, miR-126 supports cancer progression via promotion of blood vessel formation. Downregulation of miR-126 induces cancer cell proliferation, migration, and invasion via targeting specific oncogenes. Also, reduced levels of miR-126 are a significant predictor of poor survival of patients in many cancers. In addition, miR-126 can alter a multitude of cellular mechanisms in cancer pathogenesis via suppressing gene translation of numerous validated targets such as PI3K, KRAS, EGFL7, CRK, ADAM9, HOXA9, IRS-1, SOX-2, SLC7A5 and VEGF. To conclude, miR-126 is commonly down-regulated in cancer, most likely due to its ability to inhibit cancer cell growth, adhesion, migration, and invasion through suppressing a range of important gene targets. Understanding these mechanisms by which miR-126 is involved with cancer pathogenesis will be useful in the development of therapeutic targets for the management of patients with cancer.

Isolation of microvascular endothelial cells from cadaveric corneal limbus

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with fetal bovine serum (10% v/v), D-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 μg/mL), hydrocortisone (1 μg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF+ cells formed aligned cell-to-cell ‘trains’ when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32 to 80 years) or duration in eye bank corneal preservation medium prior to use (3 to 10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p<0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

Improved fabrication of melt electrospun tissue engineering scaffolds using direct writing and advanced electric field control

Direct writing melt electrospinning is an additive manufacturing technique capable of the layer-by-layer fabrication of highly ordered 3d tissue engineering scaffolds from micron-diameter fibres. The utility of these scaffolds, however, is limited by the maximum achievable height of controlled fibre deposition, beyond which the structure becomes increasingly disordered. A source of this disorder is charge build-up on the deposited polymer producing unwanted coulombic forces. In this study we introduce a novel melt electrospinning platform with dual voltage power supplies to reduce undesirable charge effects and improve fibre deposition control. We produced and characterised several 90° cross-hatched fibre scaffolds using a range of needle/collector plate voltages. Fibre thickness was found to be sensitive only to overall potential and invariant to specific tip/collector voltage. We also produced ordered scaffolds up to 200 layers thick (fibre spacing 1 mm, diameter 40 μm) and characterised structure in terms of three distinct zones; ordered, semi-ordered and disordered. Our in vitro analysis indicates successful cell attachment and distribution throughout the scaffolds, with little evidence of cell death after seven days. This study demonstrates the importance of electrostatic control for reducing destabilising polymer charge effects and enabling the fabrication of morphologically suitable scaffolds for tissue engineering.

Identification and characterization of novel proteolytic interactions of prostate cancer-expressed kallikrein-related peptidases, type II transmembrane serine proteases and matrix metalloproteinases

This study investigated interactions of protein-cleaving enzymes (or proteases) that promote prostate cancer progression. It provides the first evidence of a novel regulatory network of protease activity at the surface of cells. The proteases kallikrein-related peptidases 4 and 14, and matrix metalloproteinases 3 and 9 are cleaved at the cell surface by the cell surface proteases hepsin and TMPRSS2. These cleavage events potentially regulate activation of downstream targets of kallikrein 4 and 14 such as cell surface signalling via the protease-activated receptors (PARs) and cell growth-promoting factors such as hepatocyte-growth factor.

Three dimensional in-vitro models for studying cancer angiogenesis

Introduction Hydrogels prepared from star-shaped poly(ethylene glycol) (PEG) and maleimide-functionalized heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support the cultivation of human umbilical vein endothelial cells (HUVECs). We extend this body of work to study the ability to create an extracellular matrix (ECM)-like model to study breast and prostate cancer cell growth in 3D. Also, we investigate the ability to produce a tri-culture mimicking tumour angiogenesis with cancer spheroids, HUVECs and mesenchymal stem cells (MSCs). Materials and Methods The breast cancer cell lines, MCF-7 and MDA-MB-231, and prostate cancer cell lines, LNCaP and PC3, were seeded into starPEG-heparin hydrogels and grown for 14 Days to analyze the effects of varying hydrogel stiffness on spheroid development. Resulting hydrogel constructs were analyzed via proliferation assays, light microscopy, and immunostaining. Cancer cell lines were then seeded into starPEG-heparin hydrogels functionalized with growth factors as spheroids with HUVECs and MSCs and grown as a tri-culture. Cultures were analyzed via immunostaining and observed using confocal microscopy. Results Cultures prepared in MMP-cleavable starPEG-heparin hydrogels display spheroid formation in contrast to adherent growth on tissue culture plastic. Small differences were visualized in cancer spheroid growth between different gel stiffness across the range of cell lines. Cancer cell lines were able to be co-cultivated with HUVECs and MSC. Interaction was visualized between tumours and HUVECs via confocal microscopy. Further studies intend to further optimize and mimic the ECM environment of in-situ tumour angiogenesis. Discussion Our results confirm the suitability of hydrogels constructed from starPEG-heparin for HUVEC and MSC co-cultivation with cancer cell lines to study cell-cell and cell-matrix interactions in a 3D environment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of breast and prostate cancer.