Autocrine Pathway Research Articles

Highlights of This Issue

Gastric cancer remains a leading cause of cancer mortality worldwide. Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) is overexpressed in two-thirds of gastric cancers. In this report, Zhu and colleagues uncovered a new function for DARPP-32 by demonstrating its role in cancer cell invasion. The results indicate that DARPP-32–CXCR4 interaction stabilizes the CXCR4 protein by attenuating the CXCL-12–induced CXCR4 protein ubiquitination and degradation. The ability of DARPP-32 to bind CXCR4 and activate MT1-MMP/MMP-2 signaling provides a novel mechanism that regulates CXCR4 protein expression and cancer cell invasion.Yi and colleagues found that activated STAT3 increases RANTES expression and secreted RANTES activates STAT3 via an autocrine pathway, which promotes resistance to 4-hydroxytamoxifen. These findings suggest that combination chemotherapy with tamoxifen and a STAT3 or RANTES inhibitor may be a promising treatment for overcoming breast cancer chemoresistance in the clinic. This study therefore provides new insights that may contribute to the proposed autocrine loop of RANTES, in which tamoxifen-resistant breast cancer cells become more aggressive and maintain drug resistance through STAT3 phosphorylation and increased antiapoptotic signals.Recently, histone deacetylase 2 (HDAC2) was reported to be increased in gastric, colon, and liver cancers. However, little is known about the role of HDAC2 in carcinogenesis. The present study discovered that overexpression of HDAC2 was accompanied with the loss of cyclin-dependent kinase inhibitor CDKN2A (p16INK4a). Further studies showed that the expression of p16INK4a was transcriptionally regulated by HDAC2, as demonstrated by the direct binding of HDAC2 on the promoter of p16INK4a. Moreover, inhibition of HDAC2 restored the expression of p16INK4a; induced the apoptosis, autophagic cell death, and cell-cycle arrest in human MKN-1 gastric cancer cells in vitro; and reduced the gastric tumor growth in vivo. These findings provide molecular basis for developing novel therapeutic strategies in gastric cancer through HDAC2 inhibition.SUSD2 (Sushi Domain Containing 2) encodes a membrane surface protein with weak or no expression in normal breast epithelial cells, yet high expression in breast carcinomas. SUSD2 interacts with galectin-1, a secreted protein that promotes tumor immune evasion, angiogenesis, and metastasis. Localization of galectin-1 on the surface of cells is dependent on the presence of SUSD2. Using a syngeneic mouse model, significantly fewer CD4 tumor-infiltrating lymphocytes were present in tumors expressing Susd2 compared with the vector control. SUSD2 contributes to an immune evasion mechanism and may represent a promising therapeutic target for breast cancer.

Expression of leptin and its receptor genes in the ovarian follicles of cycling and early pregnant pigs

Leptin is a polypeptide hormone produced primarily by adipocytes. It has been implicated in the regulation of satiety and energy homeostasis. Leptin has been suggested to play a role in reproduction based on its involvement in the regulation of the hypothalamic–pituitary–gonadal axis via endocrine, paracrine and/or autocrine pathways. The aim of the present study was to localize the cellular distribution of leptin and the long isoform of leptin receptor (OB-Rb) genes in porcine ovarian antral follicles and to compare the expression levels of leptin and OB-Rb mRNAs in porcine granulosa cells (GC), theca interna (TIC) and theca externa (TEC) cells during the luteal phase of the estrous cycle and in early pregnancy. The expression of leptin and OB-Rb genes was detected in GC, TIC and TEC. Significantly higher levels of leptin gene expression in GC were observed during the mid- and late-luteal phases of the cycle than on days 30 to 32 of pregnancy. On days 14 to 16 of pregnancy, leptin mRNA expression was higher than that on days 14 to 16 of the cycle. The expression of the OB-Rb gene in GC and TEC increased during pregnancy in comparison with the analyzed luteal phases of the cycle. Our results validate the hypothesis that locally produced leptin plays a role in the regulation of porcine reproduction at the ovarian level and exerts a direct effect on porcine follicles. The differences in OB-Rb gene expression in porcine GC and theca cells also suggest that their sensitivity to leptin varies in the ovaries of pregnant and cyclic pigs.

Expression and Localization of Neuregulin-1 (Nrg1) and ErbB2/ErbB4 Receptors

BackgroundAlthough Neuregulin-1 （Nrg1）and its receptors have been indicated at the mRNA level in partial human endocrine organs and its functional roles have been evaluated in vitro, their morphological distribution in higher animals are not fully studied. The present research focused on expression of Nrg1 and its main receptors ErbB2 and ErbB4 in main endocrine organs of the rhesus monkey.Materials and MethodsThe morphological expression of Nrg1 and its receptors ErbB2 and ErbB4 as well as their potential co-localization were determined by double immunofluorescence in the pituitary, thyroid, parathyroid, pancreas and adrenal gland sample tissues. The expression level of Nrg1 on each sample was indexed by the fold of integrative fluorescence intensity (IFI) relative to that of one cortical tissue.ResultsDifferential expression of Nrg1 and their cognate receptors ErbB2 and ErbB4 were found selectively expressed in endocrine organs we tested, with higher expression levels detected in the adrenal gland (AG) and pancreas. Co-localization of Nrg1 with either ErbB2 or ErbB4 was detected in AG, thyroid and parathyroid gland, and Nrg1 was only co-localized with ErbB4 in the islet cells of the pancreas. In the pituitary, adjacent localization of Nrg1 positive cells with ErbB4 positive cells were observed.ConclusionsThis investigation morphologically profiles the differential expression of Nrg1 and its receptors ErbB2 and ErbB4 in the main endocrine organ structures, suggesting an autocrine or paracrine-directed Nrg1-ErbB signaling pathway in some of these structures.

The Role of the Intraplaque Vitamin D System in Atherogenesis

Vitamin D has been shown to play critical activities in several physiological pathways not involving the calcium/phosphorus homeostasis. The ubiquitous distribution of the vitamin D receptor that is expressed in a variety of human and mouse tissues has strongly supported research on these “nonclassical” activities of vitamin D. On the other hand, the recent discovery of the expression also for vitamin D-related enzymes (such as 25-hydroxyvitamin D-1α-hydroxylase and the catabolic enzyme 1,25-dihydroxyvitamin D-24-hydroxylase) in several tissues suggested that the vitamin D system is more complex than previously shown and it may act within tissues through autocrine and paracrine pathways. This updated model of vitamin D axis within peripheral tissues has been particularly investigated in atherosclerotic pathophysiology. This review aims at updating the role of the local vitamin D within atherosclerotic plaques, providing an overview of both intracellular mechanisms and cell-to-cell interactions. In addition, clinical findings about the potential causal relationship between vitamin D deficiency and atherogenesis will be analysed and discussed.

Pro-Neoplastic Effects of Amphiregulin in Colorectal Carcinogenesis

Epidermal growth factor (EGF) family plays critical roles in intestinal epithelial growth and transformation. Amphiregulin (AREG) is a member of the EGF family, and has been suggested to be more important to tumor versus normal growth. The precise roles of AREG in colorectal carcinogenesis have not been thoroughly elucidated. AREG expression was analyzed in colon cancer specimens using immunohistochemistry. Genetically disruption of AREG in APC (min/+) mouse was achieved by crossbreeding AREG knockout mouse with APC (min/+) mice. Knockdown AREG expression was accomplished by using plasmid-based RNA interference. Growth-stimulatory effects of AREG were determined using cell co-culture systems. AREG was expressed in both epithelial and stromal compartments in human colon cancer; however, it was regulated by different mechanisms. AREG was predominantly regulated at transcriptional level in colon cancer cells while both transcriptional and post-transcriptional mechanisms were involved in colon cancer derived myofibroblasts. Functionally, knockout of AREG strongly reduced tumorigenicity in APC (min/+) mice. Immunohistochemistry demonstrated the coordinate expression of AREG, EGF receptor activity, and cell proliferation marker in APC (min/+) mouse adenoma, indicating the growth-stimulatory function of AREG signaling in tumor development. Furthermore, we demonstrated that AREG may stimulate tumor cell growth through both autocrine and paracrine pathways in cell culture models. Knockdown of AREG impaired the ability of anchorage-independent growth of transformed intestinal epithelial cells. On the other hand, myofibroblast-produced AREG stimulated the growth of colon cancer cells when co-cultured in extracellular matrix. AREG plays pro-neoplastic roles in colorectal carcinogenesis and may be targeted for colon cancer prevention and treatment.

Differentiation-Associated Reprogramming of the Transforming Growth Factor β Receptor Pathway Establishes the Circuitry for Epithelial Autocrine/Paracrine Repair

Transforming growth factor (TGF) β has diverse and sometimes paradoxical effects on cell proliferation and differentiation, presumably reflecting a fundamental but incompletely-understood role in regulating tissue homeostasis. It is generally considered that downstream activity is modulated at the ligand:receptor axis, but microarray analysis of proliferative versus differentiating normal human bladder epithelial cell cultures identified unexpected transcriptional changes in key components of the canonical TGFβ R/activin signalling pathway associated with cytodifferentiation. Changes included upregulation of the transcriptional modulator SMAD3 and downregulation of inhibitory modulators SMURF2 and SMAD7. Functional analysis of the signalling pathway revealed that non-differentiated normal human urothelial cells responded in paracrine mode to TGFβ by growth inhibition, and that exogenous TGFβ inhibited rather than promoted differentiation. By contrast, in differentiated cell cultures, SMAD3 was activated upon scratch-wounding and was involved in promoting tissue repair. Exogenous TGFβ enhanced the repair and resulted in hyperplastic scarring, indicating a feedback loop implicit in an autocrine pathway. Thus, the machinery for autocrine activation of the SMAD3-mediated TGFβR pathway is established during urothelial differentiation, but signalling occurs only in response to a trigger, such as wounding. Our study demonstrates that the circuitry of the TGFβR pathway is defined transcriptionally within a tissue-specific differentiation programme. The findings provide evidence for re-evaluating the role of TGFβR signalling in epithelial homeostasis as an autocrine-regulated pathway that suppresses differentiation and promotes tissue repair. This provides a new paradigm to help unravel the apparently diverse and paradoxical effect of TGFβ signalling on cell proliferation and differentiation.

Modeling of Diseases of Retinal Ischemia in vitro: Possible Participation of Autocrine Vascular Endothelial Growth Factor Signaling

Purpose: To establish and evaluate a novel in vitro model of retinal ischemia, and to determine whether an autocrine pathway of retinal microvascular endothelial cells (RMVECs) by vascular endothelial growth factor (VEGF) signaling plays a role based on this model. Methods: Primary RMVECs were isolated from the retinas of C57/BL6J rats and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the biobag at the time of 12, 24, 48 and 72 h, and evaluated with a blood-gas analyzer. The control groups were incubated under normoxic conditions for the same length of time. Cell proliferation was evaluated by the CCK-8 method. Apoptosis was assayed using a flow cytometry method. RNA and protein expressions of VEGF-A, VEGFR-2 and iNOs were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Results: The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 h, the pO₂ was below 4.5 kPa, pCO₂ and pH shifted slightly. Real-time RT-PCR revealed that the expressions of VEGF-A, VEGFR-2 and iNOs mRNA in hypoxic groups increased in comparison to those in the normoxia groups (p < 0.01) and the expression of mRNA increased significantly in a time-dependent fashion in the hypoxic groups (p < 0.01), peaking at 48 h, and then decreasing. Western blot analysis revealed that the expression of relative proteins ranked in this order. CCK-8 analysis revealed that the proliferative capacity of RMVECs in the hypoxic groups was significantly higher than those in the normoxic groups at each time point (p < 0.05). At 48 h, the proliferative capacity was highest in the hypoxia groups (p < 0.05). Data acquisition from flow cytometry showed that cell survival rates in the hypoxic groups were higher than those in the normoxic groups and apoptosis rates dropped accordingly. The survival rate was highest at 48 h. Conclusion: These findings suggested that a novel in vitro model of retinal ischemia using the biobag had a good authenticity. According to the well-established in vitro hypoxia model by the biobag, RMVECs include the requisite elements for an autocrine pathway that may serve to amplify the angiogenic effects of VEGF.

Renal pericytes: regulators of medullary blood flow

Regulation of medullary blood flow (MBF) is essential in maintaining normal kidney function. Blood flow to the medulla is supplied by the descending vasa recta (DVR), which arise from the efferent arterioles of juxtamedullary glomeruli. DVR are composed of a continuous endothelium, intercalated with smooth muscle-like cells called pericytes. Pericytes have been shown to alter the diameter of isolated and in situ DVR in response to vasoactive stimuli that are transmitted via a network of autocrine and paracrine signalling pathways. Vasoactive stimuli can be released by neighbouring tubular epithelial, endothelial, red blood cells and neuronal cells in response to changes in NaCl transport and oxygen tension. The experimentally described sensitivity of pericytes to these stimuli strongly suggests their leading role in the phenomenon of MBF autoregulation. Because the debate on autoregulation of MBF fervently continues, we discuss the evidence favouring a physiological role for pericytes in the regulation of MBF and describe their potential role in tubulo-vascular cross-talk in this region of the kidney. Our review also considers current methods used to explore pericyte activity and function in the renal medulla.

Autocrine IFNγ Controls the Regulatory Function of Lymphoproliferative Double Negative T Cells

TCRαβ+ CD4−CD8−NK− double negative T cells (DN T cells) can act as regulatory T cells to inhibit allograft rejection and autoimmunity. Their role in graft-versus-host disease and mechanisms of suppression remain elusive. In this study, we demonstrate that DN T cells can inhibit CD4+ T cell-mediated GVHD in a semi-allogeneic model of bone marrow transplantation. Furthermore, we present evidence of a novel autocrine IFNγ signaling pathway in Fas-deficient C57BL/6.lpr (B6.lpr) DN T cells. B6.lpr DN T cells lacking IFNγ or its receptor were impaired in their ability to suppress syngeneic CD4+ T cells responding to alloantigen stimulation both in vitro and in vivo. Autocrine IFNγ signaling was required for sustained B6.lpr DN T cell IFNγ secretion in vivo and for upregulation of surface Fas ligand expression during TCR stimulation. Fas ligand (FasL) expression by B6.lpr DN T cells permitted lysis of activated CD4+ T cells and was required for suppression of GVHD. Collectively, our data indicate that DN T cells can inhibit GVHD and that IFNγ plays a critical autocrine role in controlling the regulatory function of B6.lpr DN T cells.

Gastric bypass surgery alters the mechanisms of insulin resistance in the adipose tissue of GK rats

Roux-en-Y gastric bypass (RYGB) has become an effective treatment for type 2 diabetes mellitus (T2DM). However, the blood glucose mechanism involved remains unclear. In this study, 60 male Goto-Kakizaki (GK) rats were randomly divided into 6 groups (each with 10 rats): the GB1/2 group (treated with gastric bypass surgery), the SO1/2 group (treated with sham operation) and the CO1/2 group (with normal water consumption only). We tested expression levels of tumor necrosis factor α (TNFα) mRNA, glucose transporter4 (GLUT4), peroxisome proliferator-activated receptorγ2 (PPARγ2) and phosphatidylinositol-3-kinase subunit p85α (PI3Kp85α) in the adipose tissues. We found that the weight, fasting blood glucose and postprandial blood glucose decreased in the GB group compared with the CO group. A total of 20 and 30 days after surgery, PPARγ2, PI3Kp85α and GLUT4 increased in the fat tissue in the GB group compared with the CO group. However, 20 days after surgery, TNFα mRNA decreased in fat tissue by 0.51 times in the GB group compared with the CO group, and serum TNFα levels showed no statistically significant difference. Fasting blood glucose and GLUT4 membrane protein were negatively correlated. In conclusion, RYGB may improve insulin resistance and treat T2DM through upregulation of the PPARγ2 protein, downregulation of TNFα mRNA transcription, through the autocrine pathway, upregulation of PI3Kp85α expression, upregulation of GLUT4 mRNA transcripts and by inducing translocation of GLUT4 in adipose tissue.

Vascular endothelial growth factor signaling in acute myeloid leukemia.

This review is designed to provide an overview of the current literature concerning vascular endothelial growth factor signaling (VEGF) in acute myeloid leukemia (AML). Aberrant VEGF signaling operates in the bone marrow of AML patients and is related to a poor prognosis. The altered signaling pathway demonstrated to interfere in several autocrine and paracrine signaling pathways. VEGF signaling promotes autocrine AML blast cell proliferation, survival, and chemotherapy resistance. In addition, VEGF signaling can mediate paracrine vascular endothelial cell-controlled angiogenesis in AML. Both effects presumably explain the association of high VEGF levels and poor therapeutic outcome. More recently, researches focusing on bone marrow stem cell niches demonstrate a role for VEGF signaling in the preservation of several cell types within these niches. The bone marrow niches are proposed to be a protective microenvironment for AML cells that could be responsible for relapses in AML patients. This implies the need of sophisticated VEGF-targeted therapeutics in AML therapy strategies. This review highlights our current understanding of aberrant VEGF signaling in AML, appoints the interference of VEGF signaling in the AML-associated microenvironment, and reflects the novelty of current VEGF-targeted therapeutics used in clinical trails for the treatment of AML.

The Hedgehog signaling pathway in ovarian teratoma is stimulated by Sonic Hedgehog which induces internalization of Patched

The Hedgehog-Gli (Hh-Gli) signaling pathway was examined in ovarian dermoids, which show characteristics of both tumors and developmental malformations. Dermoids are classified as mature teratomas that present differentiation into various tissues, mostly epidermal elements such as glands, multilayered epithelium, hair follicles and occasionally bone and cartilage. Their development is attributed to aberrant meiosis of germinal cells within the ovary. We showed activation of the Hh-Gli signaling in ovarian dermoid primary cultures. Cyclopamine treatment slows down cell proliferation, while the Sonic Hedgehog (Shh) protein stimulates cell proliferation and induces internalization of the Patched (Ptch) protein, which accumulates in the form of granules in the cytoplasm, colocalized with the Shh protein. Cyclopamine treatment decreases Gli1 localization in the nucleus compared to non-treated cells. Based on our observations, the mechanism of Hedgehog activation in the ovarian dermoids could be the ligand-dependent autocrine pathway, which can also be stimulated by paracrine signals.

Spermatogenesis in mammals: proteomic insights

Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling expression studies and/or systematic analyses of testicular proteomes in entire organs or isolated cells from various species. We consider the pros and cons of proteomics for studying the testicular germ cell gene expression program. Finally, we address the use of protein datasets, through integrative genomics (i.e., combining genomics, transcriptomics, and proteomics), bioinformatics, and modelling.

Mathematical model of heterogeneous cancer growth with an autocrine signalling pathway

Cancer is a complex biological occurrence which is difficult to describe clearly and explain its growth development. As such, novel concepts, such as of heterogeneity and signalling pathways, grow exponentially and many mathematical models accommodating the latest knowledge have been proposed. Here, we present a simple mathematical model that exhibits many characteristics of experimental data, using prostate carcinoma cell spheroids under treatment. We have modelled cancer as a two-subpopulation system, with one subpopulation representing a cancer stem cell state, and the other a normal cancer cell state. As a first approximation, these follow a logistical growth model with self and competing capacities, but they can transform into each other by using an autocrine signalling pathway. By analysing regulation behaviour of each of the system parameters, we show that the model exhibits many characteristics of actual cancer growth curves. Features reproduced in this model include delayed phase of evolving cancer under 17AAG treatment, and bi-stable behaviour under treatment by irradiation. In addition, our interpretation of the system parameters corresponds well with known facts involving 17AAG treatment. This model may thus provide insight into some of the mechanisms behind cancer.

Platelet-Mediated Inflammation in Cardiovascular Disease. Potential Role of Platelet-Endothelium Interactions

Inflammation of the vascular wall is considered as the principal underlying mechanism in the development of atherosclerosis. Besides their specific functions in haemostasis via thrombus formation after an endothelial injury, a growing body of evidence indicates that platelets play an important role in the inflammatory reactions occurring in the vascular wall as well as in the subsequent tissue repair mechanisms. Platelets interact with activated endothelium as well as with circulating leukocytes and progenitor cells. These interactions, involve direct cell-to-cell interactions as well as autocrine and paracrine pathways, which lead to activation of platelets and their respective cellular counterpart. An increasing body of evidence suggests that antiplatelet therapy may reduce vascular inflammation primarily by inhibiting platelet activation. The aim of the present review is to highlight the molecular basis of platelet-mediated inflammatory response, focusing on the mechanisms underlying the platelet-endothelial cell interaction. The anti-inflammatory effects of current antiplatelet therapies will be also discussed.

Cytokine gene expression signature in ovarian clear cell carcinoma

Cytokine expression in a tumor microenvironment can impact both host defense against the tumor and tumor cell survival. In this study, we sought to clarify whether the cytokine gene expression profile could have clinical associations with ovarian cancer. We analyzed the expression of 16 cytokine genes (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-γ, TNF-α, IL-6, HLA-DRA, HLA-DPA1 and CSF1) in 50 ovarian carcinomas. Hierarchical clustering analysis of these tumors was carried out using Cluster software and differentially expressed genes were examined between clear cell carcinoma (CCC) and other subtypes. Following this examination we evaluated the biological significance of IL-6 knockdown in CCC. Unsupervised hierarchical clustering analysis of cytokine gene expression revealed two distinct clusters. The relationship between the two clusters and clinical parameters showed statistically significant differences in CCC compared to other histologies. CCC showed a dominant Th-2 cytokine expression pattern driven largely by IL-6 expression. Inhibition of IL-6 in CCC cells suppressed Stat3 signaling and rendered cells sensitive to cytotoxic agents. The unique cytokine expression pattern found in CCC may be involved in the pathogenesis of this subtype. In particular, high IL-6 expression appears likely to be driven by the tumor cells, fueling an autocrine pathway involving IL-6 expression and Stat3 activation and may influence survival when exposed to cytotoxic chemotherapy. Modulation of IL-6 expression or its related signaling pathway may be a promising strategy of treatment for CCC.

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ≥ 98 %). All samples were analyzed following injection (100 μl) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 μm, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2–30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min−1 flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-012-9321-8) contains supplementary material, which is available to authorized users.

‘Big’-Insulin-Like Growth Factor–II Signaling Is an Autocrine Survival Pathway in Gastrointestinal Stromal Tumors

New treatment targets need to be identified in gastrointestinal stromal tumors (GISTs) to extend the treatment options for patients experiencing failure with small-molecule tyrosine kinase inhibitors, such as imatinib. Insulin-like growth factor (IGF)-II acts as an autocrine factor in several tumor types by binding to IGF receptor type 1 (IGF-1R) and/or the insulin receptor (IR) isoform A. The aim of the present study was to investigate the putative role of unprocessed pro-IGF-II, called 'big'-IGF-II, in GISTs. The imatinib-sensitive GIST882 and imatinib-resistant GIST48 cell lines secrete high levels of big-IGF-II as demonstrated by ELISA and Western blotting analyses. IR isoform A mRNA and protein expression, but not that of IGF-1R, was found in these KIT mutant cell lines and in KIT and platelet-derived growth factor receptor α-mutant GIST specimens. Down-regulation of either big-IGF-II or IR affected AKT and MAPK signaling and reduced survival in both cell lines. Disruption of big-IGF-II signaling in combination with imatinib had additive cytotoxic effects on GIST882 cells. IGF-II mRNA as determined by in situ hybridization was present in 91% of 60 primary GISTs. Immunohistochemical analysis of big-IGF-II protein expression was associated with moderate- to high-risk tumors compared with tumors with a lower risk classification (P < 0.028). Our data put forth the big-IGF-II/IR isoform A axis as an autocrine survival pathway and potential therapeutic target in GISTs.

A hedgehog survival pathway in ‘undead’ lipotoxic hepatocytes

Ballooned hepatocytes in non-alcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine whether ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro. Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using HuH-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3(-/-) mice. Ballooned hepatocytes in NASH display diminished expression of caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which induces SHH expression via AP-1. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3(-/-) cells during lipotoxic stress. Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway.

AZ64 inhibits TrkB and enhances the efficacy of chemotherapy and local radiation in neuroblastoma xenografts

Neuroblastoma is a common pediatric tumor characterized by clinical heterogeneity. Because it is derived from sympathetic neuroblasts, the NTRK family of neurotrophin receptors plays an integral role in neuroblastoma cell survival, growth, and differentiation. Indeed, high expression of NTRK1 is associated with favorable clinical features and outcome, whereas expression of NTRK2 and its ligand, brain-derived neurotrophic factor (BDNF), are associated with unfavorable features and outcome. AZ64 (Astra Zeneca) is a potent and selective inhibitor of the NTRK tyrosine kinases that blocks phosphorylation at nanomolar concentrations. To determine the preclinical activity of AZ64, we performed intervention trials in a xenograft model with NTRK2-overexpressing neuroblastomas. AZ64 alone significantly inhibited tumor growth compared to vehicle-treated animals (p = 0.0006 for tumor size). Furthermore, the combination of AZ64 with conventional chemotherapeutic agents, irinotecan and temozolomide (irino-temo), showed significantly enhanced anti-tumor efficacy compared to irino-temo alone [(p < 0.0001 for tumor size, p < 0.0005 for event-free survival (EFS)]. We also assessed the combination of AZ64 and local radiation therapy (RT) on a neuroblastoma hindlimb xenograft model, and the efficacy of local RT was significantly increased when animals were treated simultaneously with AZ64 (p < 0.0001 for tumor size, p = 0.0006 for EFS). We conclude that AZ64 can inhibit growth of NTRK-expressing neuroblastomas both in vitro and in vivo. More importantly, it can significantly enhance the efficacy of conventional chemotherapy as well as local RT, presumably by inhibition of the NTRK2/BDNF autocrine survival pathway.