Notch and NOXA-related pathways in melanoma cells

Notch receptor-mediated intracellular events represent an ancient cell signaling system, and alterations in Notch expression are associated with various malignancies in which Notch may function as an oncogene or less commonly as a tumor suppressor. Notch signaling regulates cell fate decisions in the epidermis, including influencing stem cell dynamics and growth/differentiation control of cells in skin. Because of increasing evidence that the Notch signaling network is deregulated in human malignancies, Notch receptors have become attractive targets for selective killing of malignant cells. Compared with proliferating normal human melanocytes, melanoma cell lines are characterized by markedly enhanced levels of activated Notch-1 receptor. By using a small molecule gamma-secretase inhibitor (GSI) consisting of a tripeptide aldehyde, N-benzyloxycarbonyl-Leu-Leu-Nle-CHO, which can block processing and activation of all four different Notch receptors, we identified a specific apoptotic vulnerability in melanoma cells. GSI triggers apoptosis in melanoma cells, but only G2/M growth arrest in melanocytes without subsequent cell death. Moreover, GSI treatment induced a pro-apoptotic BH3-only protein, NOXA, in melanoma cells but not in normal melanocytes. The use of GSI to induce NOXA induction overcomes the apoptotic resistance of melanoma cells, which commonly express numerous cell survival proteins such as Mcl-1, Bcl-2, and survivin. Taken together, these results highlight the concept of synthetic lethality in which exposure to GSI, in combination with melanoma cells overexpressing activated Notch receptors, has lethal consequences, producing selective killing of melanoma cells, while sparing normal melanocytes. By identifying signaling pathways that contribute to the transformation of melanoma cells (e.g. Notch signaling), and anti-cancer agents that achieve tumor selectivity (e.g., GSI-induced NOXA), this experimental approach provides a useful framework for future therapeutic strategies in cutaneous oncology.

Osteocyte-induced angiogenesis via VEGF–MAPK-dependent pathways in endothelial cells

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis- related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2–MAPK–ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK–ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis.

Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes : role of integrinβ1 and focal adhesion kinase signaling cues

INTRODUCTION: Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function. METHODS: De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies. RESULTS: The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell's attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression. CONCLUSION: This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.

The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer

Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI2/TXA2 ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer. © 2010 Elsevier B.V. All rights reserved.

Pro-osteogenic topographical cues promote early activation of osteoprogenitor differentiation via enhanced TGFβ, Wnt, and Notch signaling

Objectives Titanium implant surfaces with modified topographies have improved osteogenic properties in vivo. However, the molecular mechanisms remain obscure. This study explored the signaling pathways responsible for the pro-osteogenic properties of micro-roughened (SLA) and chemically/nanostructurally (modSLA) modified titanium surfaces on human alveolar bone-derived osteoprogenitor cells (BCs) in vitro. Materials and methods The activation of stem cell signaling pathways (TGFβ/BMP, Wnt, FGF, Hedgehog, Notch) was investigated following early exposure (24 and 72 h) of BCs to SLA and modSLA surfaces in the absence of osteogenic cell culture supplements. Results Key regulatory genes from the TGFβ/BMP (TGFBR2, BMPR2, BMPR1B, ACVR1B, SMAD1, SMAD5), Wnt (Wnt/β-catenin and Wnt/Ca2+) (FZD1, FZD3, FZD5, LRP5, NFATC1, NFATC2, NFATC4, PYGO2, LEF1) and Notch (NOTCH1, NOTCH2, NOTCH4, PSEN1, PSEN2, PSENEN) pathways were upregulated on the modified surfaces. These findings correlated with a higher expression of osteogenic markers bone sialoprotein (IBSP) and osteocalcin (BGLAP), and bone differentiation factors BMP2, BMP6, and GDF15, as observed on the modified surfaces. Conclusions These findings demonstrate that the activation of the pro-osteogenic cell signaling pathways by modSLA and SLA surfaces leads to enhanced osteogenic differentiation as evidenced after 7 and 14 days culture in osteogenic media and provides a mechanistic insight into the superior osseointegration on the modified surfaces observed in vivo.

Effect of atmospheric gas plasmas on cancer cell signaling

Cancer is one of the most life-threatening diseases with many forms still regarded as incurable. The conventional cancer treatments have unwanted side effects such as the death of normal cells. A therapy that can accurately target and effectively kill tumor cells could address the inadequacies of the available therapies. Atmospheric gas plasmas (AGP) that are able to specifically kill cancerous cells offer a promising alternative approach compared to conventional therapies. AGP have been shown to exploit tumor-specific genetic defects and a recent trial in mice has confirmed its antitumor effects. The mechanism by which the AGP act on tumor cells but not normal cells is not fully understood. A review of the current literature suggests that reactive oxygen species (ROS) generated by AGP induce death of cancer cells by impairing the function of intracellular regulatory factors. The majority of cancer cells are defective in tumor suppressors that interfere normal cell growth pathways. It appears that pro-oncogene or tumor suppressor-dependent regulation of antioxidant/or ROS signaling pathways may be involved in AGP-induced cancer cell death. The toxic effects of ROS are mitigated by normal cells by adjustment of their metabolic pathways. On the other hand, tumor cells are mostly defective in several regulatory signaling pathways which lead to the loss of metabolic balance within the cells and consequently, the regulation of cell growth. This review article evaluates the impact of AGP on the activation of cellular signaling and its importance for exploring mechanisms for safe and efficient anticancer therapies.

Modeling of protein signaling networks in clinical proteomics

Molecular interactions that underlie pathophysiological states are being elucidated using techniques that profile proteomicend points in cellular systems. Within the field of cancer research, protein interaction networks play pivotal roles in the establishment and maintenance of the hallmarks of malignancy, including cell division, invasion, and migration. Multiple complementary tools enable a multifaceted view of how signal protein pathway alterations contribute to pathophysiological states.One pivotal technique is signal pathway profiling of patient tissue specimens. This microanalysis technology provides a proteomic snapshot at one point in time of cells directly procured from the native context of a tumor micro environment. To study the adaptive patterns of signal pathway events over time, before and after experimental therapy, it is necessary to obtain biopsies from patients before, during, and after therapy. A complementary approach is the profiling of cultured cell lines with and without treatment. Cultured cell models provide the opportunity to study short-term signal changes occurring over minutes to hours. Through this type of system, the effects of particular pharmacological agents may be used to test the effects of signal pathway inhibition or activation on multiple endpoints within a pathway. The complexity of the data generated has necessitated the development of mathematical models for optimal interpretation of interrelated signaling pathways. In combination,clinical proteomic biopsy profiling, tissue culture proteomic profiling, and mathematical modeling synergistically enable a deeper understanding of how protein associations lead to disease states and present new insights into the design of therapeutic regimens.

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.

Laser capture microdissection and multiplex-tandem PCR analysis of proximal tubular epithelial cell signaling in human kidney disease

Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD) but emerging evidence is now linking many forms of acute kidney disease (AKD) with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC) are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate “real time” gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i) confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii) provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii) identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv) describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-β in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

ERK-1/2 and p38 in the Regulation of Hypertrophic Changes of Normal Articular Cartilage Chondrocytes Induced by Osteoarthritic Subchondral Osteoblasts

Objective. Previous studies have shown the influence of subchondral bone osteoblasts (SBOs) on phenotypical changes of articular cartilage chondrocytes (ACCs) during the development of osteoarthritis (OA). The molecular mechanisms involved during this process remain elusive, in particular, the signal transduction pathways. The aim of this study was to investigate the in vitro effects of OA SBOs on the phenotypical changes in normal ACCs and to unveil the potential involvement of MAPK signaling pathways during this process. Methods. Normal and arthritic cartilage and bone samples were collected for isolation of ACCs and SBOs. Direct and indirect coculture models were applied to study chondrocyte hypertrophy under the influence of OA SBOs. MAPKs in the regulation of the cell–cell interactions were monitored by phosphorylated antibodies and relevant inhibitors. Results. OA SBOs led to increased hypertrophic gene expression and matrix calcification in ACCs by means of both direct and indirect cell–cell interactions. In this study, we demonstrated for the first time that OA SBOs suppressed p38 phosphorylation and induced ERK-1/2 signal phosphorylation in cocultured ACCs. The ERK-1/2 pathway inhibitor PD98059 significantly attenuated the hypertrophic changes induced by conditioned medium from OA SBOs, and the p38 inhibitor SB203580 resulted in the up-regulation of hypertrophic genes in ACCs. Conclusion. The findings of this study suggest that the pathologic interaction of OA SBOs and ACCs is mediated via the activation of ERK-1/2 phosphorylation and deactivation of p38 phosphorylation, resulting in hypertrophic differentiation of ACCs.

Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment

Prostate cancer metastasis is reliant on the reciprocal interactions between cancer cells and the bone niche/micro-environment. The production of suitable matrices to study metastasis, carcinogenesis and in particular prostate cancer/bone micro-environment interaction has been limited to specific protein matrices or matrix secreted by immortalised cell lines that may have undergone transformation processes altering signaling pathways and modifying gene or receptor expression. We hypothesize that matrices produced by primary human osteoblasts are a suitable means to develop an in vitro model system for bone metastasis research mimicking in vivo conditions. We have used a decellularized matrix secreted from primary human osteoblasts as a model for prostate cancer function in the bone micro-environment. We show that this collagen I rich matrix is of fibrillar appearance, highly mineralized, and contains proteins, such as osteocalcin, osteonectin and osteopontin, and growth factors characteristic of bone extracellular matrix (ECM). LNCaP and PC3 cells grown on this matrix, adhere strongly, proliferate, and express markers consistent with a loss of epithelial phenotype. Moreover, growth of these cells on the matrix is accompanied by the induction of genes associated with attachment, migration, increased invasive potential, Ca2+ signaling and osteolysis. In summary, we show that growth of prostate cancer cells on matrices produced by primary human osteoblasts mimics key features of prostate cancer bone metastases and thus is a suitable model system to study the tumor/bone micro-environment interaction in this disease.

Cross-talk of subchondral bone osteoblasts and articular cartilage chondrocytes : a new insight in understanding osteoarthritis pathogenesis

Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo atypical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. Collectively these findings indicate that the pathological changes typical of OA, involve alterations of the phenotypic properties of cells in both the subchondral bone and articular cartilage. However, the mechanism(s) by which these changes occur during OA development are not completely understood. The purpose of this project was to address the question of how subchondral bone osteoblasts (SBOs) and ACCs interact with each other with respect to regulation of respective cells’ phenotypic properties and in particular the involvement of mitogen activated protein kinase (MAPK) signalling pathways under normal and OA joint condition. We also endeavoured to test the influence of cross-talk between SBOs and ACCs isolated from normal and OA joint on matrix metalloproteinase (MMP) expression. For this purpose tissues from the knees of OA patients and normal controls were collected to isolate SBOs and ACCs. The cellular cross-talk of SBOs and ACCs were studied by means of both direct and indirect co-culture systems, which made it possible to identify the role of both membrane bound and soluble factors. Histology, immunohistochemistry, qRT-PCR, zymography, ELISA and western blotting were some of the techniques applied to distinguish the changes in the co-cultured vs. non co-cultured cells. The MAPK signalling pathways were probed by using targeted MAPK inhibitors, and their activity monitored by western blot analysis using phospho MAPK specific antibodies. Our co-culture studies demonstrated that OA ACCs enhanced the SBOs differentiation compared to normal ACCs. We demonstrated that OA ACCs induced these phenotypic changes in the SBOs via activating an ERK1/2 signalling pathway. The findings from this study thus provided clear evidence that OA ACCs play an integral role in altering the SBO phenotype. In the second study, we tested the influence of normal SBOs and OA SBOs on ACCs phenotype changes. The results showed that OA SBOs increased the hypertrophic gene expression in co-cultured ACCs compared to normal SBOs, a phenotype which is considered as pathological to the health and integrity of articular cartilage. It was demonstrated that these phenotype changes occurred via de-activation of p38 and activation of ERK1/2 signaling pathways. These findings suggest that the pathological interaction of OA SBOs with ACCs is mediated by cross-talking between ERK1/2 and p38 pathways, resulting in ACCs undergoing hypertrophic differentiation. Subsequent experiments to determine the effect on MMP regulation, of SBOs and ACCs cross-talk, revealed that co-culturing OA SBOs with ACCs significantly enhanced the proteolytic activity and expression of MMP-2 and MMP-9. In turn, co-culture of OA ACCs with SBOs led to abundant MMP-2 expression in SBOs. Furthermore, we showed that the addition of ERK1/2 and JNK inhibitors reversed the elevated MMP-2 and MMP-9 production which otherwise resulted from the interactions of OA SBOs-ACCs. Thus, this study has demonstrated that the altered interactions between OA SBOs-ACCs are capable of triggering the pathological pathways leading to degenerative changes seen in the osteoarthritic joint. In conclusion, the body of work presented in this dissertation has given clear in vitro evidence that the altered bi-directional communication of SBOs and ACCs may play a role in OA development and that this process was mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA.