Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor

Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent tyrosine phosphorylation of platelet proteins, and binding studies with 125I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor gamma chain, phospholipase Cgamma2, p72(SYK), c-Cbl, and p36-38. However, unlike collagen, pp125(FAK) and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72(SYK), showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the alpha2beta1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor gamma chain and, by a mechanism previously described in T- and B-cells, activate p72(SYK) that is critical for downstream activation of platelets.

An open-label, noncomparative phase II trial to evaluate the efficacy and safety of docetaxel in combination with gefitinib in patients with hormone-refractory metastatic prostate cancer

BACKGROUND: Prostate cancer is the most common type of cancer in men, however, therapeutic options are limited. 50-90% of hormone-refractory prostate cancer cells show an overexpression of epidermal growth factor receptor (EGFR), which may contribute to uncontrolled proliferation and resistance to chemotherapy. In vitro, gefitinib, an orally administered tyrosine kinase inhibitor, has shown a significant increase in antitumor activity when combined with chemotherapy. PATIENTS AND METHODS: In this phase II study, the safety and efficacy of gefitinib in combination with docetaxel, a chemotherapeutic agent commonly used for prostate cancer, was investigated in patients with hormone-refractory prostate cancer (HRPC). 37 patients with HRPC were treated continuously with gefitinib 250 mg once daily and docetaxel 35 mg/m2 i.v. for up to 6 cycles. PSA response, defined as a =50% decrease in serum PSA compared with trial entry, was the primary efficacy parameter. PSA levels were measured at prescribed intervals. RESULTS: The response rate and duration of response were consistent with those seen with docetaxel monotherapy. The combination of docetaxel and gefitinib was reasonably well tolerated in this study. CONCLUSION: Future studies should investigate whether patients with specific tumor characteristics, e.g. EGFR protein overexpression, respond better to gefitinib than patients without, leading to a more customized therapy option.

Epidermal growth factor receptor inhibitors plus chemotherapy in non-small-cell lung cancer: biologic rationale for combination strategies

Chemotherapy continues to play an essential role in the treatment of most stages of non-small-cell lung cancer (NSCLC). In fact, within the past 5 years, this role has greatly expanded into adjuvant therapy for early-stage resected disease. Likewise, agents targeting the epidermal growth factor receptor (EGFR), particularly the tyrosine kinase inhibitors gefitinib and erlotinib, have proven to be clinically active in patients with advanced-stage NSCLC. Because of these findings, it is logical to expect that combinations of these 2 classes of antineoplastic agents would prove more efficacious than either one alone. Yet 4 large randomized phase III trials of chemotherapy with or without an EGFR tyrosine kinase inhibitor in unselected patients with advanced-stage NSCLC, altogether totaling > 4000 patients, did not demonstrate improvement in clinical outcomes with the combination. Whether these negative results will be reproduced in ongoing combination studies of chemotherapy plus monoclonal antibodies directed against EGFR remain to be determined. Herein, we review recent preclinical and clinical data addressing this topic and explore the biologic rationale for developing new combination strategies based on patient selection by molecular and clinical factors, or by pharmacodynamic parameters.

Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1beta-induced prostaglandin E2 formation

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.

FTY720 suppresses interleukin-1beta-induced secretory phospholipase A2 expression in renal mesangial cells by a transcriptional mechanism

BACKGROUND AND PURPOSE: FTY720 is a potent immunomodulatory prodrug that is converted to its active phosphorylated form by a sphingosine kinase. Here we have studied whether FTY720 mimicked the action of sphingosine-1-phosphate (S1P) and exerted an anti-inflammatory potential in renal mesangial cells. EXPERIMENTAL APPROACH: Prostaglandin E(2) (PGE(2)) was quantified by an enzyme-linked immunosorbent-assay. Secretory phospholipase A(2) (sPLA(2)) protein was detected by Western blot analyses. mRNA expression was determined by Northern blot analysis and sPLA(2)-promoter activity was measured by a luciferase-reporter-gene assay. KEY RESULTS: Stimulation of cells for 24 h with interleukin-1beta (IL-1beta) is known to trigger increased PGE(2) formation which coincides with an induction of the mRNA for group-IIA-sPLA(2) and protein expression. FTY720 dose-dependently suppressed IL-1beta-induced IIA-sPLA(2) protein secretion and activity in the supernatant. This effect is due to a suppression of cytokine-induced sPLA(2) mRNA expression which results from a reduced promoter activity. As a consequence of suppressed sPLA(2) activity, PGE(2) formation is also reduced by FTY720. Mechanistically, the FTY720-suppressed sPLA(2) expression results from an activation of the TGFbeta/Smad signalling cascade since inhibition of the TGFbeta receptor type I by a specific kinase inhibitor reverses the FTY720-mediated decrease of sPLA(2) protein expression and sPLA(2) promoter activity. CONCLUSIONS AND IMPLICATIONS: In summary, our data show that FTY720 was able to mimic the anti-inflammatory activity of TGFbeta and blocked cytokine-triggered sPLA(2) expression and subsequent PGE(2) formation. Thus, FTY720 may exert additional in vivo effects besides the well reported immunomodulation and its anti-inflammatory potential should be considered.

A central role for DOCK2 during interstitial lymphocyte motility and sphingosine-1-phosphate-mediated egress

Recent observations using multiphoton intravital microscopy (MP-IVM) have uncovered an unexpectedly high lymphocyte motility within peripheral lymph nodes (PLNs). Lymphocyte-expressed intracellular signaling molecules governing interstitial movement remain largely unknown. Here, we used MP-IVM of murine PLNs to examine interstitial motility of lymphocytes lacking the Rac guanine exchange factor DOCK2 and phosphoinositide-3-kinase (PI3K)gamma, signaling molecules that act downstream of G protein-coupled receptors, including chemokine receptors (CKRs). T and B cells lacking DOCK2 alone or DOCK2 and PI3Kgamma displayed markedly reduced motility inside T cell area and B cell follicle, respectively. Lack of PI3Kgamma alone had no effect on migration velocity but resulted in increased turning angles of T cells. As lymphocyte egress from PLNs requires the sphingosine-1-phosphate (S1P) receptor 1, a G(alphai) protein-coupled receptor similar to CKR, we further analyzed whether DOCK2 and PI3Kgamma contributed to S1P-triggered signaling events. S1P-induced cell migration was significantly reduced in T and B cells lacking DOCK2, whereas T cell-expressed PI3Kgamma contributed to F-actin polymerization and protein kinase B phosphorylation but not migration. These findings correlated with delayed lymphocyte egress from PLNs in the absence of DOCK2 but not PI3Kgamma, and a markedly reduced cell motility of DOCK2-deficient T cells in close proximity to efferent lymphatic vessels. In summary, our data support a central role for DOCK2, and to a lesser extent T cell-expressed PI3Kgamma, for signal transduction during interstitial lymphocyte migration and S1P-mediated egress.

Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.

Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.

Skeletal muscle cells from insulin-resistant (non-diabetic) individuals are susceptible to insulin desensitization by palmitate

We recently demonstrated that in vivo insulin resistance is not retained in cultured skeletal muscle cells. In the present study, we tested the hypothesis that treating cultured skeletal muscle cells with fatty acids has an effect on insulin action which differs between insulin-sensitive and insulin-resistant subjects. Insulin effects were examined in myotubes from 8 normoglycemic non-obese insulin-resistant and 8 carefully matched insulin-sensitive subjects after preincubation with or without palmitate, linoleate, and 2-bromo-palmitate. Insulin-stimulated glycogen synthesis decreased by 27 +/- 5 % after palmitate treatment in myotubes from insulin-resistant, but not from insulin-sensitive subjects (1.50 +/- 0.08-fold over basal vs. 1.81 +/- 0.09-fold, p = 0.042). Despite this observation, we did not find any impairment in the PI 3-kinase/PKB/GSK-3 pathway. Furthermore, insulin action was not affected by linoleate and 2-bromo-palmitate. In conclusion, our data provide preliminary evidence that insulin resistance of skeletal muscle does not necessarily involve primary defects in insulin action, but could represent susceptibility to the desensitizing effect of fatty acids and possibly other environmental or adipose tissue-derived factors.

Cardiac toxicity with anti-HER-2 therapies: what have we learned so far?

Trastuzumab, a monoclonal antibody that blocks HER-2 receptor, improves the survival of women with HER-2-positive early and advanced breast cancer when given with chemotherapy. Lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER-2, is approved for the treatment of metastatic breast cancer patients after failure of prior anthracycline, taxanes and trastuzumab therapies in combination with capecitabine. Importantly, cardiac toxicity, manifested as symptomatic congestive heart failure or asymptomatic left ventricular ejection fraction decline, has been reported in some of the patients receiving these novel anti-HER-2 therapies, particularly when these drugs are used following anthracyclines, whose cardiotoxic potential has been recognized for decades. This review will focus on the incidence, natural history, underlying mechanisms, management, and areas of uncertainty regarding trastuzumab-and lapatinib-induced cardiotoxicity.

Novel biomarkers for the prediction of metastasis in colorectal cancer

INTRODUCTION In patients with metastatic colorectal cancers, multimodal management and the use of biological agents such as monoclonal antibodies have had major positive effects on survival. The ability to predict which patients may be at 'high risk' of distant metastasis could have major implications on patient management. Histomorphological, immunohistochemical or molecular biomarkers are currently being investigated in order to test their potential value as predictors of metastasis. AREAS COVERED Here, the author reviews the clinical and functional data supporting the investigation of three novel promising biomarkers for the prediction of metastasis in patients with colorectal cancer: tumor budding, Raf1 kinase inhibitor protein (RKIP) and metastasis-associated in colon cancer-1 (MACC1). EXPERT OPINION The lifespan of most potential biomarkers is short as evidenced by the rare cases that have successfully made their way into daily practice such as KRAS or microsatellite instability (MSI) status. Although the three biomarkers reviewed herein have the potential to become important predictive biomarkers of metastasis, they have similar hurdles to overcome before they can be implemented into clinical management: standardization and validation in prospective patient cohorts.

Targeting class IA PI3K isoforms selectively impairs cell growth, survival, and migration in glioblastoma.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110α was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110β expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110α/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110α activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110α or PI3K p110β also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110δ did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110α can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110α/p-S6.

WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells.

Members of the WD-repeat protein interacting with phosphoinositides (WIPI) family are phosphatidylinositol 3-phosphate (PI3P) effectors that are essential for the formation of autophagosomes. Autophagosomes, unique double-membraned organelles, are characteristic for autophagy, a bulk degradation mechanism with cytoprotective and homeostatic function. Both, WIPI-1 and WIPI-2 are aberrantly expressed in several solid tumors, linking these genes to carcinogenesis. We now found that the expression of WIPI-1 was significantly reduced in a large cohort of 98 primary acute myeloid leukemia (AML) patient samples (complex karyotypes; t(8;21); t(15,17); inv(16)). In contrast, the expression of WIPI-2 was only reduced in acute promyelocytic leukemia (APL), a distinct subtype of AML (t(15,17)). As AML cells are blocked in their differentiation, we tested if the expression levels of WIPI-1 and WIPI-2 increase during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of APL. According to the higher WIPI-1 expression in granulocytes compared with immature blast cells, WIPI-1 but not WIPI-2 expression was significantly induced during neutrophil differentiation of NB4 APL cells. Interestingly, the induction of WIPI-1 expression was dependent on the transcription factor PU.1, a master regulator of myelopoiesis, supporting our notion that WIPI-1 expression is reduced in AML patients lacking proper PU-1 activity. Further, knocking down WIPI-1 in NB4 cells markedly attenuated the autophagic flux and significantly reduced neutrophil differentiation. This result was also achieved by knocking down WIPI-2, suggesting that both WIPI-1 and WIPI-2 are functionally required and not redundant in mediating the PI3P signal at the onset of autophagy in NB4 cells. In line with these data, downregulation of PI3KC3 (hVPS34), which generates PI3P upstream of WIPIs, also inhibited neutrophil differentiation. In conclusion, we demonstrate that both WIPI-1 and WIPI-2 are required for the PI3P-dependent autophagic activity during neutrophil differentiation, and that PU.1-dependent WIPI-1 expression is significantly repressed in primary AML patient samples and that the induction of autophagic flux is associated with neutrophil differentiation of APL cells.

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.

Biochanin A and prunetin improve epithelial barrier function in intestinal CaCo-2 cells via downregulation of ERK, NF-κB, and tyrosine phosphorylation

The single-layered gut epithelium represents the primary line of defense against environmental stressors; thereby monolayer integrity and tightness are essentially required to maintain gut health and function. To date only a few plant-derived phytochemicals have been described as affecting intestinal barrier function. We investigated the impact of 28 secondary plant compounds on the barrier function of intestinal epithelial CaCo-2/TC-7 cells via transepithelial electrical resistance (TEER) measurements. Apart from genistein, the compounds that had the biggest effect in the TEER measurements were biochanin A and prunetin. These isoflavones improved barrier tightness by 36 and 60%, respectively, compared to the untreated control. Furthermore, both isoflavones significantly attenuated TNFα-dependent barrier disruption, thereby maintaining a high barrier resistance comparable to nonstressed cells. In docking analyses exploring the putative interaction with the tyrosine kinase EGFR, these novel modulators of barrier tightness showed very similar values compared to the known tyrosine kinase inhibitor genistein. Both biochanin A and prunetin were also identified as potent reducers of NF-κB and ERK activation, zonula occludens 1 tyrosine phosphorylation, and metalloproteinase-mediated shedding activity, which may account for the barrier-improving ability of these isoflavones.