Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer.

The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10-50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.

Safety and pharmacokinetic studies of silodosin, a new α1A-adrenoceptor selective antagonist, in healthy Chinese male subjects.

The objectives of the study were to assess the safety and pharmacokinetics of silodosin capsules in 82 healthy male Chinese subjects. To evaluate the safety after single-dosing escalation, 40 subjects were equally divided into 4 groups (2, 4, 8, 12 mg) by a randomized, double-blind and placebo-controlled design. To assess the pharmacokinetics after single-dosing, 30 subjects were equally divided into 3 groups (4, 8, 12 mg). To assess the safety and pharmacokinetics via multiple-dosing, 12 subjects were included as a group (4 mg once daily at day 1 and day 7; 4 mg twice daily at day 2 through day 6). The safety observations showed that mild adverse events, including postural hypotension, dizziness, and headache, were observed. After single-dosing at doses of 4, 8, and 12 mg, the mean area under the concentration-time curve from 0 to 36 h (AUC(0-36)) values were 136.82±46.38, 270.17±54.66, and 474.63±108.50 µg/l·h and the mean maximal silodosin concentration in plasma (C(max)) values were 26.70±7.48, 48.47±12.35, and 94.07±22.59 µg/l, respectively. After multiple-dosing, the C(max) value at day 7 was 33.84±19.54 µg/l, and the AUC(0-24) value at day 7 was 193.19±68.96 µg/l·h. The accumulation ratio of the AUC value was 1.55 by comparing the multiple-dosing with the single-dosing. It is concluded that silodosin is safe and tolerated in healthy Chinese male subjects at the dosing levels used in this study. The mean C(max) and AUC values of silodosin increased proportionally with dose escalation, showing characteristics of linear pharmacokinetics.

Pharmacological properties of SD-282 - an alpha-isoform selective inhibitor for p38 MAP kinase.

The effects of small-molecule p38 inhibitors in numerous models of different disease states have been published, including those of SD-282, an indole-5-carboxamide inhibitor. The aim of the present study was to evaluate the pharmacological activity of SD-282 on cytokine production in vitro as well as in 2 in vivo models of inflammation in order to illuminate the role of this particular inhibitor in diverse disease states. The results presented here provide further characterization of SD-282 and provide a context in which to interpret the activity of this p38 inhibitor in models of arthritis, pain, myocardial injury, sepsis and asthma; all of which have an inflammatory component. SD-282 represents a valuable tool to elucidate the role of p38 MAP kinase in multiple models of inflammation.

Antitumor activity of TGF-beta inhibitor is dependent on the microenvironment.

Pancreatic cancer is one of the deadliest forms of cancer and effective treatment remains a clinical challenge. Transforming growth factor-beta (TGF-beta) has important roles in primary tumor progression and in promoting metastasis, and has become an attractive target for therapy. Previously, we reported that treatment of pancreatic cancer cells in vitro with SD-208, a small molecule inhibitor of the TGF-beta receptor I kinase (TGF-betaRI), inhibited expression of genes associated with tumor progression and inhibited invasiveness in a cell-based assay. In a demonstration of efficacy of TGF-beta signaling inhibition in an in vivo model of pancreatic cancer, we showed significantly reduced primary tumor weight and decreased incidence of metastasis in the Panc-1 orthotopic xenograft model of established pancreatic cancer. In this report, we extend these in vivo findings to examine the mechanistic consequences of TGF-betaRI inhibition on Panc-1 primary tumors and their microenvironment in situ. In a longitudinal study of TGF-betaRI inhibition in the Panc-1 orthotopic model, we show that SD-208 treatment significantly reduced tumor growth measured as bioluminescence intensity throughout the study. Histological evaluation revealed that SD-208 treatment reduced proliferation and induced apoptosis in the primary tumors, and reduced fibrosis in the tumor microenvironment. An immune contribution (greater B-cell infiltration in SD-208-treated tumors) was also suggested by the histological analyses. SD-208 not only blocked direct TGF-beta signaling in Panc-1 primary tumors (reduced phospho SMAD2/3), but also down-regulated the expression of TGF-beta-regulated genes (PAI-1 and COL7A1). Taken together, our results indicate that a TGF-betaRI kinase inhibitor has a potential therapeutic benefit for pancreatic cancer patients.

Transforming growth factor-beta receptor type 1 (TGFbetaRI) kinase activity but not p38 activation is required for TGFbetaRI-induced myofibroblast differentiation and profibrotic gene expression.

Transforming growth factor-beta (TGFbeta) is a major mediator of normal wound healing and of pathological conditions involving fibrosis, such as idiopathic pulmonary fibrosis. TGFbeta also stimulates the differentiation of myofibroblasts, a hallmark of fibrotic diseases. In this study, we examined the underlying processes of TGFbetaRI kinase activity in myofibroblast conversion of human lung fibroblasts using specific inhibitors of TGFbetaRI (SD-208) and p38 mitogen-activated kinase (SD-282). We demonstrated that SD-208, but not SD-282, inhibited TGFbeta-induced SMAD signaling, myofibroblast transformation, and collagen gel contraction. Furthermore, we extended our findings to a rat bleomycin-induced lung fibrosis model, demonstrating a significant decrease in the number of myofibroblasts at fibroblastic foci in animals treated with SD-208 but not those treated with SD-282. SD-208 also reduced collagen deposition in this in vivo model. Microarray analysis of human lung fibroblasts identified molecular fingerprints of these processes and showed that SD-208 had global effects on reversing TGFbeta-induced genes involved in fibrosis, inflammation, cell proliferation, cytoskeletal organization, and apoptosis. These studies also revealed that although the p38 pathway may not be needed for appearance or disappearance of the myofibroblast, it can mediate a subset of inflammatory and fibrogenic events of the myofibroblast during the process of tissue repair and fibrosis. Our findings suggest that inhibitors such as SD-208 may be therapeutically useful in human interstitial lung diseases and pulmonary fibrosis.

Preventive and therapeutic potential of p38 alpha-selective mitogen-activated protein kinase inhibitor in nonobese diabetic mice with type 1 diabetes.

Mitogen-activated protein kinases (MAPKs) and heat shock proteins (HSPs) are ubiquitous proteins that function within T cells in both normal and stress-related pathophysiological states, including type 1 diabetes. The nonobese diabetic (NOD) mouse spontaneously develops T cell-mediated autoimmune pancreatic beta cell destruction that is similar to type 1 diabetes in humans. Because p38 MAPKs have been shown to modulate T cell function, we studied the effects of a p38alpha MAPK-selective inhibitor, indole-5-carboxamide (SD-169), on the development and progression of type 1 diabetes in the NOD mouse. In preventive treatment studies, SD-169 significantly reduced p38 and HSP60 expression in T cells of the pancreatic beta islets. Following treatment, the incidence of diabetes as determined by blood glucose levels was significantly lower, and immuno-histochemistry of pancreatic beta islet tissue demonstrated significant reduction in CD5+ T cell infiltration in the SD-169 treatment group as compared with untreated NOD mice. In therapeutic studies using mildly and moderately hyperglycemic NOD mice, SD-169 treatment lowered blood glucose and improved glucose homeostasis. Furthermore, following cessation of SD-169 treatment, NOD mice showed significant arrest of diabetes. In conclusion, we report that this p38alpha-selective inhibitor prevents the development and progression of diabetes in NOD mice by inhibiting T cell infiltration and activation, thereby preserving beta cell mass via inhibition of the p38 MAPK signaling pathway. These results have bearing on current prophylactic and therapeutic protocols using p38alpha-selective inhibitors in the prediabetic period for children at high risk of type 1 diabetes, in the honeymoon period, and for adults with latent autoimmune diabetes.

SD-208, a novel transforming growth factor beta receptor I kinase inhibitor, inhibits growth and invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo.

The cytokine transforming growth factor (TGF)-beta, by virtue of its immunosuppressive and promigratory properties, has become a major target for the experimental treatment of human malignant gliomas. Here we characterize the effects of a novel TGF-beta receptor (TGF-betaR) I kinase inhibitor, SD-208, on the growth and immunogenicity of murine SMA-560 and human LN-308 glioma cells in vitro and the growth of and immune response to intracranial SMA-560 gliomas in syngeneic VM/Dk mice in vivo. SD-208 inhibits the growth inhibition of TGF-beta-sensitive CCL64 cells mediated by recombinant TGF-beta1 or TGF-beta2 or of TGF-beta-containing glioma cell supernatant at an EC(50) of 0.1 mumol/L. SD-208 blocks autocrine and paracrine TGF-beta signaling in glioma cells as detected by the phosphorylation of Smad2 or TGF-beta reporter assays and strongly inhibits constitutive and TGF-beta-evoked migration and invasion, but not viability or proliferation. Peripheral blood lymphocytes or purified T cells, cocultured with TGF-beta-releasing LN-308 glioma cells in the presence of SD-208, exhibit enhanced lytic activity against LN-308 targets. The release of interferon gamma and tumor necrosis factor alpha by these immune effector cells is enhanced by SD-208, whereas the release of interleukin 10 is reduced. SD-208 restores the lytic activity of polyclonal natural killer cells against glioma cells in the presence of recombinant TGF-beta or of TGF-beta-containing glioma cell supernatant. The oral bioavailability of SD-208 was verified by demonstrating the inhibition of TGF-beta-induced Smad phosphorylation in spleen and brain. Systemic SD-208 treatment initiated 3 days after the implantation of SMA-560 cells into the brains of syngeneic VM/Dk mice prolongs their median survival from 18.6 to 25.1 days. Histologic analysis revealed no difference in blood vessel formation, proliferation, or apoptosis. However, animals responding to SD-208 showed an increased tumor infiltration by natural killer cells, CD8 T cells, and macrophages. These data define TGF-beta receptor I kinase inhibitors such as SD-208 as promising novel agents for the treatment of human malignant glioma and other conditions associated with pathological TGF-beta activity.

Positive and negative regulation of APP amyloidogenesis by sumoylation.

Amyloid beta peptide (Abeta) generated from amyloid precursor protein (APP) is central to Alzheimer's disease (AD). Signaling pathways affecting APP amyloidogenesis play critical roles in AD pathogenesis and can be exploited for therapeutic intervention. Here, we show that sumoylation, covalent modification of cellular proteins by small ubiquitin-like modifier (SUMO) proteins, regulates Abeta generation. Increased protein sumoylation resulting from overexpression of SUMO-3 dramatically reduces Abeta production. Conversely, reducing endogenous protein sumoylation with dominant-negative SUMO-3 mutants significantly increases Abeta production. We also show that mutant SUMO-3, K11R, which can only be monomerically conjugated to target proteins, has an opposite effect on Abeta generation to that by SUMO-3, which can form polymeric chains on target proteins. In addition, SUMO-3 immunoreactivity is predominantly detected in neurons in brains from AD, Down's syndrome, and nondemented humans. Therefore, polysumoylation reduces whereas monosumoylation or undersumoylation enhances Abeta generation. These findings provide a regulatory mechanism in APP amyloidogenesis and suggest that components in the sumoylation pathway may be critical in AD onset or progression.