Using the Chick Embryo Model to Examine the Effects of Hypoxia Pre-conditioning of Uveal Melanoma Cells on Tumor Growth and Metastasis.

PURPOSE: Highly dynamic oxygen gradients occur within tumors that can result in a hypoxic response, contributing to tumor progression and metastasis. Evidence in uveal melanoma (UM) suggests an upregulated hypoxia response in some poor prognosis UM characterized by HIF1α signaling. We aimed to investigate the effects of exposure to hypoxia on tumor growth and dissemination in the chick embryo chorioallantoic membrane (CAM) model. METHODS: UM cell lines (MP41, 92.1, MP46, and OMM1) were grown in two-dimensional culture and pre-exposed to hypoxic (1% O2) conditions for 72 h. The effects of this hypoxia pre-conditioning on cell number and clonogenicity as compared with 21% O2 ("normoxia") were investigated prior to transplantation of the cells onto the CAM. Nodule-forming efficiency (NFE), nodule size, and the presence/absence of tumor cell dissemination were determined macroscopically and histologically. RESULTS: Exposure of UM cell lines to hypoxia upregulated HIF1α expression compared to cells cultured in normoxia. A 72-h pre-exposure to hypoxia significantly reduced cell number and clonogenicity in the MP41 and OMM1 cell lines while it had little effect in 92.1 and MP46 cells. When 72-h hypoxia pre-conditioned cells were grown in three-dimensions on the CAM, a reduction in NFE and nodule size was observed when compared with normoxic UM cells. All nodules were composed of proliferating (Ki-67+) Melan-A + cells and displayed chick blood vessel recruitment. Spread of UM cells into the adjacent CAM was observed; however, dissemination to the chick liver was only seen with 92.1 cells grown under normoxia. CONCLUSIONS: Hypoxia pre-conditioning does not appear to drive a metastatic phenotype in UM; however, further understanding of how oxygen dynamics within the tumor microenvironment regulates HIF1 signaling is needed to determine whether inhibitors of HIF signaling represent a therapeutic option in metastatic UM.

Carboplatin- and Etoposide-Loaded Lactoferrin Protein Nanoparticles for Targeting Cancer Stem Cells in Retinoblastoma In Vitro.

Purpose: Cancer stem cells (CSCs) are known to contribute to tumor relapses by virtue of their chemoresistance. With the knowledge that nanoformulations can overcome drug resistance, we evaluated the efficacy and cytotoxicity of clinical-grade carboplatin (CPT)- and etoposide (ETP)-loaded lactoferrin nanoparticles (Lf-Nps) on total, CD133-enriched (non-CSC), and CD133-depleted (CSC) populations of retinoblastoma (Rb) Y79 cells. Methods: Physicochemical properties of drug-loaded Lf-Nps were measured with transmission electron microscopy and attenuated total reflectance-Fourier transform infrared. The encapsulation efficiency, uptake, and release of drug-loaded Lf-Nps were measured using high-performance liquid chromatography and a UV-visible spectrophotometer. Cytotoxicity of the standard and drug-loaded Lf-Nps was evaluated by the MTT assay. Results: The mean (SD) size and encapsulation efficiency of Lf-CPT and Lf-ETP were 61.2 (3.94) nm, 60% and 45.15 (5.85) nm, 38%, respectively, and the drug release efficiency was highest at pH 6. The increased drug uptake and lower release of drug-loaded Lf-Nps were observed in CSC and non-CSC populations compared to their standard forms. The relative increase of drug uptake and sustained intracellular retention of the drug-loaded Lf-Nps compared to standard drugs showed an enhanced cytotoxicity up to 50%, especially in Rb Y79 CSCs (IC50: CPT, 230.3; Lf-CPT, 118.2; ETP, 198.1; and Lf-ETP, 129) compared to non-CSCs. Conclusions: Our study documents an increase in drug uptake, retention, and cytotoxicity of Lf-CPT and Lf-ETP on Y79 CSCs and non-CSCs as compared to their standard drugs in vitro. The reversal of chemoresistance in the CSC population by nanoformulation appears promising with the potential to pave the way for improved targeted therapy and better clinical outcomes.

Pigment Dispersing Factors and Their Cognate Receptors in a Crustacean Model, With New Insights Into Distinct Neurons and Their Functions.

Pigment dispersing factors (PDFs, or PDHs in crustaceans) form a structurally related group of neuropeptides found throughout the Ecdysozoa and were first discovered as pigmentary effector hormones in crustaceans. In insects PDFs fulfill crucial neuromodulatory roles, most notably as output regulators of the circadian system, underscoring their central position in physiological and behavioral organization of arthropods. Intriguingly, decapod crustaceans express multiple isoforms of PDH originating from separate genes, yet their differential functions are still to be determined. Here, we functionally define two PDH receptors in the crab Carcinus maenas and show them to be selectively activated by four PDH isoforms: PDHR 43673 was activated by PDH-1 and PDH-2 at low nanomolar doses whilst PDHR 41189 was activated by PDH-3 and an extended 20 residue e-PDH. Detailed examination of the anatomical distribution of all four peptides and their cognate receptors indicate that they likely perform different functions as secreted hormones and/or neuromodulators, with PDH-1 and its receptor 43,673 implicated in an authentic hormonal axis. PDH-2, PDH-3, and e-PDH were limited to non-neurohemal interneuronal sites in the CNS; PDHR 41189 was largely restricted to the nervous system suggesting a neuromodulatory function. Notably PDH-3 and e-PDH were without chromatophore dispersing activity. This is the first report which functionally defines a PDHR in an endocrine system in a crustacean and to indicate this and other putative roles of this physiologically pivotal peptide group in these organisms. Thus, our findings present opportunities to further examine the endocrine and circadian machinery in this important arthropod phylum.

Transcriptomic analysis of crustacean neuropeptide signaling during the moult cycle in the green shore crab, Carcinus maenas.

BACKGROUND: Ecdysis is an innate behaviour programme by which all arthropods moult their exoskeletons. The complex suite of interacting neuropeptides that orchestrate ecdysis is well studied in insects, but details of the crustacean ecdysis cassette are fragmented and our understanding of this process is comparatively crude, preventing a meaningful evolutionary comparison. To begin to address this issue we identified transcripts coding for neuropeptides and their putative receptors in the central nervous system (CNS) and Y-organs (YO) within the crab, Carcinus maenas, and mapped their expression profiles across accurately defined stages of the moult cycle using RNA-sequencing. We also studied gene expression within the epidermally-derived YO, the only defined role for which is the synthesis of ecdysteroid moulting hormones, to elucidate peptides and G protein-coupled receptors (GPCRs) that might have a function in ecdysis. RESULTS: Transcriptome mining of the CNS transcriptome yielded neuropeptide transcripts representing 47 neuropeptide families and 66 putative GPCRs. Neuropeptide transcripts that were differentially expressed across the moult cycle included carcikinin, crustacean hyperglycemic hormone-2, and crustacean cardioactive peptide, whilst a single putative neuropeptide receptor, proctolin R1, was differentially expressed. Carcikinin mRNA in particular exhibited dramatic increases in expression pre-moult, suggesting a role in ecdysis regulation. Crustacean hyperglycemic hormone-2 mRNA expression was elevated post- and pre-moult whilst that for crustacean cardioactive peptide, which regulates insect ecdysis and plays a role in stereotyped motor activity during crustacean ecdysis, was elevated in pre-moult. In the YO, several putative neuropeptide receptor transcripts were differentially expressed across the moult cycle, as was the mRNA for the neuropeptide, neuroparsin-1. Whilst differential gene expression of putative neuropeptide receptors was expected, the discovery and differential expression of neuropeptide transcripts was surprising. Analysis of GPCR transcript expression between YO and epidermis revealed 11 to be upregulated in the YO and thus are now candidates for peptide control of ecdysis. CONCLUSIONS: The data presented represent a comprehensive survey of the deduced C. maenas neuropeptidome and putative GPCRs. Importantly, we have described the differential expression profiles of these transcripts across accurately staged moult cycles in tissues key to the ecdysis programme. This study provides important avenues for the future exploration of functionality of receptor-ligand pairs in crustaceans.

Functional Identification and Characterization of the Diuretic Hormone 31 (DH31) Signaling System in the Green Shore Crab, Carcinus maenas.

The functional characterization of crustacean neuropeptides and their cognate receptors has not kept pace with the recent advances in sequence determination and, therefore, our understanding of the physiological roles of neuropeptides in this important arthropod sub-phylum is rather limited. We identified a candidate receptor-ligand pairing for diuretic hormone 31 (DH31) in a neural transcriptome of the crab, Carcinus maenas. In insects, DH31 plays species -specific but central roles in many facets of physiology, including fluid secretion, myoactivity, and gut peristalsis but little is known concerning its functions in crustaceans. The C. maenas DH31 transcript codes for a 147 amino acid prepropeptide, and a single receptor transcript translates to a secretin-like (Class B1) G protein-coupled receptor (GPCR). We used an in vitro aequorin luminescence Ca2+ mobilization assay to demonstrate that this candidate DH31R is activated byCarcinus and insect DH31s in a dose-dependent manner (EC50 15-30 nM). Whole mount immunohistochemical and in situ hybridization localization revealed extensive DH31 expressing neurons throughout the central nervous system, most notably in the abdominal ganglion where large, unpaired cells give rise to medial nerves, which terminate in extensive DH31 immunopositive dendritic fields intimately associated with oesophageal musculature. This system constitutes a large and hitherto undescribed neurohemal area adjacent to key muscle groups associated with the gastric system. DH31 expressing neurons were also seen in the cardiac, commissural, oesophageal, and stomatogastric ganglia and intense labeling was seen in dendrites innervating fore- and hindgut musculature but not with limb muscles. These labeling patterns, together with measurement of DH31R mRNA in the heart and hindgut, prompted us test the effects of DH31 on semi-isolated heart preparations. Cardiac superfusion with peptide evoked increased heart rates (10-100 nM). The neuroanatomical distribution of DH31 and its receptor transcripts, particularly that associated with gastric and cardiac musculature, coupled with the cardio- acceleratory effects of the peptide implicate this peptide in key myoactive roles, likely related to rhythmic coordination.

Functional Characterization and Signaling Systems of Corazonin and Red Pigment Concentrating Hormone in the Green Shore Crab, Carcinus maenas.

Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides.