Rat Pancreatic Cells Research Articles

Melatonin potentiates chemotherapy‐induced cytotoxicity and apoptosis in rat pancreatic tumor cells

Melatonin has antitumor activity via several mechanisms including its antiproliferative and proapoptotic effects in addition to its potent antioxidant action. Thus, melatonin has proven useful in the treatment of tumors in association with chemotherapeutic drugs. This study was performed to evaluate the effect of melatonin on the cytotoxicity and apoptosis induced by three different chemotherapeutic agents, namely 5-fluorouracil (5-FU), cisplatin, and doxorubicin in the rat pancreatic tumor cell line AR42J. We found that both melatonin and the three chemotherapeutic drugs induce a time-dependent decrease in AR42J cell viability, reaching the highest cytotoxic effect after 48 hr of incubation. Furthermore, melatonin significantly augmented the cytotoxicity of the chemotherapeutic agents. Consistently, cotreatment of AR42J cells with each of the chemotherapeutic agents in the presence of melatonin increased the population of apoptotic cells, elevated mitochondrial membrane depolarization, and augmented intracellular reactive oxygen species (ROS) production compared to treatment with each chemotherapeutic agent alone. These results provide evidence that in vitro melatonin enhances chemotherapy-induced cytotoxicity and apoptosis in rat pancreatic tumor AR42J cells and, therefore, melatonin may be potentially applied to pancreatic tumor treatment as a powerful synergistic agent in combination with chemotherapeutic drugs.

Rat Pancreatic Stromal Cells (PSC) affect Differentiation of Human Mesenchymal Stem Cells (hMSC) into Insulin-Producing Cells (IPCs) In vitro

The use of different sources of stem cells including embryonic and mesenchymal stem cells is a novel therapy for diabetic’s patients. However the efficiency of differentiation is not enough to complete treatment. An important point in the induction of stem cells into IPCs in vitro is the role of the pancreatic niche (which includes the stromal and epithelial niche). It can physically contact to adjacent cells and influence stem cell behavior via close range signaling. In this respect, we hypnotized that Pancreatic Stromal Cell (PSC) as a fundamental factor of the stromal niche may have an effective role in the generation IPCs (i.e, the efficiency of differentiation and function of newlyformed β-cells) in vitro. Therefore in this study, MSCs derived from umbilical cord (UC-MSCs) vain and bone marrow (BM-MSCs) was selected to differentiate into IPCs in co-culture with rat PSCs. Our results have demonstrated that only BM-MSCs were able to differentiate into IPCs. Cells in Islet-like clusters with (out) co-cultured with rat pancreatic stromal cells, produced insulin and C-peptide and released them to culture medium at the end of the induction protocol; however they did not respond to glucose challenges very well. The presence of rat pancreatic stromal cells, up-regulated the expressions of insulin, Glut2, and Nkx2.2 were at the mRNA level in IPCs. These results suggested that rat PSCs possibly affect MSCs differentiation into IPCs by increasing the number of immature β-cells.

One-step purification of functional human and rat pancreatic alpha cells

Pancreatic alpha cells contribute to glucose homeostasis by the regulated secretion of glucagon, which increases glycogenolysis and hepatic gluconeogenesis in response to hypoglycemia. Alterations of glucagon secretion are observed in diabetic patients and exacerbate the disease. The restricted availability of purified primary alpha cells has limited our understanding of their function in health and disease. This study was designed to establish convenient protocols for the purification of viable alpha cells from rat and human pancreatic islets by FACS, using intrinsic cellular properties. Islets were isolated from the pancreata of Wistar rats or deceased human organ donors. Dispersed islet cells were separated by FACS based on light scatter and autofluorescence. Purity of sorted cells was evaluated by immunocytochemistry using hormone specific antibodies. Relative hormone expression was further determined by quantitative RT-PCR. Viability was determined by Annexin V and propidium iodide staining and function was assessed by monitoring cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) using Fura-2/AM. We developed species-specific FACS gating strategies that resulted in populations consisting mainly of alpha cells (96.6 ± 1.4%, n = 3 for rat; 95.4 ± 1.7%, n = 4 for human, mean ± SEM). These cell fractions showed ~5-fold and ~4-fold enrichment (rat and human, respectively) of glucagon mRNA expression compared to total ungated islet cells. Most of the sorted cells were viable and functional, as they responded with an increase in [Ca(2+)](i) upon stimulation with L-arginine (10 mM). The majority of the sorted human alpha cells responded also to stimulation with kainate (100 μM), whereas this response was infrequent in rat alpha cells. Using the same sample preparation, but a different gating strategy, we were also able to sort rat and human populations enriched in beta cells. In conclusion, we have simplified and optimized a method for the purification of rat alpha cells, as well as established a novel approach to separate human alpha cells using neither antibodies nor dyes possibly interfering with cellular functions.

Rab3D regulates amylase levels, not agonist-induced amylase release, in AR42J cells

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.

RAS oncogenic signal upregulates EZH2 in pancreatic cancer

The neoplastic transformation by mutant RAS is thought to require remodeling of expression of an entire set of genes. However, the underlying mechanism for initiation of gene expression remodeling in tumorigenesis remains elusive. This study was aimed to define the oncogenic role of EZH2, a histone modifier protein that is induced by oncogenic mutant RAS, using pancreatic cancers of transgenic rats exogenously expressing human mutant RAS. Immunohistochemical observation of preneoplastic or cancerous lesions in the animal model suggested that upregulation of Ezh2 protein is an initiating event in pancreatic carcinogenesis. MEK-inhibition or Elk-1-knockdown downregulated EZH2, and MEK-inhibition or EZH2-knockdown restored expression of a tumor suppressor, RUNX3 in human and rat pancreatic cancer cells activated by the oncogenic RAS. Furthermore, Elk-1- or EZH2-knockdown inhibited growth of the cancer cells. These results strongly suggested that the oncogenic RAS upregulates EZH2 through MEK-ERK signaling, resulted in downregulation of tumor suppressors including RUNX3 in pancreatic carcinogenesis.

Comparison of receptor affinity of natSc-DOTA-TATE versus natGa-DOTA-TATE

44Sc as a positron emitter can be an interesting alternative to 68Ga (T½=67.71 min) due to its longer half-life (T½=3.97 h). Moreover, the b-emitter 47Sc can be used for therapy when attached to the same biomolecule vectors. DOTA as a chelating agent has been proven suitable for the radiolabelling of peptides recognising tumour cell receptors in vivo with M3+ radiometals. DOTA-derivatized peptides have been successfully labelled with 90Y and 177Lu for therapy, and with 68Ga for PET imaging. However, published data on 44Sc-labelled DOTA-biomolecules as potential PET radiotracers are still very limited. The aim of this study was to compare the affinity of natGa- and natSc-labelled DOTA-TATE to somatostatin receptors subtype 2 expressed in rat pancreatic cancer cell line AR42J. The cold complexes of DOTA-TATE with natGa and natSc were synthesized and identified by HPLC and MS analysis and evaluated in vitro for competitive binding to cancer cell line AR42J expressing somatostatin receptors subtype 2 (sstr2). The IC50 values calculated from the displacement curve of {125I-Tyr11}-SST-14 were: 0.20±0.18, 0.70±0.20, 0.64±0.22 and 0.67±0.12 for natGa-DOTA-TATE, natSc-DOTA-TATE, DOTA-TATE, and {Tyr11}-SST-14 complexes, respectively, with the affinity lowering in the decreasing order: natGa-DOTA-TATE>DOTA-TATE>Tyr11-SST-14>natSc-DOTA-TATE. The binding affinity of natGa-DOTA-TATE appeared higher than that of natSc-DOTA-TATE. Further in vitro and in vivo studies are needed to verify the influence of the chelated metal on the affinity and uptake of the respective radiolabelled compounds. This information might be crucial when the in vivo applications of peptides labelled with 68Ga and 44Sc for PET, as well as the use of 47Sc for radiotherapy are considered.

Modulation of pancreatic islets-stress axis by hypothalamic releasing hormones and 11β-hydroxysteroid dehydrogenase

Corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone (GHRH), primarily characterized as neuroregulators of the hypothalamic-pituitary-adrenal axis, directly influence tissue-specific receptor-systems for CRH and GHRH in the endocrine pancreas. Here, we demonstrate the expression of mRNA for CRH and CRH-receptor type 1 (CRHR1) and of protein for CRHR1 in rat and human pancreatic islets and rat insulinoma cells. Activation of CRHR1 and GHRH-receptor significantly increased cell proliferation and reduced cell apoptosis. CRH stimulated both cellular content and release of insulin in rat islet and insulinoma cells. At the ultrastructural level, CRHR1 stimulation revealed a more active metabolic state with enlarged mitochondria. Moreover, glucocorticoids that promote glucose production are balanced by both 11b-hydroxysteroid dehydrogenase (11β-HSD) isoforms; 11β-HSD-type-1 and 11β-HSD-type-2. We demonstrated expression of mRNA for 11β-HSD-1 and 11β-HSD-2 and protein for 11β-HSD-1 in rat and human pancreatic islets and insulinoma cells. Quantitative real-time PCR revealed that stimulation of CRHR1 and GHRH-receptor affects the metabolism of insulinoma cells by down-regulating 11β-HSD-1 and up-regulating 11β-HSD-2. The 11β-HSD enzyme activity was analyzed by measuring the production of cortisol from cortisone. Similarly, activation of CRHR1 resulted in reduced cortisol levels, indicating either decreased 11β-HSD-1 enzyme activity or increased 11β-HSD-2 enzyme activity; thus, activation of CRHR1 alters the glucocorticoid balance toward the inactive form. These data indicate that functional receptor systems for hypothalamic-releasing hormone agonists exist within the endocrine pancreas and influence synthesis of insulin and the pancreatic glucocorticoid shuttle. Agonists of CRHR1 and GHRH-receptor, therefore, may play an important role as novel therapeutic tools in the treatment of diabetes mellitus.

Targeting properties of peptide-modified radiolabeled liposomal nanoparticles

Radiolabeled PEGylated liposomal nanoparticles (NPs) open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and monitoring treatment effects. Here we describe the characterization of liposomal NPs (liposomes and micelles) derivatized with the somatostatin analogue tyrosine-3-octreotide as a proof of concept for tumor targeting. NPs were radiolabeled with indium-111, and targeting properties were evaluated in vitro on rat pancreatic tumor cells (AR42J), demonstrating specific binding and IC 50 values in the low nanomolar range. Biodistribution studies were performed in Lewis rats and compared to single-photon emission computed tomography images. Moderate tumor uptake was found in xenografted nude mice (<2.5% ID/g tissue) as compared to control. Micelles and liposomes revealed comparable pharmacokinetics and targeting properties. This study provides insight into tumor-targeting characteristics of peptide-derivatized liposomal NPs and can serve as a basis for further improvement of these constructs. From the Clinical Editor The authors investigated tumor-targeting characteristics of peptide-derivatized liposomal NPs. Similar radiolabeled PEGylated liposomal NPs open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and treatment monitoring.

Expression of C4.4A, a structural uPAR homolog, reflects squamous epithelial differentiation in the adult mouse and during embryogenesis.

The glycosylphosphatidylinositol (GPI)-anchored C4.4A was originally identified as a metastasis-associated protein by differential screening of rat pancreatic carcinoma cell lines. C4.4A is accordingly expressed in various human carcinoma lesions. Although C4.4A is a structural homolog of the urokinase receptor (uPAR), which is implicated in cancer invasion and metastasis, no function has so far been assigned to C4.4A. To assist future studies on its function in both physiological and pathophysiological conditions, the present study provide a global survey on C4.4A expression in the normal mouse by a comprehensive immunohistochemical mapping. This task was accomplished by staining paraffin-embedded tissues with a specific rabbit polyclonal anti-C4.4A antibody. In the adult mouse, C4.4A was predominantly expressed in the suprabasal layers of the squamous epithelia of the oral cavity, esophagus, non-glandular portion of the rodent stomach, anus, vagina, cornea, and skin. This epithelial confinement was particularly evident from the abrupt termination of C4.4A expression at the squamo-columnar transition zones found at the ano-rectal and utero-vaginal junctions, for example. During mouse embryogenesis, C4.4A expression first appears in the developing squamous epithelium at embryonic day 13.5. This anatomical location of C4.4A is thus concordant with a possible functional role in early differentiation of stratified squamous epithelia.

Effect of EGCG on Expression of Neurogenin 3 via the MAP Kinase Signaling Pathway in AR42J Cells, a Rat Pancreatic Tumor Cell Line

Epigallocatechin gallate (EGCG), or epigallocatechin 3-gallate. is the ester of epigallocatcchin and gallic acid, and is a type of catec hin. EGCG may be therapeutic for many disorders including diabetic s and some types of cancer. Howev-er it is unknown whet her EGCG ca n induce transdifferentiation of pancreatic cells in pancreatitis. The aim of this study was to investigate the effects of EGCG on the expression of pancreatic regenerating related markers in pancreatic AR42J cells, a model of pancreatic progenitor cells. AR42J cells. differentiated with betacellulin and activin A, were cultured with/without EGCG in a time-dependent manner. Cell growth rate, levels of mRNA. and protein expression were examined with the MTT assay, quantitative PCR, and Western blots, respectively. The results showed that AR42J cell growth rates were inhibited by EGCG in a dose-dependent manner. mRNA and protein expression of amylase, insulin and neurogenin 3 (ngn 3) increased in AR42J cells treated with EGCG. Additionally, we demonstrated that the signal transduction pathway of mitogen-activated protein (MA P) kinase is active in EGCG-treated AR42J cells. ERK and JNK phosphorylation decreased in cells treated with EGCG but not p38 phosphorylation. Activation of the p38 MAP kinase pathway was confirmed by specific MAP kinase pathways inhibitors: U0126 for ERK, SP600126 for INK, and SB203580 for p38. Activated p38 phosphorylation was inhibited by the specific p38 inhibitor SB203580 but p38 phosphorylation was inhibit ed with increased EGCG treatment. The ERK and JNK MAP kinase pathways were not affected by EGCG treatment. Although further studies are needed, these result s suggest that EGCG affects the induction of pancreatic cell regeneration by increasing the ngn 3 protein and mRNA expression and activating the p38 MAP kinase pathway.

CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet β-cells mediated by hepatocyte growth factor

We have previously reported that the PI3K/Akt signaling pathway is involved in hepatocyte growth factor (HGF)-induced differentiation of adult rat pancreatic ductal epithelial cells (PDECs) into islet β-cells in vitro. The transcription factor CREB is one of the downstream key effectors of the PI3K/Akt signaling pathway. Recent studies showing that CREB is required for the survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the HGF-dependent Ser/Thr kinase Akt/PKB in the differentiation of pancreatic duct cell into islet β-cells. In this study, we first attempted to examine whether HGF modulates the Akt-dependent activation of target gene CREB and then investigated whether CREB activity affects the differentiation of HGF-induced PDECs. Finally, we studied the role of CREB in modulating the expression of transcription factors in PDECs during the differentiation of HGF-induced PDECs. Our results demonstrated that CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet β-cells mediated by HGF.

Pancreatic Islet Derived Stem Cells Can Express Co-stimulatory Molecules of Antigen-Presenting Cells

Background Antigen-presenting cells (APCs) are crucial intermediates in the generation of both innate and specific immune responses. It has long been understood that some APCs are resident in islets in situ as well as after isolation. Our aim was to investigate the presence of molecules involved in antigen presentation in rat pancreatic islet-derived stem cells (PI-SCs). Methods We used immunocytochemistry and reverse transcription polymenzation chain reaction to study immunophenotypic characteristics; pluripotent-related gene expressions; transcripts coding for antigen-presenting surface proteins CD40, CD80, CD86; and major histocompatibility complex class II in addition to genes with known antiapoptotic functions including mitogen-activated protein kinase-activated protein kinase 2 ( MAPKAPK2), tumor necrosis factor alpha-induced protein 3 (TNFAIP3) interacting protein 1 ( TNIP1) and BCL3 of the PI-SCs. Results Rat PI-SCs were negative for CD45 as demonstrated by flow cytometry and for CD31, CD34, and CD71 as demonstrated by immunocytochemistry. Therefore, there was no evidence of hematopoietic precursors in the cultures. OCT4, SOX2, and REX1 were expressed by rat PI-SCs. We determined the expression of genes for antigen-presenting surface proteins CD40 and CD80, and genes with known antiapoptotic functions including MAPKAPK2, TNIP1 and BCL3, besides the surface protein, CD80, by flow cytometry. Conclusion Expression of these genes by rat PI-SCs implied that they could be involved in the regulation of immunity in islets, highlighting the influence of protective role-playing antiapoptotic mechanisms on pancreatic islet cells. This study offers the potential to understand the molecular mechanisms of a devastating disease, type-1 diabetes mellitus.

Distribution patterns of calcium-binding proteins in pancreatic tissue of non-diabetic as well as type 2 diabetic rats and in rat insulinoma β-cells (INS-1)

The present study dealt with the localization of different calcium-binding proteins (CaBPs) in the pancreatic tissue of non-diabetic and diabetic rats and in rat insulinoma beta-cells (INS-1). Transcripts of CaBPs displayed different expression levels in rat pancreatic tissue and INS-1 cells. Immunohistochemistry demonstrated that three of these proteins, calmodulin, calreticulin and calbindin-D28k, were located predominantly in the pancreatic islets (in both alpha- and beta-cells) of rats, showing weaker labeling of exocrine tissue. Secretagogin was exclusively found within islets. All CaBPs were also immunohistochemically detected in INS-1 cells. Immunohistochemical analysis demonstrates differences in CaBP distributions when comparing the pancreatic tissues of diabetic Goto-Kakizaki rats and non-diabetic Wistar rats. Pancreatic tissue in type 2 diabetic Goto-Kakizaki rats showed significantly higher transcript levels of all CaBPs compared to those in Wistar rats. These results indicate that alterations of CaBPs in pancreatic islets are associated with metabolic disturbances related to type 2 diabetes.

In vitro structure–activity relationship of Re-cyclized octreotide analogues

Development of radiolabeled octreotide analogues is of interest for targeting somatostatin receptor (SSTR)-positive tumors for diagnostic and therapeutic purposes. We are investigating a direct labeling approach for incorporation of a Re ion into octreotide analogues, where the peptide sequences are cyclized via coordination to Re rather than through a disulfide bridge. Various octreotide analogue sequences and coordination systems (e.g., S(2)N(2) and S(3)N) were synthesized and cyclized with nonradioactive Re. In vitro competitive binding assays with (111)In-DOTA-Tyr(3)-octreotide in AR42J rat pancreatic tumor cells yielded IC(50) values as a measure of SSTR affinity of the Re-cyclized analogues. Three-dimensional structures of Re-cyclized Tyr(3)-octreotate and its disulfide-bridged analogue were calculated from two-dimensional NMR experiments to visualize the effect of metal cyclization on the analogue's pharmacophore. Only two of the 11 Re-cyclized analogues investigated showed moderate in vitro binding affinity toward somatostatin subtype 2 receptors. Three-dimensional molecular structures of Re- and disulfide-cyclized Tyr(3)-octreotate were calculated, and both of their pharmacophore turns appear to be very similar with minor differences due to metal coordination to the amide nitrogen of one of the pharmacophore amino acids. Various Re-cyclized analogues were developed and analogue 4 had moderate affinity toward somatostatin subtype 2 receptors. In vitro stable studies that are in progress showed stable radiometal cyclization of octreotide analogues via NS(3) and N(2)S(2) coordination forming five- and six-membered chelate rings. In vivo biodistribution studies are underway of (99m)Tc-cyclized analogue 4.

Synthesis of DOTA-Conjugated Multimeric [Tyr3]Octreotide Peptides via a Combination of Cu(I)-Catalyzed “Click” Cycloaddition and Thio Acid/Sulfonyl Azide “Sulfo-Click” Amidation and Their in Vivo Evaluation

Herein, we describe the design, synthesis, and biological evaluation of a series of DOTA-conjugated monomeric, dimeric, and tetrameric [Tyr(3)]octreotide-based analogues as a tool for tumor imaging and/or radionuclide therapy. These compounds were synthesized using a Cu(I)-catalyzed 1,3-dipolar cycloaddition ("click" reaction) between peptidic azides and dendrimer-derived alkynes and a subsequent metal-free introduction of DOTA via the thio acid/sulfonyl azide amidation ("sulfo-click" reaction). In a competitive binding assay using rat pancreatic AR42J tumor cells, the monomeric [Tyr(3)]octreotide conjugate displayed the highest binding affinity (IC(50) = 1.32 nM) followed by dimeric [Tyr(3)]octreotide (2.45 nM), [DOTA(0),Tyr(3)]octreotide (2.45 nM), and tetrameric [Tyr(3)]octreotide (14.0 nM). Biodistribution studies with BALB/c nude mice with subcutaneous AR42J tumors showed that the (111)In-labeled monomeric [Tyr(3)]octreotide conjugate had the highest tumor uptake (42.3 +/- 2.8 %ID/g) at 2 h p.i., which was better than [(111)In-DOTA(0),Tyr(3)]octreotide (19.5 +/- 4.8 %ID/g). The (111)In-labeled dimeric [Tyr(3)]octreotide conjugate showed a long tumor retention (25.3 +/- 5.9 %ID/g at 2 h p.i. and 12.1 +/- 1.3 %ID/g at 24 h p.i.). These promising results can be exploited for therapeutic applications.

Expression of fatty acid binding proteins 3 and 5 genes in rat pancreatic islets and INS-1E cells: Regulation by fatty acids and glucose

Fatty acids binding proteins (FABPs) are involved in uptake, binding, transport and metabolism of fatty acids (FAs). Although FAs are known to stimulate insulin secretion from pancreatic islets when transiently elevated, while contributing to islet loss of function, lipotoxicity and apoptosis when chronically elevated, almost nothing is known regarding the FABPs in this tissue. The present study aimed at exploring the expression pattern and regulation of FABPs in rat islets and the insulin-secreting INS-1E cells. Rat islets and INS-1E cells expressed the heart/muscle type (FABP3) and the epidermal type (FABP5) genes. Different FAs significantly enhanced the expression of both FABPs. High glucose concentration induced a similar elevation of both FABP mRNA levels, and similarly to its effect on insulin 1 mRNA. Addition of oleic or palmitic acids to glucose did not render a further effect. FABP3 gene expression increased in response to PPARα agonist, while FABP5 increased in response to PPARα and PPARγ agonists, and decreased in response to a PPARβ agonist. Beta-oxidation of FAs is required for the gene expression of both FABP genes in INS-1E cells. Inhibition of CPT-1 by etomoxir inhibited the oleic acid-induced FABP 3 and 5 gene expression, while activation of AMPK by metformin amplified the oleic-induced expression of both FABPs. FABP3 and 5 gene transcription required de novo protein synthesis, since inhibition by cycloheximide significantly decreased both FABP mRNAs. These data show a complex interrelationship between glucose and FAs in the control of FABP gene expression and that FABP3 and 5 may play a role in insulin secretion.

Abstract 5770: MDM2 protein variants expression in the plasma membrane of cancer cells: A target for anticancer peptide PNC-27

Abstract PNC-27 anti-cancer peptide, derived from the MDM2 binding site of p53 and linked to a membrane residency peptide (MRP), has been shown to cause necrosis of cancer cells without affecting untransformed cells. PNC-27 was also able to eradicate pancreatic tumor xenografts in mice (Michl et al, Int. J of Cancer, 2006). Recently, we have identified the mechanism of action of this peptide as due to formation of oligomeric pores in the plasma membrane (PM) of cancer cells but not in the PM of untransformed cells. The mechanism of pore-formation by PNC-27 closely resembles the pore-formation process by streptolysin-O, melittin and similar pore-forming agents. We have shown MDM2 as a targeting molecule that leads to PNC-27 selectivity towards cancer cells by its mis-localization to cancer cell PM (Sarafraz-Yazdi et al, PNAS, in press). Examining purified PM of a variety of cancer cells by immunoblotting, we now provide evidence for multiple MDM2 protein variants, and that were absent in the PM of normal untransformed cells. To confirm the purity of the isolated PM, fractions were also immunoblotted for the PM markers Na+/K+-ATPase, E-Cadherin and ß-Catenin, all of which were enriched in the PM fractions. In contrast, Abs against membrane markers specific for intracellular organelles, COX IV and Cytochrome C for mitochondrial membrane, showed no reaction in the PM fractions while they reacted with total membrane fractions. Three protein variants of MDM2 with MW of 27kD, 40kD and 57kD were consistently expressed in the PM of human and rat pancreatic cancer cells, human melanoma, breast cancer. We also confirmed these observations in isolated PM from freshly obtained primary ovarian tumors from human patients with aggressive tumor. Remarkably, no MDM2 variant was detectable in the PM fractions of primary human fibroblasts and untransformed pancreatic HPNE cells. Our finding of differently sized MDM2 variants complies with other, previously reported MDM2 protein variants. Of the different MDM2 mRNA splice variants published, 5 have been shown to be translated into protein including proteins of 27kD, 40kD and 57kD. Our present study demonstrates the localization of the 3 variant proteins in the PM of cancer cells, suggesting a possible role for these membrane-localized MDM2 variant in the action of PNC-27. The notion of this specific MDM2-PNC-27 interaction at the PM level was strongly supported by the effective competition by a monoclonal MDM2-specific Ab, reducing PNC-27-mediated cytotoxicity by &amp;gt;80% as measured by LDH cytotoxicity assay and propidium iodide staining of the nuclei of dead cells in real-time spinning disc confocal microscopy. These findings for the first time demonstrate not only the novel localization of different MDM2 variants in PM of different cancer cells but they also suggest a potentially wider applicability of PNC-27 as a novel selective anti-cancer drug for clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5770.

Effects of Aminoguanidine (AG) in a Rat Pancreatic Tumor Cell line

Aminoguanidine is a compound that reportedly exerts both pro‐ and antioxidant effects. In animal models and in humans it was shown as an antioxidant against lipid peroxidation. The current study evaluated the direct effect of AG on rat pancreatic tumor cell.MethodsAR42J cells were grown in F‐12 nutrient medium at 370C and 5% CO2. At log phase, they were separated, sub cultured for 3 days at specified dose and time with hydrogen peroxide and aminoguanidine. The cellular extracts were subjected to Western blotting employing antibodies to ERK ½ and analyzed further by immunohistochemistry. Cell proliferation lipid peroxidation assay was also conducted. Cell functional assay was conducted with CCK‐8 (10–10 M) incubated for 30 min at 370C and was assessed by the amount of amylase released in the media.ResultsIncubation of AR42J cells with aminoguanidine induced 3–5 fold activation of phospho ERK‐1/2. This was confirmed by immunohistochemistry with FITC conjugated antibody to ERK1/2. CCK‐stimulated amylase release was enhanced in the presence of aminoguanidine.ConclusionThese data suggest that ERK‐1/2, a proliferative signal of MAPK pathway, is induced and enhanced in AR42J cells by aminoguanidine. This is coupled with enhanced cell function and proliferation suggesting that aminoguanidine acts as a pro‐oxidant in this cell line.

CD44, a therapeutic target for metastasising tumours

Members of the CD44 family of transmembrane glycoproteins, in particular CD44v6 isoforms, were shown to be metastatic determinants of rat pancreatic tumour cells back in the early 1990s. Furthermore, the expression of several CD44 proteins correlates with aggressive stages of various human cancers. Because of the frequent and homogeneous expression of CD44v6 isoforms in squamous cell carcinoma, antibodies recognising these proteins were used in clinical trials for patients suffering from head and neck squamous cell carcinoma (HNSCC). Although the phase I clinical trials looked promising, the studies were abruptly ended after the death of a patient. Despite the termination of the trials, CD44 certainly remains a valid target for anti-cancer therapy. In this review, alternative strategies targeting CD44 functions are presented. These functions include the binding to hyaluronan (HA), the collaboration with osteopontin and the contribution of CD44 isoforms to receptor tyrosine kinase (RTKs) activation. These new attempts led to the development of peptides that interfere for example with HA binding and that might be used to induce apoptosis in mammary carcinoma or to prevent homing of leukaemia stem cells. Other peptides block RTK activation and thereby inhibit tumour angiogenesis and metastatic spread.

In Vivo Molecular Imaging of Somatostatin Receptors in Pancreatic Islet Cells and Neuroendocrine Tumors by Miniaturized Confocal Laser-Scanning Fluorescence Microscopy

The aim of the study was to evaluate real time in vivo molecular imaging of somatostatin receptors (sstrs) using a handheld miniaturized confocal laser scan microscope (CLM) in conjunction with fluorescein-labeled octreotate (OcF) in healthy mice and murine models of neuroendocrine tumors. For CLM a small rigid probe (diameter 7 mm) with an integrated single line laser (488 nm) was used (optical slice thickness 7 mum; lateral resolution 0.7 mum). OcF was synthesized via Fmoc solid-phase peptide synthesis and purified by HPLC showing high-affinity binding to the sstr2 (IC(50) 6.2 nmol). For in vitro evaluation, rat and human pancreatic cancer cells were used and characterized with respect to its sstr subtype expression and functional properties. For in vivo confocal imaging, healthy mouse pancreatic islet and renal tubular cells as well as immunoincompetent nude mice harboring sstr-expressing tumors were evaluated. Incubation of sstr-positive cells with OcF showed a specific time- and dose-dependent staining of sstr-positive cells. CLM showed rapid internalization and homogenous cytoplasmatic distribution. After systemic application to mice (n = 8), specific time-dependent internalization and cytoplasmatic distribution into pancreatic islet cells and tubular cells of the renal cortex was recorded. After injection in tumor-harboring nude mice (n = 8), sstr-positive cells selectively displayed a cell surface and cytoplasmatic staining. CLM-targeted biopsies detected sstr-positive tumor cells with a sensitivity of 87.5% and a specificity of 100% as correlated with ex vivo immunohistochemistry. CLM with OcF permits real-time molecular, functional, and morphological imaging of sstr-expressing cell structures, allowing the specific visualization of pancreatic islet cells and neuroendocrine tumors in vivo.