Nesfatin-1 is an inhibitor of the growth hormone-insulin-like growth factor axis in goldfish (Carassius auratus).

Nesfatin-1, an 82 amino acid peptide cleaved from the N-terminal of its precursor nucleobindin-2 (NUCB2), is emerging as a multifunctional peptide in fish. The present study aimed to determine whether nesfatin-1 plays a role in fish somatic growth by modulating the growth hormone (GH)/insulin-like growth factor (IGF) axis, using a representative teleost model, the goldfish (Carassius auratus). The results demonstrated that a single i.p. injection of synthetic goldfish nesfatin-1 significantly decreased the expression of hypothalamic pacap (approximately 90%) and pituitary Gh (approximately 90%) mRNAs at 15 minutes post-injection. Serum GH levels were also reduced as a result of nesfatin-1 administration, by approximately 45% and 55% at 15 and 30 minutes post-injection, respectively. Likewise, in vitro treatment of goldfish dispersed pituitary cells with nesfatin-1 reduced Gh secretion, suggesting that nesfatin-1 acts directly on pituitary somatotrophs to inhibit Gh release. Exposure of cultured liver fragments to nesfatin-1 (0.1, 1 and 10 nmol L-1 ) led to a significant reduction in igf-1 mRNA at 120 minutes and of igf-II mRNA at 30 and 60 minutes post-incubation. Collectively, these results indicate a suppressive role for nesfatin-1 on the goldfish GH/IGF axis. Immunohistochemical studies demonstrated that NUCB2/nesfatin-1-like immunoreactivity, although present in the goldfish pituitary, is not colocalised with GH in goldfish somatotrophs. Thus, nesfatin-1 does not appear to act in an autocrine manner to regulate GH secretion. Taken together, this research found that the pituitary gland is an important source of endogenous NUCB2/nesfatin-1 and also that nesfatin-1 directly suppresses the Gh/IGF axis in goldfish.

Disrupting Insulin and IGF Receptor Function in Cancer.

The insulin and insulin-like growth factor (IGF) system plays an important role in regulating normal cell proliferation and survival. However, the IGF system is also implicated in many malignancies, including breast cancer. Preclinical studies indicate several IGF blocking approaches, such as monoclonal antibodies and tyrosine kinase inhibitors, have promising therapeutic potential for treating diseases. Uniformly, phase III clinical trials have not shown the benefit of blocking IGF signaling compared to standard of care arms. Clinical and laboratory data argue that targeting Type I IGF receptor (IGF1R) alone may be insufficient to disrupt this pathway as the insulin receptor (IR) may also be a relevant cancer target. Here, we review the well-studied role of the IGF system in regulating malignancies, the limitations on the current strategies of blocking the IGF system in cancer, and the potential future directions for targeting the IGF system.

Is there a role for insulin-like growth factor inhibition in the treatment of COVID-19-related adult respiratory distress syndrome?

Adult respiratory distress syndrome (ARDS) is the leading cause of death associated with SARS-CoV-2 infection and COVID-19. IGF-1 has been implicated in ARDS, yet its role in relation to COVID-19-related lung injury has not been investigated. We hypothesize that blockage of the IGF-1 receptor (IGF-1R) mitigates lung injury and decreases the risk of death in patients COVID-19-related ARDS. Patients with fibroproliferative ARDS have been shown to have increased IGF-1 and IGF-1R staining in lung tissue specimens. Rising levels of IGF-1 in bronchioalveolar fluid (BAL) and increased IGF-1 mRNA expression in lung tissues (but declining serum IGF-1 levels) have been found in late stage ARDS compared with early lung injury. Blockage of IGF-1R decreases lung tissue damage and increases survival in bleomycin-induced as well as H1N1 influenza-related lung injury in animal models. Teprotumumab is a monoclonal antibody directed against the IGF-1R that was FDA-approved in 2020 for the treatment of Graves' orbitopathy. In order to determine if teprotumumab may reduce lung injury and death related to ARDS in the setting of COVID-19, preliminary clinical data is needed. IGF-1 levels in serum and BAL fluid must be measured in patients with COVID-19-related ARDS. Histopathology from lung samples from patients with COVID-19-related ARDS must be examined for increased expression of the IGF-1R. Once these are ascertained, and if the data support IGF-1 involvement, a randomized, placebo-controlled phase 2A trial of teprotumumab therapy in the setting of COVID-19-related ARDS and non-COVID-19-related ARDS designed to generate initial data on short-term efficacy, safety, dosing and administration should be performed.

Potential targeted therapy and diagnosis based on novel insight into growth factors, receptors, and downstream effectors in acute kidney injury and acute kidney injury-chronic kidney disease progression.

Acute kidney injury (AKI) is defined as a rapid decline in renal function and is characterized by excessive renal inflammation and programmed death of resident cells. AKI shows high morbidity and mortality, and severe or repeated AKI can transition to chronic kidney disease (CKD) or even end-stage renal disease (ESRD); however, very few effective and specific therapies are available, except for supportive treatment. Growth factors, such as epidermal growth factor (EGF), insulin-like growth factor (IGF), and transforming growth factor-ß (TGF-ß), are significantly altered in AKI models and have been suggested to play critical roles in the repair process of AKI because of their roles in cell regeneration and renal repair. In recent years, a series of studies have shown evidence that growth factors, receptors, and downstream effectors may be highly involved in the mechanism of AKI and may function in the early stage of AKI in response to stimuli by regulating inflammation and programmed cell death. Moreover, certain growth factors or correlated proteins act as biomarkers for AKI due to their sensitivity and specificity. Furthermore, growth factors originating from mesenchymal stem cells (MSCs) via paracrine signaling or extracellular vesicles recruit leukocytes or repair intrinsic cells and may participate in AKI repair or the AKI-CKD transition. In addition, growth factor-modified MSCs show superior therapeutic potential compared to that of unmodified controls. In this review, we summarized the current therapeutic and diagnostic strategies targeting growth factors to treat AKI in clinical trials. We also evaluated the possibilities of other growth factor-correlated molecules as therapeutic targets in the treatment of AKI and the AKI-CKD transition.

Therapeutic Targeting of the IGF Axis.

The insulin like growth factor (IGF) axis plays a fundamental role in normal growth and development, and when deregulated makes an important contribution to disease. Here, we review the functions mediated by ligand-induced IGF axis activation, and discuss the evidence for the involvement of IGF signaling in the pathogenesis of cancer, endocrine disorders including acromegaly, diabetes and thyroid eye disease, skin diseases such as acne and psoriasis, and the frailty that accompanies aging. We discuss the use of IGF axis inhibitors, focusing on the different approaches that have been taken to develop effective and tolerable ways to block this important signaling pathway. We outline the advantages and disadvantages of each approach, and discuss progress in evaluating these agents, including factors that contributed to the failure of many of these novel therapeutics in early phase cancer trials. Finally, we summarize grounds for cautious optimism for ongoing and future studies of IGF blockade in cancer and non-malignant disorders including thyroid eye disease and aging.

The role of vascular endothelial growth factor, interleukin 8, and insulinlike growth factor in sustaining autophagic DIRAS3-induced dormant ovarian cancer xenografts.

BACKGROUND: Re-expression of the imprinted tumor suppressor gene DIRAS family GTPase 3 (DIRAS3) (aplysia ras homology member I [ARHI]) induces autophagy and tumor dormancy in ovarian cancer xenografts, but drives autophagic cancer cell death in cell culture. The current study explored the tumor and host factors required to prevent autophagic cancer cell death in xenografts and the use of antibodies against those factors or their receptors to eliminate dormant autophagic ovarian cancer cells. METHODS: Survival factors (insulinlike growth factor 1 [IGF-1], vascular endothelial growth factor [VEGF], and interleukin 8 [IL-8]) were detected with growth factor arrays and measured using enzyme-linked immunoadsorbent assay analysis. Phosphorylation of protein kinase B (AKT), phosphorylation of extracellular signal-regulated kinase (ERK), nuclear localization of translocation factor EB (TFEB) or forkhead box O3a (FOXo3a), and expression of microtubule-associated proteins 1A/1B light chain 3B (MAPLC3B; LC3B) were examined using Western blot analysis. The effect of treatment with antibodies against survival factors or their receptors was studied using DIRAS3-induced dormant xenograft models. RESULTS: Ovarian cancer cells grown subcutaneously in nude mice exhibited higher levels of phosphorylated ERK/AKT activity and lower levels of nuclear TFEB/FOXo3a, MAPLC3B, and autophagy compared with cells grown in culture. Induction of autophagy and dormancy with DIRAS3 was associated with decreased ERK/AKT signaling. The addition of VEGF, IGF-1, and IL-8 weakened the inhibitory effect of DIRAS3 on ERK/AKT activity and reduced DIRAS3-mediated TFEB or FOXo3a nuclear localization and MAPLC3B expression in ovarian cancer cells. Treatment with antibodies against VEGF, IL-8, and IGF receptor inhibited the growth of dormant xenografts, thereby prolonging survival from 99 to >220 days (P < .05) and curing a percentage of mice. CONCLUSIONS: Treatment with a combination of anti-VEGF, anti-IL-8, and anti-IGF receptor antibodies prevented the outgrowth of dormant cells and prolonged survival in a preclinical model.

Prominin-like, a homolog of mammalian CD133, suppresses di lp6 and TOR signaling to maintain body size and weight in Drosophila.

CD133 (AC133/prominin-1) has been identified as a stem cell marker and a putative cancer stem cell marker in many solid tumors. Its biologic function and molecular mechanisms remain largely elusive. Here, we show that a fly mutant for prominin-like, a homolog of mammalian CD133, shows a larger body size and excess weight accompanied with higher fat deposits as compared with the wild type. The expression levels of prominin-like are mediated by ecdysone signaling where its protein levels increase dramatically in the fat body during metamorphosis. Prominin-like mutants exhibit higher Drosophila insulin-like peptide 6 (di lp6) levels during nonfeeding stages and increased Akt/ Drosophila target of rapamycin (dTOR) signaling. On an amino acid-restricted diet, prominin-like mutants exhibit a significantly larger body size than the wild type does, similar to that which occurs upon the activation of the dTOR pathway in the fat body. Our data suggest that prominin-like functions by suppressing TOR and dilp6 signaling to control body size and weight. The identification of the physiologic function of prominin-like in Drosophila may provide valuable insight into the understanding of the metabolic function of CD133 in mammals.-Zheng, H., Zhang, Y., Chen, Y., Guo, P., Wang, X., Yuan, X., Ge, W., Yang, R., Yan, Q., Yang, X., Xi, Y. Prominin-like, a homolog of mammalian CD133, suppresses di lp6 and TOR signaling to maintain body size and weight in Drosophila.

Insulin-like growth factor axis targeting in cancer and tumour angiogenesis - the missing link.

Numerous molecular players in the process of tumour angiogenesis have been shown to offer potential for therapeutic targeting. Initially denoted to be involved in malignant transformation and tumour progression, the insulin-like growth factor (IGF) signalling axis has been subject to therapeutic interference, albeit with limited clinical success. More recently, IGFs and their receptors have received attention for their contribution to tumour angiogenesis, which offers novel therapeutic opportunities. Here we review the contribution of this signalling axis to tumour angiogenesis, the mechanisms of resistance to therapy and the interplay with other pro-angiogenic pathways, to offer insight in the renewed interest in the application of IGF axis targeting agents in anti-cancer combination therapies.

Tumour initiating cells and IGF/FGF signalling contribute to sorafenib resistance in hepatocellular carcinoma.

OBJECTIVE: Sorafenib is effective in hepatocellular carcinoma (HCC), but patients ultimately present disease progression. Molecular mechanisms underlying acquired resistance are still unknown. Herein, we characterise the role of tumour-initiating cells (T-ICs) and signalling pathways involved in sorafenib resistance. DESIGN: HCC xenograft mice treated with sorafenib (n=22) were explored for responsiveness (n=5) and acquired resistance (n=17). Mechanism of acquired resistance were assessed by: (1) role of T-ICs by in vitro sphere formation and in vivo tumourigenesis assays using NOD/SCID mice, (2) activation of alternative signalling pathways and (3) efficacy of anti-FGF and anti-IGF drugs in experimental models. Gene expression (microarray, quantitative real-time PCR (qRT-PCR)) and protein analyses (immunohistochemistry, western blot) were conducted. A novel gene signature of sorafenib resistance was generated and tested in two independent cohorts. RESULTS: Sorafenib-acquired resistant tumours showed significant enrichment of T-ICs (164 cells needed to create a tumour) versus sorafenib-sensitive tumours (13 400 cells) and non-treated tumours (1292 cells), p<0.001. Tumours with sorafenib-acquired resistance were enriched with insulin-like growth factor (IGF) and fibroblast growth factor (FGF) signalling cascades (false discovery rate (FDR)<0.05). In vitro, cells derived from sorafenib-acquired resistant tumours and two sorafenib-resistant HCC cell lines were responsive to IGF or FGF inhibition. In vivo, FGF blockade delayed tumour growth and improved survival in sorafenib-resistant tumours. A sorafenib-resistance 175 gene signature was characterised by enrichment of progenitor cell features, aggressive tumorous traits and predicted poor survival in two cohorts (n=442 patients with HCC). CONCLUSIONS: Acquired resistance to sorafenib is driven by T-ICs with enrichment of progenitor markers and activation of IGF and FGF signalling. Inhibition of these pathways would benefit a subset of patients after sorafenib progression.

Clinical prospects of IGF-signaling system components study in ovarian cancer patients.

Among various auto/paracrine growth-regulating signaling pathways an important role belongs to that of insulin-like growth factors (IGFs) and insulin. IGF-signaling system is actively involved in the regulation of both normal ovarian function and ovarian tumor growth. On the one hand, all members of this system are expressed in malignant ovarian epithelial cells, and the prognostic significance of this expression has been revealed for some of them in ovarian cancer patients in several studies. On the other hand, circulating IGFs/IGFBPs levels have not been undoubtedly associated with ovarian cancer risk or disease progression, but some of them can be regarded as supplementary serological ovarian cancer markers. An important route to the clinical application of IGF-signaling system studies in ovarian cancer is the growing possibility of using specific molecular targeted agents to suppress its growth-stimulating and other activities. However, the introduction of such agents to practical oncology has met serious problems, with the main difficulties resulting from the absence of reliable predictive molecular markers and metabolic side effects due to the tight connection between IGF-signaling and insulin-regulated processes. The prognostic and diagnostic values of various IGF system components and the current state of corresponding molecular targeted therapies development for ovarian cancer are reviewed.

Inhibition of the insulin-like growth factor system is a potential therapy for rheumatoid arthritis.

OBJECTIVE: We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). Insulin-like growth factor binding proteins (IGFBPs) are modules of CTGF. IGFBPs bind IGF-I and IGF-II. IGF-I plays a role in the regulation of immunity, bone metabolism and inflammation. Therefore, we investigated how the IGF system is associated with RA disease progression. METHODS: Serum samples were collected from RA patients. IGF-I and IGFBP-3 production were evaluated by enzyme-linked immunosorbent assay, real-time RT-PCR and indirect immunofluorescence microscopy. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase staining, a bone resorption assay and osteoclast-specific enzyme production. Angiogenesis was examined by a tube formation assay using human umbilical vein endothelial cells. RESULTS: The serum concentrations of IGFBP-3 in RA patients were greater than those in normal controls. IGF-I and IGFBP-3 were produced primarily by macrophages in the RA synovium. Furthermore, tumor necrosis factor-α could induce aberrant IGF-I and IGFBP-3 production in synovial fibroblasts. IGF-I and IGFBP-3 promoted the induction of osteoclast generation and morphological changes, in combination with M-colony stimulating factor and the receptor activator of NF-κB ligand. In addition, IGF-I and IGFBP-3 induced angiogenesis, as determined by the tube formation assay. These effects were neutralized by anti-IGF-IR monoclonal antibody (mAb). CONCLUSIONS: These results indicate that aberrant IGF-I and IGFBP-3 production plays a role in abnormal osteoclastic activation and angiogenesis in RA. This work supports future clinical exploration of anti-IGF-IR mAb in drug repositioning as a new treatment for RA.

Bioactive insulin-like growth factors as a possible molecular target for non-islet cell tumor hypoglycemia.

Non-islet cell tumor hypoglycemia (NICTH) is a paraneoplastic syndrome characterized by persistent, severe hypoglycemia with a wide variety of solid tumors. It is considered to cause hypoglycemia by increasing the insulin-like bioactivity of the circulating insulin-like growth factor (IGF) system, however, the precise mechanism of hypoglycemia remains unclear. In this manuscript, we report on a patient suffering from NICTH caused by a small cell carcinoma of the colon. This is the first report focusing on the role of bioactive IGFs for this pathological condition. First, we demonstrated that the IGF signal pathway has been activated in this tumor in an autocrine and/or paracrine manner using immunohistochemical analysis. Second, we confirmed that bioactive IGFs in the patient's serum were increased using a modified kinase receptor activation assay, thus bioactive IGFs (mainly IGF-2) could be considered to play a major pathogenic role in enhanced hypoglycemic insulin-like activity. Third, increased IGF bioactivity in the patient's serum was completely inhibited by an anti-IGF neutralizing antibody in vitro. These results suggest that neutralization of bioactive IGFs might become a novel therapeutic strategy for NICTH to relieve the hypoglycemic symptoms together with the tumor suppressive effect.

The IGF signalling pathway in Wilms tumours--a report from the ENCCA Renal Tumours Biology-driven drug development workshop.

It is hypothesised that Wilms tumour (WT) results from aberrant renal development due to its embryonic morphology, associated undifferentiated precursor lesions (termed nephrogenic rests) and embryonic kidney-like chromatin and gene expression profiles. From the study of overgrowth syndrome-associated WT, germline dysregulation was identified in the imprinted region at 11p15 affecting imprinted genes IGF2 and H19. This is also detected in ~70% sporadic cases, making this the most common somatic molecular aberration in WT. This review summarises the critical discussion at an international workshop held under the auspices of The European Network for Cancer Research in Children and Adolescents (ENCCA) consortium, where the potential for drug development to target IGF2 and the WT epigenome was debated. Here, we consider current cancer treatments which include targeting the IGF pathway and the use of methylation agents alone or in combination with other drugs in clinical trials of paediatric cancers. Finally, we discuss the possibility of the use of these drugs to treat patients with WT.

Prospects and challenges for the development of new therapies for Ewing sarcoma.

The Ewing sarcoma family of tumors or Ewing sarcoma (ES) is the second most common malignant bone tumor of childhood. The prognosis for localized Ewing sarcoma has improved through the development of intense multimodal therapy over the past several decades. Unfortunately, patients with recurrent or metastatic disease continue to have a poor prognosis. Therefore, a number of complementary approaches are being developed in both the preclinical and clinical arenas to improve these outcomes. In this review, we will discuss efforts to directly target the biologic drivers of this disease and relate these efforts to the experience with several different agents both in the clinic and under development. We will review the data for compounds that have shown excellent activity in the clinic, such as the camptothecins, and summarize the biological data that supports this activity. In addition, we will review the clinical experience with IGF1 targeted agents, ET-743 and epigenetically targeted therapies, the substantial amount of literature that supports their activity in Ewing sarcoma and the challenges remaining translating these therapies to the clinic. Finally, we will highlight recent work aimed at directly targeting the EWS-FLI1 transcription factor with small molecules in Ewing tumors.

Deletion of muscle GRP94 impairs both muscle and body growth by inhibiting local IGF production.

Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were ∼30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.

Effect of insulin-like growth factor blockade on hyperoxia-induced lung injury.

Insulin-like growth factor (IGF)-1 is increased in different models of acute lung injury, and is an important determinant of survival and proliferation in many cells. We previously demonstrated that treatment of mice with IGF-1 receptor-blocking antibody (A12) improved early survival in bleomycin-induced lung injury. We have now examined whether administration of A12 improved markers of lung injury in hyperoxia model of lung injury. C57BL/6 mice underwent intraperitoneal administration of A12 or control antibody (keyhole limpet hemocyanin [KLH]), then were exposed to 95% hyperoxia for 88-90 hours. Mice were killed and bronchoalveolar lavage (BAL) and lung tissue were obtained for analysis. Hyperoxia caused a significant increase in IGF levels in BAL and lung lysates. Peripheral blood neutrophils expressed IGF-1R at baseline and after hyperoxia. BAL neutrophils from hyperoxia-treated mice and patients with acute lung injury also expressed cell surface IGF-1R. A12-treated mice had significantly decreased polymorphonuclear cell (PMN) count in BAL compared with KLH control mice (P = 0.02). BAL from A12-treated mice demonstrated decreased PMN chemotactic activity compared with BAL from KLH-treated mice. Pretreatment of PMNs with A12 decreased their chemotactic response to BAL from hyperoxia-exposed mice. Furthermore, IGF-1 induced a dose-dependent chemotaxis of PMNs. There were no differences in other chemotactic cytokines in BAL, including CXCL1 and CXCL2. In summary, IGF blockade decreased PMN recruitment to the alveolar space in a mouse model of hyperoxia. Furthermore, the decrease in BAL PMNs was at least partially due to a direct effect of A12 on PMN chemotaxis.

Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells.

Curcumin (diferuloylmethane) is a phenolic compound present in turmeric and is ingested daily in many parts of the world. Curcumin has been reported to cause inhibition on proliferation and induction of apoptosis in many human cancer cell lines, including non-small cell lung cancer cells (NSCLC). However, the clinical application of curcumin is restricted by its low bioavailability. In this report, it was observed that combined treatment of a low dosage of curcumin (5-10 µM) with a low concentration (0.1-2.5 µM) of small molecule inhibitors, including AG1478, AG1024, PD173074, LY294002 and caffeic acid phenethyl ester (CAPE) increased the growth inhibition in two human NSCLC cell lines: A549 and H1299 cells. The observation suggested that combined treatment of a low dosage of curcumin with inhibitors against epidermal growth factor receptor (EGFR), insulin-like growth factor 1 (IGF-1R), fibroblast growth factors receptor (FGFR), phosphatidylinositol 3-kinases (PI3K) or NF-κB signaling pathway may be a potential adjuvant therapy beneficial to NSCLC patients.

Insulin-like growth factor: current concepts and new developments in cancer therapy.

The insulin-like growth factor (IGF) family and the IGF-1 receptor (IGF-1R) play an important role in cancer. This intricate and complex signaling pathway provides many opportunities for therapeutic intervention, and several novel therapeutics aimed at the IGF-1R, particularly monoclonal antibodies and small molecule tyrosine kinase inhibitors, are under clinical investigation. This article provides a patent overview of the IGF signaling pathway and its complexity, addresses the justification for the use of IGF-1R-targeted therapy, and reviews the results of in vivo and in vitro novel therapeutics. Over the past year, the completion of several phase I, II, and III trials have provided interesting new information about the clinical activity of these novel compounds, particularly CP-751,871, IMC-A12, R1507, AMG-479, AVE-1642, MK-0646, XL-228, OSI-906, and BMS-754807. We review the important preliminary results from clinical trials with these compounds and conclude with a discussion about future therapeutic efforts.

Pharmacodynamic modeling of sequence-dependent antitumor activity of insulin-like growth factor blockade and gemcitabine.

Agents that block insulin-like growth factor (IGF) signaling are under investigation in clinical trials. Antitumor effects are likely to be enhanced when combined with other agents, but administration sequence effects on activity are not well-described. Three breast cancer cell lines (MCF-7, MDA-MB-231, and Hs-578T) were treated with Gemcitabine and small molecule receptor tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]1-(2-phenyl-quinolin-7-yl)-imidazo [1,5-a]pyrazin-8-ylamine (PQIP) as single agents and then in combination in the forward (Gemcitabine followed by PQIP) and reverse (PQIP followed by Gemcitabine) sequences. Antitumor effects were assessed longitudinally by Bayesian analysis using WinBUGS. The pharmacodynamic model adequately predicted the observed data. The differences in the cell-kill rate constants for the forward vs. reverse sequence ranged from 0.11 to 0.64 (day(-1)), and statistical significance was generally dependent on cell line and PQIP concentration. These data indicate that treatment with Gemcitabine first, followed by PQIP is superior to the reverse sequence in vitro.

Regulation of temporal and spatial organization of newborn GnRH neurons by IGF signaling in zebrafish.

When and how newborn neurons are organized to form a functional network in the developing brain remains poorly understood. An attractive model is the gonadotropin-releasing hormone (GnRH) neuron system, master regulator of the reproductive axis. Here we show that blockage of IGF signaling, a central growth-promoting signaling pathway, by the induced expression of a dominant-negative form of IGF1 receptor (IGF1R) or specific IGF1R inhibitors delayed the emergence of GnRH2 neurons in the midbrain and GnRH3 neurons in the olfactory bulb region. Blockage of IGF signaling also resulted in an abnormal appearance of GnRH3 neurons outside of the olfactory bulb region, although it did not change the locations of other olfactory neurons, GnRH2 neurons, or brain patterning. This IGF action is developmental stage-dependent because the blockade of IGF signaling in advanced embryos had no such effect. An application of phosphatidylinositol 3-kinase (PI3K) inhibitors phenocopied the IGF signaling deficient embryos, whereas the MAPK inhibitors had no effect, suggesting that this IGF action is mediated through the PI3K pathway. Real-time in vivo imaging studies revealed that the ectopic GnRH3 neurons emerged at the same time as the normal GnRH3 neurons in IGF-deficient embryos. Further experiments suggest that IGF signaling affects the spatial distribution of newborn GnRH3 neurons by influencing neural crest cell migration and/or differentiation. These results suggest that the IGF-IGF1R-PI3K pathway regulates the precise temporal and spatial organization of GnRH neurons in zebrafish and provides new insights into the regulation of GnRH neuron development.