The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity

Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n = 4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n = 4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6 h (n = 4), 12 h (n = 4), 24 h (n = 4) and 48 h (n = 4), and in normal control horses (n = 4). The only change in gene expression observed was an upregulation of MMP-9 (p < 0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p < 0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11–19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours.

Differential expression of chemokine and matrix re-modelling genes is associated with contrasting schistosome-induced hepatopathology in murine models

The pathological outcomes of schistosomiasis are largely dependent on the molecular and cellular mechanisms of the host immune response. In this study, we investigated the contribution of variations in host gene expression to the contrasting hepatic pathology observed between two inbred mouse strains following Schistosoma japonicum infection. Whole genome microarray analysis was employed in conjunction with histological and immunohistochemical analysis to define and compare the hepatic gene expression profiles and cellular composition associated with the hepatopathology observed in S. japonicum-infected BALB/c and CBA mice. We show that the transcriptional profiles differ significantly between the two mouse strains with high statistical confidence. We identified specific genes correlating with the more severe pathology associated with CBA mice, as well as genes which may confer the milder degree of pathology associated with BALB/c mice. In BALB/c mice, neutrophil genes exhibited striking increases in expression, which coincided with the significantly greater accumulation of neutrophils at granulomatous regions seen in histological sections of hepatic tissue. In contrast, up-regulated expression of the eosinophil chemokine CCL24 in CBA mice paralleled the cellular influx of eosinophils to the hepatic granulomas. Additionally, there was greater down-regulation of genes involved in metabolic processes in CBA mice, reflecting the more pronounced hepatic damage in these mice. Profibrotic genes showed similar levels of expression in both mouse strains, as did genes associated with Th1 and Th2 responses. However, imbalances in expression of matrix metalloproteinases (e.g. MMP12, MMP13) and tissue inhibitors of metalloproteinases (TIMP1) may contribute to the contrasting pathology observed in the two strains. Overall, these results provide a more complete picture of the molecular and cellular mechanisms which govern the pathological outcome of hepatic schistosomiasis. This improved understanding of the immunopathogenesis in the murine model schistosomiasis provides the basis for a better appreciation of the complexities associated with chronic human schistosomiasis.

Vertical Inhibition of the PI3K/Akt/mTOR Pathway for the Treatment of Osteoarthritis

Osteoarthritis is characterized by degenerative alterations of articular cartilage including both the degradation of extracellular matrix and the death of chondrocytes. The PI3K/Akt pathway has been demonstrated to involve in both processes. Inhibition of its downstream target NF-kB reduces the degradation of extracellular matrix via decreased production of matrix metalloproteinases while inhibition of mTOR increased autophagy to reduce chondrocyte death. However, mTOR feedback inhibits the activity of the PI3K/Akt pathway and inhibition of mTOR could result in increased activity of the PI3K/Akt/NF-kB pathway. We proposed that the use of dual inhibitors of PI3K and mTOR could be a promising approach to more efficiently inhibit the PI3K/Akt pathway than rapamycin or PI3K inhibitor alone and produce better treatment outcome.

The roles of the preproghrelin-derived peptides - ghrelin, desacyl ghrelin and obestatin - in prostate cancer

Prostate cancer is the second most common cause of cancer related deaths in Western men. Despite the significant improvements in current treatment techniques, there is no cure for advanced metastatic, castrate-resistant disease. Early detection and prevention of progression to a castrate-resistant state may provide new strategies to improve survival. A number of growth factors have been shown to act in an autocrine/paracrine manner to modulate prostate cancer tumour growth. Our laboratory has previously shown that ghrelin and its receptors (the functional GHS-R1a and the non-functional GHS-R1b) are expressed in prostate cancer specimens and cell lines. We have shown that ghrelin increases cell proliferation in the PC3 and LNCaP prostate cancer cell lines through activation of ERK1/2, suggesting that ghrelin could regulate prostate cancer cell growth and play a role in the progression of the disease. Ghrelin is a 28 amino-acid peptide hormone, identified to be the natural ligand of the growth hormone secretagogue receptor (GHS-R1a). It is well characterised as a growth hormone releasing and as an orexigenic peptide that stimulates appetite and feeding and regulates energy expenditure and bodyweight. In addition to its orexigenic properties, ghrelin has been shown to play a regulatory role in a number of systems, including the reproductive, immune and cardiovascular systems and may play a role in a number of pathological conditions such as chronic heart failure, anorexia, cachexia, obesity, diabetes and cancer. In cancer, ghrelin and its receptor are expressed in a range of tumours and cancer cell lines and ghrelin has been demonstrated to modulate cell proliferation, apoptosis, migration and invasion in some cell types. The ghrelin gene (GHRL) encodes preproghrelin peptide, which is processed to produce three currently known functional peptides - ghrelin, desacyl ghrelin and obestatin. Prohormone convertases (PCs) have been shown to cleave the preproghrelin peptide into two primary products - the 28 amino acid peptide, ghrelin, and the remaining 117 amino acid C-terminal peptide, C-ghrelin. C-ghrelin can then be further processed to produce the 23 amino acid peptide, obestatin. Ghrelin circulates in two different forms - an octanoylated form (known as ghrelin) and a non-octanoylated form, desacyl ghrelin. The unique post-translational addition of octanoic acid to the serine 3 residue of the propeptide chain to form acylated ghrelin is catalysed by ghrelin O-acyltransferase (GOAT). This modification is necessary for binding of ghrelin to its only known functional receptor, the GHS-R1a. As desacyl ghrelin cannot bind and activate the GHS-R1a, it was initially thought to be an inactive peptide, despite the fact that it circulates at much higher levels than ghrelin. Further research has demonstrated that desacyl ghrelin is biologically active and shares some of the actions of ghrelin, as well as having some opposing and distinct roles. Interestingly, both ghrelin and desacyl ghrelin have been shown to modulate apoptosis, cell differentiation and proliferation in some cell types, and to stimulate cell proliferation through activation of ERK1/2 and PI3K/Akt pathways. The third known peptide product of the ghrelin preprohormone, obestatin, was initially thought to oppose the actions of ghrelin in appetite regulation and food intake and to mediate its effects through the G protein-coupled receptor 39 (GPR39). Subsequent research failed to reproduce the initial findings, however, and the possible anorexigenic effects of obestatin, as well as the identity of its receptor, remain unclear. Obestatin plays some important physiological roles, including roles in improving memory, the inhibition of thirst and anxiety, increased secretion of pancreatic juice, and regulation of cell proliferation, survival, apoptosis and differentiation. Preliminary studies have also shown that obestatin stimulates cell proliferation in some cell types through activation of ERK1/2, Akt and PKC pathways. Overall, however, at the commencement of this PhD project, relatively little was known regarding the functions and mechanisms of action of the preproghrelin-derived functional peptides in modulating prostate cancer cell proliferation. The roles of obestatin, and desacyl ghrelin as potential growth factors had not previously been investigated, and the potential expression and regulation of the preproghrelin processing enzymes, GOAT and prohormone convertases was unknown in prostate cancer cell lines. Therefore, the overall objectives of this study were to: 1. investigate the effects of obestatin on cell proliferation and signaling in prostate cancer cell lines 2. compare the effects of desacyl ghrelin and ghrelin on cell proliferation and signaling in prostate cancer cell lines 3. investigate whether prostate cancer cell lines possess the necessary enzymatic machinery to produce ghrelin and desacyl ghrelin and if these peptides can regulate GOAT expression Our laboratory has previously shown that ghrelin stimulates cell proliferation in the PC3 and LNCaP prostate cancer cell line through activation of the ERK1/2 pathway. In this study it has been demonstrated that treatments with either ghrelin, desacyl ghrelin or obestatin over 72 hours significantly increased cell proliferation in the PC3 prostate cancer cell line but had no significant effect in the RWPE-1 transformed normal prostate cell line. Ghrelin (1000nM) stimulated cell proliferation in the PC3 prostate cancer cell line by 31.66 6.68% (p<0.01) with the WST-1 method, and 13.55 5.68% (p<0.05) with the CyQUANT assay. Desacyl ghrelin (1000nM) increased cell proliferation in PC3 cells by 21.73 2.62% (p<0.01) (WST-1), and 15.46 7.05% (p<0.05) (CyQUANT) above untreated control. Obestatin (1000nM) induced a 28.37 7.47% (p<0.01) (WST-1) and 12.14 7.47% (p<0.05) (CyQUANT) significant increase in cell proliferation in the PC3 prostate cancer cell line. Ghrelin and desacyl ghrelin treatments stimulated Akt and ERK phosphorylation across a range of concentrations (p<0.01). Obestatin treatment significantly stimulated Akt, ERK and PKC phosphorylation (p<0.05). Through the use of specific inhibitors, the MAPK inhibitor U0126 and the Akt1/2 kinase inhibitor, it was demonstrated that ghrelin- and obestatin-induced cell proliferation in the PC3 prostate cancer cell line is mediated through activation of ERK1/2 and Akt pathways. Although desacyl ghrelin significantly stimulated Akt and ERK phosphorylation, U0126 failed to prevent desacyl ghrelin-induced cell proliferation suggesting ghrelin and desacyl ghrelin might act through different mechanisms to increase cell proliferation. Ghrelin and desacyl ghrelin have shown a proliferative effect in osteoblasts, pancreatic -cells and cardiomyocytes through activation of ERK1/2 and PI3K/Akt pathways. Here it has been shown that ghrelin and its non-acylated form exert the same function and stimulate cell proliferation in the PC3 prostate cancer cell line through activation of the Akt pathway. Ghrelin-induced proliferation was also mediated through activation of the ERK1/2 pathway, however, desacyl ghrelin seems to stimulate cell proliferation in an ERK1/2-independent manner. As desacyl ghrelin does not bind and activate GHSR1a, the only known functional ghrelin receptor, the finding that both ghrelin and desacyl ghrelin stimulate cell proliferation in the PC3 cell line suggests that these peptides could be acting through the yet unidentified alternative ghrelin receptor in this cell type. Obestatin treatment also stimulated PKC phosphorylation, however, a direct role for this pathway in stimulating cell proliferation could not be proven using available PKC pathway inhibitors, as they caused significant cell death over the extended timeframe of the cell proliferation assays. Obestatin has been shown to stimulate cell proliferation through activation of PKC isoforms in human retinal epithelial cells and in the human gastric cancer cell line KATO-III. We have demonstrated that all of the prostate-derived cell lines examined (PC3, LNCaP, DU145, 22Rv1, RWPE-1 and RWPE-2) expressed GOAT and at least one of the prohormone convertases, which are known to cleave the proghrelin peptide, PC1/3, PC2 and furin, at the mRNA level. These cells, therefore, are likely to possess the necessary machinery to cleave the preproghrelin protein and to produce the mature ghrelin and desacyl ghrelin peptides. In addition to prohormone convertases, the presence of octanoic acid is essential for acylated ghrelin production. In this study octanoic acid supplementation significantly increased cell proliferation in the PC3 prostate cancer cell line by over 20% compared to untreated controls (p<0.01), but surprisingly, not in the DU145, LNCaP or 22Rv1 prostate cancer cell lines or in the RWPE-1 and RWPE-2 prostate-derived cell lines. In addition, we demonstrated that exogenous ghrelin induced a statistically significant two-fold decrease in GOAT mRNA expression in the PC3 cell line (p<0.05), suggesting that ghrelin could pontentially downregulate its own acylation and, therefore, regulate the balance between ghrelin and desacyl ghrelin. This was not observed, however, in the DU145 and LNCaP prostate cancer cell lines. The GOAT-ghrelin system represents a direct link between ingested nutrients and regulation of ghrelin production and the ghrelin/desacyl ghrelin ratio. Regulation of ghrelin acylation is a potentially attractive and desirable tool for the development of better therapies for a number of pathological conditions where ghrelin has been shown to play a key role. The finding that desacyl ghrelin stimulates cell proliferation in the PC3 prostate cancer cell line, and responds to ghrelin in the same way, suggests that this cell line expresses an alternative ghrelin receptor. Although all the cell lines examined expressed both GHS-R1a and GHS-R1b mRNA, it remains uncertain whether these cell lines express the unidentified alternative ghrelin receptor. It is possible that the varied responses seen could be due to the expression of different ghrelin receptors in different cell lines. In addition to GOAT, prohormone convertases and octanoic acid availability may regulate the production of different peptides from the ghrelin preprohormone. The studies presented in this thesis provide significant new information regarding the roles and mechanisms of action of the preproghrelin-derived peptides, ghrelin, desacyl ghrelin and obestatin, in modulating prostate cancer cell line proliferation. A number of key questions remain to be resolved, however, including the identification of the alternative ghrelin/desacyl ghrelin receptor, the identification of the obestatin receptor, a clarification of the signaling mechanisms which mediate cell proliferation in response to obestatin treatment and a better understanding of the regulation at both the gene and post-translational levels of functional peptide generation. Further studies investigating the role of the ghrelin axis using in vivo prostate cancer models may be warranted. Until these issues are determined, the potential for the ghrelin axis, to be recognised as a novel useful target for therapy for cancer or other pathologies will be uncertain.

The expression of plasminogen activator system in a rat model of periodontal wound healing

BACKGROUND: The plasminogen activator system has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling, including wound healing. The aim of this study was to elucidate the presence of components of the plasminogen activator system during different stages of periodontal wound healing. METHODS: Periodontal wounds were created around the molars of adult rats and healing was followed for 28 days. Immunohistochemical analyses of the healing tissues and an analysis of the periodontal wound healing fluid by ELISA were carried out for the detection of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and 2 plasminogen activator inhibitors (PAI-1 and PAI-2). RESULTS: During the early stages (days 1 to 3) of periodontal wound healing, PAI-1 and PAI-2 were found to be closely associated with the deposition of a fibrin clot in the gingival sulcus. These components were strongly associated with the infiltrating inflammatory cells around the fibrin clot. During days 3 to 7, u-PA, PAI-1, and PAI-2 were associated with cells (particularly monocytes/macrophages, fibroblasts, and endothelial cells) in the newly formed granulation tissue. During days 7 to 14, a new attachment apparatus was formed during which PAI-1, PAI-2, and u-PA were localized in both periodontal ligament fibroblasts (PDL) and epithelial cells at sites where these cells were attaching to the root surface. In the periodontal wound healing fluid, the concentration for t-PA increased and peaked during the first week. PAI-2 had a similar expression to t-PA, but at a lower level over the entire wound-healing period. CONCLUSIONS: These findings indicate that the plasminogen activator system is involved in the entire process of periodontal wound healing, in particular with the formation of fibrin matrix on the root surface and its replacement by granulation tissue, as well as the subsequent formation of the attachment of soft tissue to the root surface during the later stages of wound repair.

More light at the end of the tunnel – apixaban in atrial fibrillation

Introduction: Subjects with atrial fibrillation are at risk of thromboembolic events. The vitamin K antagonists (e.g., warfarin) are useful at preventing coagulation in atrial fibrillation, but are difficult to use. One of the FXa inhibitors, oral apixaban, has been tested as an anticoagulant in atrial fibrillation. Areas covered: In ARISTOTLE (Apixaban for reduction in stroke and other thromboembolic events in atrial fibrillation) apixaban was compared to warfarin in subjects with atrial fibrillation, and shown to cause a lower rate of stroke or systemic embolism and of major bleeding, than warfarin. In the AVERROES (Apixaban versus acetylsalicylic acid [ASA] to prevent stroke in atrial fibrillations patients who have failed or are unsuitable for vitamin K antagonist treatment) trial, stroke or systemic embolism occurred less often with apixaban than aspirin, whereas the occurrence of major bleeding was similar in the groups. Expert opinion: Apixaban is much easier for subjects with atrial fibrillation to use than warfarin, as it does not require regular monitoring by a health professional, with dosage adjustment. In addition to replacing warfarin in subjects with atrial fibrillation who are unable or not prepared to use warfarin, apixaban has the potential to replace warfarin more widely in the prevention of thromboembolism in subjects with atrial fibrillation.

Drugs and local chemical mediators

10.1 Histamine and cytokines 10.1.1 Actions of histamine 10.1.2 Drugs that modify the actions of histamine 10.1.3 Cytokines 10.2 Eicosanoids 10.2.1 Cyclooxygenase (COX) and lipooxygenase system 10.2.2 Actions of eicosanoids 10.2.3 Drugs that modify the actions of eicosanoids 10.2.3.1 Inhibit phospholipase A2 10.2.3.2 Non-selective cyclooxygenase inhibitors 10.2.3.3 Selective COX-2 inhibitors 10.2.3.4 Agonists at prostaglandin receptors 10.2.3.5 Leukotriene receptor antagonists 10.3. 5-Hydroxtryptamine (serotonin), nitric oxide, and endothelin 10.3.1 5-HT and migraine 10.3.2 5-HT and the gastrointestinal tract 10.3.3 Nitric oxide and angina 10.3.4 Nitric oxide and erectile dysfunction 10.3.5 Endothelin and pulmonary hypertension

Drugs for hypertension, angina and heart failure

12.1 Drugs for hypertension 12.1.1 Epidemiology and pathophysiology 12.1.2 Diuretics for hypertension 12.2.3 Vasodilators for hypertension 12.4.4 β-Adrenoceptor blockers for hypertension 12.2. Drugs for angina 12.2.1 Typical angina 12.2.2 Drugs to treat an attack of typical angina 12,2.3 Drugs to prevent an attack of typical angina 12.2.4 Atypical angina 12.3 Drugs for heart failure 12.3.1 The heart failure epidemic 12.3.2 Compensatory changes in heart failure 12.3.3 Diuretics for heart failure 12.3.4 ACE inhibitors and AT1-receptor antagonists 12.3.5 β-adrenoceptor antagonists 12.3.6 Digoxin

Drugs and the gastrointestinal tract

16.1. Agents to control acidity 16.1.1 Antacids 16.1.2 Proton pump inhibitors and antibiotics for Helicobacter pylori 16.1.3 Histamine H2 receptor antagonists 16.1.4 Misoprostol 16.1.5 Sucralfate 16.2. Prokinetics and emetics 16.2.1 Introduction to prokinetics 16.2.2 Prokinetic agents 16.2.3 Emesis with cytotoxic drugs and drugs for 16.2.4 Motion sickness and drugs for 16.2.5 Drugs for post-operative emesis 16.3. Agents used for diarrhea, constipation, irritable bowel syndrome 16.3.1 Treatment for diarrhea 16.3.2 Treatment for constipation 16.3.3 Treatment for opioid-induced constipation 16.4. Drugs for inflammatory bowel disease 16.4.1 Mesalazine 16.4.2 Glucocorticoids 16.4.3 Infliximab

Anti-infectives

18.1 Antibiotics 18.1.1 Introduction to bacteria 18.1.2 Introduction to antibiotics 18.1.3 Inhibitors of bacterial cell wall synthesis 18.1.3.1 β-Lactams 18.1.3.2 Glycopeptides 18.1.4 Inhibitors of bacterial protein synthesis 18.1.4.1 Tetracyclines 18.1.4.2 Aminoglycosides 18.1.4.3 Chloramphenicol 18.1.4.4 Macrolides 18.1.4.5 Lincosamides 18.1.4.6 Oxalazidones 18.1.5 Inhibitors of DNA synthesis 18.2. Anti-tuberculotic drugs 18.2.1 Introduction 18.2.2 Isoniazid 18.2.3 Ethambutol 18.2.4 Rifamycin 18.2.5 Pyrazinamide 18.3. Anti-viral drugs 18.3.1 Introduction to viruses 18.3.2 Drugs used to treat herpesviruses 18.3.3 Drugs used to treat the flu 18.3.4 Drugs used to treat HIV/AIDS 18.4. Antifungal drugs 18.4.1 Introduction to Fungi 18.4.2 Antifungal drugs

Psychotropic drugs

19.1 Depression and Antidepressants 19.1.1 Depression 19.1.2 Neurochemistry of Depression and the Monoamine Theory 19.1.3 Antidepressant Indications and Drug Classes 19.1.4 General Considerations with the use of Antidepressants 19.1.5 Tricyclic Antidepressants 19.1.6 Monoamine Oxidase Inhibitors 19.1.7 Selective Serotonin Reuptake Inhibitors 19.1.8 Combined Serotonin and Noradrenaline Reuptake Inhibitors 19.1.9 Long Term Adaptive Changes with Antidepressants 19.2 Psychosis, Schizophrenia, and Antipsychotics 19.2.1 Psychosis and Schizophrenia 19.2.2 Neurochemistry of Psychosis and the Dopamine Theory 19.2.3 Antipsychotic Drug Indications and Drug Classes 19.2.4 Antipsychotic Mechanisms of Action 19.2.5 Typical Antipsychotics (First Generation) 19.2.6 Atypical Antipsychotics (Second Generation) 19.3 Anxiety and Anxiolytics 19.3.1 Fear, Anxiety and Anxiety Disorders 19.3.2 Neurochemistry of Anxiety 19.3.3 Anxiolytic Drug Indications and Drug Classes 19.3.4 Benzodiazepines 19.3.5 Antidepressants 19.3.6 Buspirone

An introduction to anticancer drugs

An introduction to anticancer drugs 24.1 Introduction 24.2 The rationale behind anticancer drug therapy 24.3 Drugs used in cancer 24.3.1 Alkylating agents 24.3.2 Cytotoxic antibiotics 24.3.3 Antimetabolites 24.3.4 Microtubule inhibitors 24.3.5 Monoclonal antibodies 24.3.6 Steroid hormones and their antagonists 24.3.7 Other treatments

Drugs affecting blood

25. Drugs affecting blood 25.1 Introduction 25.2 Important dysfunctions of the blood system 25.3 Drugs used in to correct dysfunctions of the blood 25.3.1 Anti-thrombosis treatments 25.3.1.1 Platelet aggregation inhibitors 25.3.1.2 Anticoagulants 25.3.1.3 Thrombolytics 25.3.2 Treatments for anaemia 25.3.3 Treatments for bleeding disorders

Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status

BACKGROUND: Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines. RESULTS: The majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201. This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1. CONCLUSIONS: Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.