Exogenous noggin binds the BMP-2 receptor and induces alkaline phosphatase activity in osteoblasts.

Osteogenesis is an important process in bone remodeling and is under strict cellular signaling governed by growth factors and antagonists. Bone morphogenetic protein 2 (BMP-2) is an important osteogenic factor involved in the transcription of key osteogenic genes such as alkaline phosphatase (ALP). While, antagonists such as noggin effectively restrict osteoblast differentiation through binding to BMP-2. In this study, we sought to understand the effect of exogenous noggin in osteoblasts and its role in BMP-2 activation of osteogenesis. Enzymatic activity of ALP was monitored to ascertain the effect of the noggin. Fluorescently labelled noggin was used to determine the binding of noggin to the BMP-2 receptor. The results demonstrated that noggin significantly increases the activity of ALP at concentrations of 50 to 400 ng/mL. While, it inhibited the activity of exogenous BMP-2. Furthermore, fluorescently labelled noggin showed strong binding to osteoblasts which were perturbed when cells were preincubated with BMP-2 suggesting that noggin shares a common receptor with BMP-2. These results suggest that exogenous noggin facilitates osteogenic differentiation and provide a novel mechanism for its interplay with BMP-2.

Investigating the role of CRIPTO-1 (TDGF-1) in glioblastoma multiforme U87 cell line.

Cripto-1 has been implicated in a number of human cancers. Although there is high potential for a role of Cripto-1 in glioblastoma multiforme (GBM) pathogenesis and progression, few studies have tried to define its role in GBM. These studies were limited in that Cripto-1 expression was not studied in detail in relation to markers of cancer initiation and progression. Therefore, these correlative studies allowed limited interpretation of Criptos-1's effect on the various aspects of GBM development using the U87 GBM cell line. In this study, we sought to delineate the role of Cripto-1 in facilitating pathogenesis, stemness, proliferation, invasion, migration and angiogenesis in GBM. Our findings show that upon overexpressing Cripto-1 in U87 GBM cells, the stemness markers Nanog, Oct4, Sox2, and CD44 increased expression. Similarly, an increase in Ki67 was observed demonstrating Cripto-1's potential to induce cellular proliferation. Likewise, we report a novel finding that increased expression of the markers of migration and invasion, Vimentin and Twist, correlated with upregulation of Cripto-1. Moreover, Cripto-1 exposure led to VEGFR-2 overexpression along with higher tube formation under conditions promoting endothelial growth. Taken together our results support a role for Cripto-1 in the initiation, development, progression, and maintenance of GBM pathogenesis. The data presented here are also consistent with a role for Cripto-1 in the re-growth and invasive growth in GBM. This highlights its potential use as a predictive and diagnostic marker in GBM as well as a therapeutic target.

The effect of decellularized tissue engineered constructs on periodontal regeneration.

AIM: To evaluate the effect of decellularized tissue engineered constructs on cell differentiation in vitro and periodontal regeneration in vivo. MATERIALS AND METHODS: Periodontal ligament cell (PDLC) sheets were loaded on polycaprolactone (PCL) scaffolds and then decellularized. Constructs were assessed for their effect on allogenic PDLC and mesenchymal stem cell (MSC) differentiation in vitro, as evaluated by gene expression of bone and periodontal ligament tissue markers post-seeding. Expression of MSC marker STRO-1 was assessed by immunostaining. Decellularized constructs were evaluated in a rat periodontal defect model to assess their biocompatibility and tissue integration. Microcomputed topography (µCT) and histological assessment were performed to assess the regenerative potential of the constructs at 2 and 4 weeks postoperatively. RESULTS: There was upregulation of bone marker gene expression by PDLCs especially on the 14th day. MSCs lacked bone markers expression, but showed increased collagen I marker expression on day 14. STRO-1 expression by the MSCs decreased over the three timepoints when seeded on decellularized sheets. Histological assessment demonstrated the biocompatibility of the decellularized constructs in vivo. More new attachment formation was observed on the decellularized constructs compared to scaffold only controls. CONCLUSION: Decellularized tissue engineered constructs are capable of inducing cell differentiation in vitro and have the potential to facilitate periodontal regeneration in vivo.

Cane Toad Skin Extract-Induced Upregulation and Increased Interaction of Serotonin 2A and D2 Receptors via Gq/11 Signaling Pathway in CLU213 Cells.

Recent evidences show that activation of serotonin 2A receptors (5-HT2A R) by agonists is significant in improving therapeutic activity of disease conditions, such as obsessive-compulsive disorder (OCD). Though the exact molecular mechanism is still not well understood, it is thought to involve agonist-driven, enhanced expression of 5-HT2A R in certain areas of brain, such as the pre-frontal cortex (PFC). Several other reports have also demonstrated association of OCD with lower dopamine receptor (D2 R) availability, primarily in the striatum of the brain along with dysfunction of 5-HT2A R-D2 R heteromer regulation. We thus hypothesized that compound(s) interacting with this molecular mechanism could be developed as drugs for long-term beneficial effects against OCD. In the present study, we have obtained experimental evidence in cultured neuronal cells (CLU213) that aqueous extract (AE, 50 µg/mL, P < 0.05) of the Australian cane toad skin significantly increased the levels of 5-HT2A R and D2 R protein and mRNA expression. AE was also found to enhance the interaction between 5-HT2A R and D2 R and formation of expression of 5-HT2A R-D2 R heteromer using co-immunoprecipitation and Western blot. Further investigation showed the involvement of classical signaling pathway (Gq/11 -PLCß) along with c-FOS transcription factor preferentially in 5-HT2A -mediated agonist activation. These results obtained demonstrated that AE upregulates 5-HT2A R by a mechanism that appears to involve Gq/11 -PLCß signaling pathway and c-FOS transcription factor activation. We indicate this enhanced 5-HT2A R and D2 R expression and their interaction to induce increased 5-HT2A R-D2 R heteromer formation by exposure to AE might provide a molecular mechanism to develop potential novel drug candidates to ameliorate OCD symptoms. J. Cell. Biochem. 118: 979-993, 2017. © 2016 Wiley Periodicals, Inc.

Gene expression profiles in guided bone regeneration using combinations of different biomaterials: a pilot animal study.

OBJECTIVES: The aim of this study was to investigate the gene expression profile related to guided bone regeneration (GBR) at the early healing stage while using combinations of different biomaterials. MATERIALS AND METHODS: Cranial defects in 4 New Zealand rabbits were filled with A) biphasic calcium phosphate/experimental pericardium-derived collagen membrane, B) Bio-Oss® /Bio-Gide® , C) biphasic calcium phosphate/strontium hydroxyapatite-containing collagen membrane and D) Bio-Oss® /strontium hydroxyapatite-containing collagen membrane. Seven days after surgery, one animal was subjected to histological observation and histomorphometric analysis, and three animals to real-time quantitative reverse transcription polymerase chain reaction (PCR). An RT2 Profiler PCR Array (PANZ-026Z, QIAGEN, QIAGEN Sciences, Germantown, MD, USA) was conducted to observe the gene expression profile of groups A, C and D as compared with the control group B. RESULTS: The analysis showed 9 of the 84 genes on the array to be significantly different in the three experimental groups (six genes in group D, four in group C and one in group A). Group D demonstrated the most changes in gene expression profile at day 7. Genes that were significantly down-regulated (AHSG, EGF) or up-regulated (CDH11, MMP13, GLI1 and MCSF) are responsible for early-stage bone formation, bone remodeling and pre-osteoclast development. The gene expression profile of this group correlated with the histological findings, as this group showed the higher formation of osteoid as compared with the other groups. CONCLUSION: Gene expression patterns at early-stage healing of GBR-treated defects appear to be related to the biomaterial used. The combination of Bio-Oss® and strontium hydroxyapatite-containing collagen membrane showed the most pro-osteogenic gene regulation profile (group D), implying the stimulation of key transcriptional factors, which appeared to translate into the up-regulation of the osteogenic process and earlier bone formation.

Aqueous and Ethanol Extracts of Australian Cane Toad Skins Suppress Pro-Inflammatory Cytokine Secretion in U937 Cells via NF-κB Signaling Pathway.

Toad skin extracts, such as aqueous extracts (AE) of Chinese toad skins, have demonstrated therapeutic benefits for a range of diseases including pain, inflammation, swelling, heart failure, and various types of cancers. In this study, we investigated the anti-inflammatory potential of an AE (0.1-10 µg/mL) and a 60% ethanol extract (EE; 0.1-10 µg/mL) from Australian cane toad (Bufo marinus) skins and the known bioactive compound, bufotenine (BT; 0.1-10 nM). The assay employed a model of the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) for the release of tumor necrosis factor (TNF)-α and interleukin (IL)-6. We demonstrated that AE, EE, and BT significantly inhibited the release and expression of TNF-α and IL-6 in a dose-dependent manner when the cells were pre-treated at non-cytotoxic concentrations. Further investigation revealed that the inhibition of TNF-α and IL-6 release and expression was associated with the suppression of nuclear factor (NF)-kappa (κ)B activation. These results indicate that AE, EE, and BT are strong inflammation inhibitors, thus have the potential for further development as anti-inflammatory therapeutic agents from a natural source regarded as a feral pest in Australia. J. Cell. Biochem. 117: 2769-2780, 2016. © 2016 Wiley Periodicals, Inc.

CCL2 and CCR2 are Essential for the Formation of Osteoclasts and Foreign Body Giant Cells.

Osteoclasts are multinucleated cells responsible for bone resorption. They are derived from the fusion of cells in the monocyte/macrophage lineage. Monocytes and macrophages can also fuse to form foreign body giant cells (FBGC). Foreign body giant cells are observed at the interface between a host and a foreign body such as implants during a foreign body reaction. Macrophages are attracted to the site of bone resorption and foreign body reactions by different cytokines. Chemokine (C-C) ligand-2 (CCL2) is an important chemotactic factor and binds to a receptor CCR2. In this study we investigated the importance of CCL2 and the receptor CCR2 in the formation of osteoclasts and FBGC. CCL2 mRNA was more highly expressed in giant cell culture than macrophages, being 9-fold and 16-fold more abundant in osteoclasts and FBGC respectively. Significantly fewer osteoclasts and FBGC were cultured from the bone marrow of CCL2 and CCR2 knockout mice, when compared to wild type. Not only were the number of giant cells reduced but there was a significant reduction in the number of nuclei and the size of these cells in the cultures of CCL2 and CCR2 knockout mice. Formation of osteoclasts and FBGC were recovered in cultures by addition of exogenous CCL2 to the media containing marrow cells from CCL2-/- mice. We conclude that CCL2 and its receptor CCR2 are important for the formation of osteoclasts and FBGC and absence of these genes causes inhibition of osteoclast and FBGC formation.

Chansu inhibits the expression of cortactin in colon cancer cell lines in vitro and in vivo.

BACKGROUND: Chansu is a transitional Chinese medicine that has been used for centuries as therapy for inflammation, anaesthesia and arrhythmia in China and other Asian countries. Recently, it has also been used for anti-cancer purposes. We have previously shown that Chansu has a huge pro-apoptotic potential on colon cancer cells, but to date the detailed mechanism of this action is not well understood. METHODS: One of the major components of Chansu, Cinobufagin (CBF) was used to treat cancer cells. The expressions of levels of cortactin, an important factor in tumour progression and cancer invasion, were assessed in in vitro and in vivo experiments. Additional analyses were performed in subcellular protein fractions and immune-fluorescent staining was used to define cortactin protein expression and the changes of location in CBF-treated cells. RESULTS: CBF strongly inhibited the expression of cortactin in HCT116 cells. There were reductions of both mRNA transcription and protein synthesis, which were more significant in the absence of oxygen in vitro. In addition, nuclear translocation of cortactin was observed in HCT116 cells post CBF exposure but not in the negative control, indicating that CBF is likely to interrupt co-localisation of cortactin to cytoskeletal proteins. Most importantly, CBF could diminish the expression of cortactin in human HCT116 xenograft tumours in nude mouse in vivo. CONCLUSIONS: CBF inhibits cortactin expression and nuclear translocation in colon cancer cells in vitro and in mouse models bearing human colon tumour in vivo, suggesting it might disrupt actin-regulated cell movement. Thus, CBF or Chansu could be developed as an effective anti-cancer therapy to stop local invasion and metastasis.

Differential expression of chemokines, chemokine receptors and proteinases by foreign body giant cells (FBGCs) and osteoclasts.

Osteoclasts and foreign body giant cells (FBGCs) are both derived from the fusion of macropahges. These cells are seen in close proximity during foreign body reactions, therefore it was assumed that they might interact with each other. The aim was to identify important genes that are expressed by osteoclasts and FBGCs which can be used to understand peri-implantitis and predict the relationship of these cells during foreign body reactions. Bone marrow macrophages (BMM) were treated with receptor activator of nuclear factor kappa B ligand (RANKL) to produce osteoclasts. Quantitative PCR (qPCR) was used to identify the genes that were expressed by osteoclasts and FBGCs compared to macrophage controls. TRAP staining was used to visualise the cells while gelatine zymography and western blots were used for protein expression. Tartrate-resistant acid phosphatase (TRAP), matrix metallo proteinase 9 (MMP9), nuclear factor of activated T cells 1 (NFATc1), cathepsin K (CTSK) and RANK were significantly lower in FBGCs compared to osteoclasts. Inflammation specific chemokines such as monocyte chemotactic protein (MCP1 also called CCL2), macrophage inflammatory protein 1 alpha (MIP1α), MIP1ß and MIP1γ, and their receptors CCR1, CCR3 and CCR5, were highly expressed by FBGCs. FBGCs were negative for osteoclast specific markers (RANK, NFATc1, CTSK). FBGCs expressed chemokines such as CCL2, 3, 5 and 9 while osteoclasts expressed the receptors for these chemokines i.e. CCR1, 2 and 3. Our findings show that osteoclast specific genes are not expressed by FBGCs and that FBGCs interact with osteoclasts during foreign body reaction through chemokines.

Foreign body giant cells and osteoclasts are TRAP positive, have podosome-belts and both require OC-STAMP for cell fusion.

Macrophages have the ability to fuse and form multinucleated giant cells such as Osteoclast (OCs) and FBGCs. Osteoclast stimulatory transmembrane protein (OC-STAMP) is an important cell surface protein involved in the formation of OCs. This study sought to determine if OC-STAMP also regulates formation of FBGCs using expression analysis and subsequent inhibition studies. qPCR and Western blot analysis showed that OC-STAMP expression is significantly higher in FBGCs compared to control monocytes (P < 0.05). Four days following cell culture, OCs were positive for TRAP and F-actin ring formation, but FBGCs were not. In contrast, FBGCs were positive for TRAP and showed podosome belts comprised of F-actin on Day 8. FBGCs were subsequently plated onto dentine, but despite presenting some morphologic features of OCs (OC-STAMP expression, TRAP reactivity, and podosome belts) they failed to resorb bone. To evaluate a role for OC-STAMP in FBGCs, we inhibited this cell surface protein with anti-OC-STAMP antibody and observed that cell fusion and podosome belt formation was inhibited in both OCs and FBGCs. Our data support the hypothesis that OC-STAMP is a regulatory molecule for FBGCs; and that they are functionally distinct from OCs, despite similarities in gene expression profile, podosome belt formation, and TRAP expression.

Chemical Inhibitors and microRNAs (miRNA) Targeting the Mammalian Target of Rapamycin (mTOR) Pathway: Potential for Novel Anticancer Therapeutics.

The mammalian target of rapamycin (mTOR) is a critical regulator of many fundamental features in response to upstream cellular signals, such as growth factors, energy, stress and nutrients, controlling cell growth, proliferation and metabolism through two complexes, mTORC1 and mTORC2. Dysregulation of mTOR signalling often occurs in a variety of human malignant diseases making it a crucial and validated target in the treatment of cancer. Tumour cells have shown high susceptibility to mTOR inhibitors. Rapamycin and its derivatives (rapalogs) have been tested in clinical trials in several tumour types and found to be effective as anticancer agents in patients with advanced cancers. To block mTOR function, they form a complex with FKBP12 and then bind the FRB domain of mTOR. Furthermore, a new generation of mTOR inhibitors targeting ATP-binding in the catalytic site of mTOR showed potent and more selective inhibition. More recently, microRNAs (miRNA) have emerged as modulators of biological pathways that are essential in cancer initiation, development and progression. Evidence collected to date shows that miRNAs may function as tumour suppressors or oncogenes in several human neoplasms. The mTOR pathway is a promising target by miRNAs for anticancer therapy. Extensive studies have indicated that regulation of the mTOR pathway by miRNAs plays a major role in cancer progression, indicating a novel way to investigate the tumorigenesis and therapy of cancer. Here, we summarize current findings of the role of mTOR inhibitors and miRNAs in carcinogenesis through targeting mTOR signalling pathways and determine their potential as novel anti-cancer therapeutics.

Apoptosis and microRNA aberrations in cancer.

Carcinogenesis arises from the malfunction of genes that control cell growth and division. Therefore, the most effective method of hindering tumourigenesis is to induce the death of immortalized cancer cells. Apoptosis or programmed cell death has shown the most promises in impairing cancer growth. A variety of proteins is involved in the regulation of apoptosis and the malfunction of any these regulators may cause cell proliferation. The microRNAs have been shown to play a central role in the regulation of the cell cycle, including apoptosis. The microRNAs are involved in post-transcriptional gene suppression and have been implicated in the regulation of cell differentiation and development. Aberrations in the microRNA regulation of apoptosis lead to tumourigenesis. The present review assesses the current knowledge of apoptotic regulation in cancer and the effect of microRNA aberrations in tumourigenesis.

Conserved bacterial de novo guanine biosynthesis pathway enables microbial survival and colonization in the environmental niche of the urinary tract.

In bacteria, guaA encodes guanosine monophosphate synthetase that confers an ability to biosynthesize guanine nucleotides de novo. This enables bacterial colonization in different environments and, while guaA is widely distributed among Bacteroidetes and Firmicutes, its contribution to the inhabitation of the human microbiome by commensal bacteria is unclear. We studied Streptococcus as a commensal urogenital tract bacterium and opportunistic pathogen, and explored the role of guaA in bacterial survival and colonization of urine. Analysis of guaA-deficient Streptococcus revealed guanine utilization is essential for bacterial colonization of this niche. The genomic location of guaA in other commensals of the human urogenital tract revealed substantial cross-phyla diversity and organizational structures of guaA that are divergent across phyla. Essentiality of guaA for Streptococcus colonization in the urinary tract establishes that purine biosynthesis is a critical element of the ability of this bacterium to survive and colonize in the host as part of the resident human microbiome.

Functional analysis of genes for benzoate metabolism in the albicidin biosynthetic region of Xanthomonas albilineans.

Albicidins are potent DNA-gyrase-inhibiting antibiotics and phytotoxins synthesised by Xanthomonas albilineans. Functions have been deduced for some clustered biosynthetic genes, including a PKS-NRPS megasynthase, methyltransferases and regulatory genes, and resistance genes including a transporter and a gyrase-binding protein. More puzzling is the presence in this cluster of apparent aromatic metabolism genes. Here, we describe functional analysis of several such genes and propose a model for their role. An apparent benzoate CoA ligase (xabE) proved essential for albicidin production and pathogenicity. A neighbouring operon includes genes for p-aminobenzoate (PABA) metabolism. A PABA synthase fusion (pabAB) restored prototrophy in pabA and pabB mutants of Escherichia coli, proving functionality. Inactivation of pabAB increased susceptibility to sulphanilamide but did not block albicidin production. X. albilineans contains a remote pabB gene which evidently supplies enough PABA for albicidin biosynthesis in culture. Additional capacity from pabAB may be advantageous in more demanding environments such as infected plants. Downstream from pabAB are a known resistance gene (albG) and ubiC which encodes a p-hydroxybenzoate (PHBA) synthase. PHBA protects X. albilineans from inhibition by PABA. Therefore, coordinated expression may protect X. albilineans against toxicity of both the PABA intermediate and the albicidin product, under conditions that induce high-level antibiotic biosynthesis.

Cripto-1 overexpression in U87 glioblastoma cells activates MAPK, focal adhesion and ErbB pathways.

Discovering the underlying signalling pathways that control cancer cells is crucial for understanding their biology and to develop therapeutic regimens. Thus, the aim of the present study was to determine the effect of Cripto-1 on pathways controlling glioblastoma (GBM) cell function. To this end, changes in protein phosphorylation in cells overexpressing Cripto-1 were analysed using the Kyoto Encyclopedia of Genes and Genomes pathway analysis tool, as well as the Uniprot resource to identify the functions of Cripto-1-dependent phosphorylated proteins. This revealed that proteins affected by Cripto-1 overexpression are involved in multiple signalling pathways. The mitogen-activated protein kinase (MAPK), focal adhesion (FA) and ErbB pathways were found to be enriched by Cripto-1 overexpression with 35, 27 and 24% of pathway proteins phosphorylated, respectively. These pathways control important cellular processes in cancer cells that correlate with the observed functional changes described in earlier studies. More specifically, Cripto-1 may regulate MAPK cellular proliferation and survival pathways by activating epithelial growth factor receptor (EGFR; Ser1070) or fibroblast GFR1 (Tyr654). Its effect on cellular proliferation and survival could be mediated through Src (Tyr418), FA kinase (FAK; Tyr396), p130CAS (Tyr410), c-Jun (Ser63), Paxillin (PXN; Tyr118) and BCL2 (Thr69) of the FA pathway. Cripto-1 may also control cellular motility and invasion by activating Src (Tyr418), FAK (Tyr396) and PXN (Tyr118) of the FA pathway. However, Cripto-1 regulation of cellular invasion and migration might be not limited to the FA pathway, it may also control these cellular mechanisms through signalling via EGFR (Ser1070)/Her2 (Tyr877) to mediate the Src (Tyr418) and FAK (Tyr396) cascade activation of the ErbB signalling pathway. Angiogenesis could be mediated by Cripto-1 by activating c-Jun (Ser63) through EGFR (Ser1070)/Her2 (Tyr877) of the ErbB pathway. To conclude, the present study has augmented and enriched our current knowledge on the crucial roles that Cripto-1 may play in controlling different cellular mechanisms in GBM cells.

miR-496, miR-1185, miR-654, miR-3183 and miR-495 are downregulated in colorectal cancer cells and have putative roles in the mTOR pathway.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by suppressing the target mRNA and inhibiting translation in order to regulate multiple biological processes. miRNAs play important roles as oncogenes or tumor suppressors in the development of various types of human cancer. The regulation of mammalian target of rapamycin (mTOR) by miRNAs has been studied in several types of cancer, including colorectal cancer (CRC). However, to the best of our knowledge, only limited information regarding the function of miRNAs in human CRC is available. In the present study, the expression of 22 miRNAs in CRC cell lines were investigated in regard to key genes in the mTOR pathway. Initially, it was revealed that mTOR, regulatory-associated protein of mTOR complex I and rapamycin-intensive companion of mTOR were overexpressed in CRC cell lines when compared with a normal colorectal cell line. Subsequently, putative miRNA-mRNA associations were identified via multiple miRNA target prediction programs. The expression levels for the candidate miRNAs were validated using quantitative real-time polymerase chain reaction. Expression analysis revealed that, among 20 miRNAs, five miRNAs (miR-496, miR-1185, miR-654, miR-3183 and miR-495) exhibited significant downregulation in association with the mTOR signaling pathway. Taken together, the results from the present study suggest that several miRNAs that are associated with CRC, with possible roles in mTOR signaling, may have potential therapeutic or diagnostic benefits in CRC treatment.

EGF ligand fused to truncated Pseudomonas aeruginosa exotoxin A specifically targets and inhibits EGFR­positive cancer cells.

Cancer cells have been known to overexpress the epidermal growth factor receptor (EGFR) and hence relevant multiple­targeted therapies have been developed, with a recent clinical application of the antibody­mediated inhibition of the EGFR. However, this strategy is not useful in cancer cells with mutations in KRAS; a GTPase downstream of EGFR which constitutively activates the pathway without EGF stimulation. Furthermore, mutations in EGFR also reduce the binding of monoclonal antibodies and thereby render them ineffective. In the present study, we designed a chimeric EGF protein fused to the truncated N­terminal domain fragment of Pseudomonas aeruginosa exotoxin A (EGF­ETA), which has ADP­ribosylation activity and induces apoptosis. The EGF­ETA protein was expressed in E. coli as a His­tagged fusion. Our results showed that EGF­ETA significantly inhibited the proliferation of EGFR­positive A431 epidermoid carcinoma (IC50 27 ng/ml) and HN5 head and neck squamous cell carcinoma (IC50 36 ng/ml) cells. However, its effect on cancer cells with little or no EGFR expression was limited (A549­IC50 1,000 ng/ml; MCF­7­IC50 >10,000 ng/ml). Compared to cetuximab, EGF­ETA was highly potent in its killing capacity of HN5 cancer cells at 1,000 ng/ml, while cetuximab had little effect at 1,000 ng/ml. Furthermore, EGF­ETA was just as potent in HCT116 (KRAS G13D) and SW480 (KRAS G12V) colon cancer cell lines harbouring KRAS hyperactivating mutations when compared to KRAS wild­type HT29 colon cancer cells. Finally, co­incubation of EGF­ETA with an anti­EGF antibody abrogated its effect on the EGFR­positive A431 cells. Our results show that the chimeric EGF­ETA toxin is extremely effective against EGFR­positive cancers and raises the potential to further develop this chimera for use in targeting EGFR­positive tumours resistant to monoclonal antibodies.

Dual mTOR/PI3K inhibitor NVP­BEZ235 arrests colorectal cancer cell growth and displays differential inhibition of 4E­BP1.

The mammalian target of rapamycin (mTOR), a downstream effector of the PI3K/Akt signalling pathway, is a critical regulator of cell metabolism, growth and survival in response to oncogenic factors. Activation of mTOR frequently occurs in human tumours making it a crucial and validated target in the treatment of cancer. mTOR inhibitors such as rapamycin and its analogues decrease cancer progression in experimental models including colorectal cancer (CRC). Recently, the second generation ATP­competitive mTOR kinase (such as PP242) and dual mTOR/PI3K (such as NVP­BEZ235) inhibitors have entered clinical trials as anticancer agents. However, in CRC, the efficacy of these novel drugs needs to be fully investigated. In the present study, we examined five human CRC cell lines, HT29, HCT116, SW480, SW620 and CSC480 to evaluate their sensitivity to three mTOR inhibitors, RAD001, PP242 and NVP­BEZ235. We observed that compared to RAD001 and PP242, NVP­BEZ235 markedly reduced cell proliferation of CRC cells. Furthermore, we found that the reduced cell proliferation caused by NVP­BEZ235 was not achieved through the disruption of mitochondrial potential. Using an mTOR­specific signalling pathway phospho array we revealed that NVP­BEZ235 significantly decreased phosphorylation of 4E­BP1 (Thr70), the downstream target of mTORC1. In addition, NVP­BEZ235 decreased phosphorylation of AKT (Ser473), the downstream target of mTORC2. Immunoblotting analysis revealed that NVP­BEZ235 effectively inhibited 4E­BP1 phosphorylation, while PP242 had a weak inhibitory effect. However, PP242 and NVP­BEZ235 decreased AKT levels in all cell lines. RAD001 demonstrated no effect on 4E­BP1. Based on the above­mentioned results, the dual PI3K/mTOR and ATP­competitive mTOR inhibitors have demonstrated high potential for targeting the mTOR pathway in CRC.

Assessment of static and perfusion methods for decellularization of PCL membrane-supported periodontal ligament cell sheet constructs.

OBJECTIVES: Decellularization aims to harness the regenerative properties of native extracellular matrix. The objective of this study was to evaluate different methods of decellularization of periodontal ligament cell sheets whilst maintaining their structural and biological integrity. DESIGN: Human periodontal ligament cell sheets were placed onto melt electrospun polycaprolactone (PCL) membranes that reinforced the cell sheets during the various decellularization protocols. These cell sheet constructs (CSCs) were decellularized under static/perfusion conditions using a) 20 mM ammonium hydroxide (NH4OH)/Triton X-100, 0.5% v/v; and b) sodium dodecyl sulfate (SDS, 0.2% v/v), both +/- DNase besides Freeze-thaw (F/T) cycling method. CSCs were assessed using a collagen quantification assay, immunostaining and scanning electron microscopy. Residual fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were assessed with Bio-plex assays. RESULTS: DNA removal without DNase was higher under static conditions. However, after DNase treatment, there were no differences between the different decellularization methods with virtually 100% DNA removal. DNA elimination in F/T was less efficient even after DNase treatment. Collagen content was preserved with all techniques, except with SDS treatment. Structural integrity was preserved after NH4OH/Triton X-100 and F/T treatment, while SDS altered the extracellular matrix structure. Growth factor amounts were reduced after decellularization with all methods, with the greatest reduction (to virtually undetectable amounts) following SDS treatment, while NH4OH/Triton X-100 and DNase treatment resulted in approximately 10% retention. CONCLUSIONS: This study showed that treatment with NH4OH/Triton X-100 and DNase solution was the most efficient method for DNA removal and the preservation of extracellular matrix integrity and growth factors retention.

Violacein anticancer activity is enhanced under hypoxia.

Current cancer treatments of solid tumours such as chemotherapy and radiotherapy, have yet to produce effective therapeutic results due to non-specific targeting. This has led to many complications, such as toxicities in cancer patients. The ability of natural compounds in inducing programmed cell death (apoptosis), a process dysregulated in cancer cells, has been extensively studied in recent studies. This study assessed the anti-proliferative activity of violacein in a number of human cancer cell lines under normal and hypoxic conditions. Furthermore, we investigated its effects in a tumour­bearing subcutaneous mouse model. We also examined the ability of a tumour­targeting Salmonella strain to produce violacein for local delivery within the tumour microenvironment. The results showed that hypoxia significantly increased the cytotoxic effects of violacein. The most significant reduction in the IC50 was in the HT29 (12.6-fold) and HCT116 (4.8-fold) colon cancer cell lines, HN5 head and neck squamous carcinoma cell line (6.5-fold), and MCF-7 breast ductal carcinoma cell line (4-fold). Among the cell lines tested for active caspase-3/7 activity, violacein only increased caspase-3/7 activity in the A549 non-small lung cancer cell line. In vivo efficacy of violacein showed that HN5 tumour­bearing mice had regressed tumours during the treatment period and survival increased. The results also showed that transfer of the violacein biosynthetic cluster into the oncolytic strain VNP20009 of Salmonella resulted in the production of active violacein, suggesting targeted delivery of violacein by VNP20009. Taken together, our study has shown that hypoxia synergises the effects of violacein and the results from the in vivo administration of violacein require further investigation of violacein as an anticancer chemotherapeutic.