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Sample records for insulinoma cell xenograft

  1. Insulinoma

    MedlinePlus

    ... behavior changes, or confusion Clouded vision Loss of consciousness or coma Convulsions or tremor Dizziness or headache ... develop any symptoms of insulinoma. Seizures and losing consciousness are an emergency. Call 911 or your local ...

  2. Effect of Carbidopa on 18F-FDOPA Uptake in Insulinoma: From Cell Culture to Small-Animal PET Imaging.

    PubMed

    Detour, Julien; Pierre, Alice; Boisson, Fréderic; Kreutter, Guillaume; Lavaux, Thomas; Namer, Izzie Jacques; Kessler, Laurence; Brasse, David; Marchand, Patrice; Imperiale, Alessio

    2017-01-01

    Patient premedication with carbidopa seems to improve the accuracy of 6-(18)F-fluoro-3,4-dihydroxy-l-phenylalanine ((18)F-FDOPA) PET for insulinoma diagnosis. However, the risk of PET false-negative results in the presence of carbidopa is a concern. Consequently, we aimed to evaluate the effect of carbidopa on (18)F-FDOPA uptake in insulinoma β-cells and an insulinoma xenograft model in mice. (18)F-FDOPA in vitro accumulation was assessed in the murine β-cell line RIN-m5F. In vivo small-animal PET experiments were performed on tumor-bearing nude mice after subcutaneous injection of RIN-m5F cells. Experiments were conducted with and without carbidopa pretreatment. Incubation of RIN-m5F cells with 80 μM carbidopa did not significantly affect the cellular accumulation of (18)F-FDOPA. Tumor xenografts were clearly detectable by small-animal PET in all cases. Insulinoma xenografts in carbidopa-treated mice showed significantly higher (18)F-FDOPA uptake than those in nontreated mice. Regardless of carbidopa premedication, the xenografts were characterized by an early increase in (18)F-FDOPA uptake and then a progressive reduction over time. Carbidopa did not influence in vitro (18)F-FDOPA accumulation in RIN-m5F cells but improved insulinoma imaging in vivo. Our findings increase current knowledge about the (18)F-FDOPA uptake profile of RIN-m5F cells and a related xenograft model. To our knowledge, the present work represents the first preclinical research specifically focused on insulinomas, with potential translational implications. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

  3. Differential expression of cell-cycle regulators in human beta-cells derived from insulinoma tissue.

    PubMed

    Ueberberg, Sandra; Tannapfel, Andrea; Schenker, Peter; Viebahn, Richard; Uhl, Waldemar; Schneider, Stephan; Meier, Juris J

    2016-05-01

    The low frequency of beta-cell replication in the adult human pancreas limits beta-cell regeneration. A better understanding of the regulation of human beta-cell proliferation is crucial to develop therapeutic strategies aiming to enhance beta-cell mass. To identify factors that control beta-cell proliferation, cell-cycle regulation was examined in human insulinomas as a model of increased beta-cell proliferation (n=11) and healthy pancreatic tissue from patients with benign pancreatic tumors (n=9). Tissue sections were co-stained for insulin and cell-cycle proteins. Transcript levels of selected cell-cycle factors in beta-cells were determined by qRT-PCR after performing laser-capture microdissection. The frequency of beta-cell replication was 3.74±0.92% in the insulinomas and 0.11±0.04% in controls (p=0.0016). p21 expression was higher in insulinomas (p=0.0058), and Rb expression was higher by trend (p=0.085), whereas p16 (p<0.0001), Cyclin C (p<0.0001), and p57 (p=0.018) expression levels were lower. The abundance of Cyclin D3 (p=0.62) and p27 (p=0.68) was not different between the groups. The reduced expression of p16 (p<0.0001) and p57 (p=0.012) in insulinomas and the unchanged expression of Cyclin D3 (p=0.77) and p27 (p=0.55) were confirmed using qRT-PCR. The expression of certain cell-cycle factors in beta-cells derived from insulinomas and healthy adults differs markedly. Targeting such differentially regulated cell-cycle proteins may evolve as a future strategy to enhance beta-cell regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Colocalization of insulin and glucagon in insulinoma cells and developing pancreatic endocrine cells.

    PubMed

    Wang, Zai; You, Jia; Xu, Shiqing; Hua, Zhan; Zhang, Wenjian; Deng, Tingting; Fang, Ni; Fang, Qing; Liu, Honglin; Peng, Liang; Wang, Peigang; Lou, Jinning

    2015-06-12

    A significant portion of human and rat insulinomas coexpress multiple hormones. This character termed as multihormonality is also observed in some early pancreatic endocrine cells which coexpress insulin and glucagon, suggesting an incomplete differentiation status of both cells. Here we demonstrate that insulinoma cells INS-1 and INS-1-derived single cell clone INS-1-15 coexpressed insulin and glucagon in a portion of cells. These two hormones highly colocalized in the intracellular vesicles within a cell. Due to the existence of both PC1/3 and PC2 in INS-1-derived cells, proglucagon could be processed into glucagon, GLP-1 and GLP-2. These glucagon-family peptides and insulin were secreted simultaneously corresponding to the elevating glucose concentrations. The coexpression and partial colocalization of insulin and glucagon was also observed in rat fetal pancreatic endocrine cells, but the colocalization rate was generally lower and more diverse, suggesting that in the developing pancreatic endocrine cells, insulin and glucagon may be stored in nonidentical pools of secreting vesicles and might be secreted discordantly upon stimulus.

  5. Metastatic Insulinoma Following Resection of Nonsecreting Pancreatic Islet Cell Tumor

    PubMed Central

    Gordon, Ilyssa O.; Van Ha, Thuong G.; Kaplan, Edwin L.; Philipson, Louis H.

    2013-01-01

    A 56-year-old woman presented to our clinic for recurrent hypoglycemia after undergoing resection of an incidentally discovered nonfunctional pancreatic endocrine tumor 6 years ago. She underwent a distal pancreatectomy and splenectomy, after which she developed diabetes and was placed on an insulin pump. Pathology showed a pancreatic endocrine neoplasm with negative islet hormone immunostains. Two years later, computed tomography scan of the abdomen showed multiple liver lesions. Biopsy of a liver lesion showed a well-differentiated neuroendocrine neoplasm, consistent with pancreatic origin. Six years later, she presented to clinic with 1.5 years of recurrent hypoglycemia. Laboratory results showed elevated proinsulin, insulin levels, and c-peptide levels during a hypoglycemic episode. Computed tomography scan of the abdomen redemonstrated multiple liver lesions. Repeated transarterial catheter chemoembolization and microwave thermal ablation controlled hypoglycemia. The unusual features of interest of this case include the transformation of nonfunctioning pancreatic endocrine tumor to a metastatic insulinoma and the occurrence of atrial flutter after octreotide for treatment. PMID:26425568

  6. Preventing T cell rejection of pig xenografts.

    PubMed

    Higginbotham, Laura; Ford, Mandy L; Newell, Kenneth A; Adams, Andrew B

    2015-11-01

    Xenotransplantation is a potential solution to the limited supply of donor organs. While early barriers to xenograft acceptance, such as hyperacute rejection, are now largely avoided through genetic engineering, the next frontier in successful xenograft survival will require prevention of T cell-mediated rejection. Most successful immunosuppressive regimens in xenotransplantation utilize T cell depletion with antibody therapy. Additionally, the use of T cell costimulatory blockade - specifically blockade of the CD40-CD154 pathway - shows promise with several reports of long-term xenograft survival. Additional therapies, such as transgenic expression of T cell coinhibitory molecules or transfer of immunomodulatory cells to promote tolerance, may be necessary to achieve reliable long-term xenograft acceptance. Further studies in pre-clinical models are essential in order to optimize these regimens prior to trials in patients. Copyright © 2015. Published by Elsevier Ltd.

  7. Insulinoma or non-insulinoma pancreatogenous hypoglycemia? A diagnostic dilemma

    PubMed Central

    Anderson, Blaire; Nostedt, Jordan; Girgis, Safwat; Dixon, Tara; Agrawal, Veena; Wiebe, Edward; Senior, Peter A.; Shapiro, A.M. James

    2016-01-01

    Insulinoma is the most common cause of endogenous hyperinsulinemic hypoglycemia in adults. An alternate etiology, non-insulinoma pancreatogenous hypoglycemia (NIPH), is rare. Clinically, NIPH is characterized by postprandial hyperinsulinemic hypoglycemia, negative 72-h fasts, negative preoperative localization studies for insulinoma and positive selective arterial calcium infusion tests. Histologically, diffuse islet hyperplasia with increased number and size of islet cells is present and confirms the diagnosis. Differentiating NIPH from occult insulinoma preoperatively is challenging. Partial pancreatectomy is the procedure of choice; however, recurrence of symptoms, although less debilitating, occurs commonly. Medical management with diazoxide, verapamil and octreotide can be used for persistent symptoms. Ultimately, near-total or total pancreatectomy may be necessary. We report a case of a 67-year-old male with hypoglycemia in whom preoperative workup, including computerized tomography abdomen, suggested insulinoma, but whose final diagnosis on pathology was NIPH instead. PMID:27887024

  8. Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells.

    PubMed

    Azzu, Vian; Affourtit, Charles; Breen, Eamon P; Parker, Nadeene; Brand, Martin D

    2008-10-01

    Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0-4.4 ng/million cells (2.7-14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.

  9. Effects of Intercellular Junction Protein Expression on Intracellular Ice Formation in Mouse Insulinoma Cells

    PubMed Central

    Higgins, Adam Z.; Karlsson, Jens O.M.

    2013-01-01

    The development of cryopreservation procedures for tissues has proven to be difficult in part because cells within tissue are more susceptible to intracellular ice formation (IIF) than are isolated cells. In particular, previous studies suggest that cell-cell interactions increase the likelihood of IIF by enabling propagation of ice between neighboring cells, a process thought to be mediated by gap junction channels. In this study, we investigated the effects of cell-cell interactions on IIF using three genetically modified strains of the mouse insulinoma cell line MIN6, each of which expressed key intercellular junction proteins (connexin-36, E-cadherin, and occludin) at different levels. High-speed video cryomicroscopy was used to visualize the freezing process in pairs of adherent cells, revealing that the initial IIF event in a given cell pair was correlated with a hitherto unrecognized precursor phenomenon: penetration of extracellular ice into paracellular spaces at the cell-cell interface. Such paracellular ice penetration occurred in the majority of cell pairs observed, and typically preceded and colocalized with the IIF initiation events. Paracellular ice penetration was generally not observed at temperatures >−5.65°C, which is consistent with a penetration mechanism via defects in tight-junction barriers at the cell-cell interface. Although the maximum temperature of paracellular penetration was similar for all four cell strains, genetically modified cells exhibited a significantly higher frequency of ice penetration and a higher mean IIF temperature than did wild-type cells. A four-state Markov chain model was used to quantify the rate constants of the paracellular ice penetration process, the penetration-associated IIF initiation process, and the intercellular ice propagation process. In the initial stages of freezing (>−15°C), junction protein expression appeared to only have a modest effect on the kinetics of propagative IIF, and even cell

  10. PPARalpha suppresses insulin secretion and induces UCP2 in insulinoma cells.

    PubMed

    Tordjman, Karen; Standley, Kara N; Bernal-Mizrachi, Carlos; Leone, Teresa C; Coleman, Trey; Kelly, Daniel P; Semenkovich, Clay F

    2002-06-01

    Fatty acids may promote type 2 diabetes by altering insulin secretion from pancreatic beta cells, a process known as lipotoxicity. The underlying mechanisms are poorly understood. To test the hypothesis that peroxisome proliferator-activated receptor alpha (PPARalpha) has a direct effect on islet function, we treated INS-1 cells, an insulinoma cell line, with a PPARalpha adenovirus (AdPPARalpha) as well as the PPARalpha agonist clofibric acid. AdPPARalpha-infected INS-1 cells showed PPARalpha agonist- and fatty acid-dependent transactivation of a PPARalpha reporter gene. Treatment with either AdPPARalpha or clofibric acid increased both catalase activity (a marker of peroxisomal proliferation) and palmitate oxidation. AdPPARalpha induced carnitine-palmitoyl transferase-I (CPT-I) mRNA, but had no effect on insulin gene expression. AdPPARalpha treatment increased cellular triglyceride content but clofibric acid did not. Both AdPPARalpha and clofibric acid decreased basal and glucose-stimulated insulin secretion. Despite increasing fatty acid oxidation, AdPPARalpha did not increase cellular ATP content suggesting the stimulation of uncoupled respiration. Consistent with these observations, UCP2 expression doubled in PPARalpha-treated cells. Clofibric acid-induced suppression of glucose-simulated insulin secretion was prevented by the CPT-I inhibitor etomoxir. These data suggest that PPARalpha-stimulated fatty acid oxidation can impair beta cell function.

  11. Thioredoxin-mimetic peptides (TXM) reverse auranofin induced apoptosis and restore insulin secretion in insulinoma cells.

    PubMed

    Cohen-Kutner, Moshe; Khomsky, Lena; Trus, Michael; Aisner, Yonatan; Niv, Masha Y; Benhar, Moran; Atlas, Daphne

    2013-04-01

    The thioredoxin reductase/thioredoxin system (TrxR/Trx1) plays a major role in protecting cells from oxidative stress. Disruption of the TrxR-Trx1 system keeps Trx1 in the oxidized state leading to cell death through activation of the ASK1-Trx1 apoptotic pathway. The potential mechanism and ability of tri- and tetra-oligopeptides derived from the canonical -CxxC- motif of the Trx1-active site to mimic and enhance Trx1 cellular activity was examined. The Trx mimetics peptides (TXM) protected insulinoma INS 832/13 cells from oxidative stress induced by selectively inhibiting TrxR with auranofin (AuF). TXM reversed the AuF-effects preventing apoptosis, and increasing cell-viability. The TXM peptides were effective in inhibiting AuF-induced MAPK, JNK and p38(MAPK) phosphorylation, in correlation with preventing caspase-3 cleavage and thereby PARP-1 dissociation. The ability to form a disulfide-bridge-like conformation was estimated from molecular dynamics simulations. The TXM peptides restored insulin secretion and displayed Trx1 denitrosylase activity. Their potency was 10-100-fold higher than redox reagents like NAC, AD4, or ascorbic acid. Unable to reverse ERK1/2 phosphorylation, TXM-CB3 (NAc-Cys-Pro-Cys amide) appeared to function in part, through inhibiting ASK1-Trx dissociation. These highly effective anti-apoptotic effects of Trx1 mimetic peptides exhibited in INS 832/13 cells could become valuable in treating adverse oxidative-stress related disorders such as diabetes. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Regulation of ATP-sensitive K sup + channels in insulinoma cells: Activation by somatostatin and protein kinase C and the role of cAMP

    SciTech Connect

    De Weille, J.R.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M. )

    1989-04-01

    The actions of somatostatin and of the phorbol ester 4{beta}-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and {sup 86}Rb{sup +} flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K{sup +} channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive {sup 86}Rb{sup +} efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, of protein kinase C, and of cAMP in the regulation of ATP-sensitive K{sup +} channels are discussed.

  13. Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice.

    PubMed Central

    Kulkarni, R N; Wang, Z L; Wang, R M; Hurley, J D; Smith, D M; Ghatei, M A; Withers, D J; Gardiner, J V; Bailey, C J; Bloom, S R

    1997-01-01

    Obesity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism. PMID:9389736

  14. Osseous metaplasia within a canine insulinoma.

    PubMed

    Pieczarka, Emily M; Russell, Duncan S; Santangelo, Kelly S; Aeffner, Famke; Burkhard, Mary Jo

    2014-03-01

    An 11-year-old male castrated mixed-breed dog was presented for exercise intolerance, tetraparesis, and persistent hypoglycemia. Abdominal ultrasound examination revealed 2 nodules within the right limb of the pancreas. Cytology from one nodule was consistent with a carcinoma of neuroendocrine origin, with a primary differential diagnosis of insulinoma. Histologic evaluation and immunohistochemistry for synaptophysin and insulin confirmed the diagnosis of insulinoma. Additionally, there was a solitary nodule of mineralized compact bone composing approximately 60% of the mass. To the authors' knowledge, this is the first report of osseous metaplasia within an insulinoma (islet cell carcinoma).

  15. Knockdown of Pyruvate Carboxylase or Fatty Acid Synthase Lowers Numerous Lipids and Glucose-Stimulated Insulin Release in Insulinoma Cells

    PubMed Central

    MacDonald, Michael J.; Hasan, Noaman M.; Dobrzyn, Agnieszka; Stoker, Scott W.; Ntambi, James M.; Liu, Xueqing; Sampath, Harini

    2013-01-01

    We previously showed that knockdown of the anaplerotic enzyme pyruvate carboxylase in the INS-1 832/13 insulinoma cell line inhibited glucose-stimulated insulin release and glucose carbon incorporation into lipids. We now show that knockdown of fatty acid synthase (FAS) mRNA and protein also inhibits glucose-stimulated insulin release in this cell line. Levels of numerous phospholipids, cholesterol esters, diacylglycerol, triglycerides and individual fatty acids with C14-C24 side chains were acutely lowered about 20% in glucose-stimulated pyruvate carboxylase knockdown cells over a time course that coincides with insulin secretion. In FAS knockdown cells glucose carbon incorporation into lipids and the levels of the subclasses of phospholipids and cholesterol ester species were lower by 20–30% without inhibition of glucose oxidation. These studies suggest that rapid lipid modification is essential for normal glucose-stimulated insulin secretion. PMID:23357280

  16. Pregnenolone sulfate activates basic region leucine zipper transcription factors in insulinoma cells: role of voltage-gated Ca2+ channels and transient receptor potential melastatin 3 channels.

    PubMed

    Müller, Isabelle; Rössler, Oliver G; Thiel, Gerald

    2011-12-01

    The neurosteroid pregnenolone sulfate activates a signaling cascade in insulinoma cells involving activation of extracellular signal-regulated protein kinase and enhanced expression of the transcription factor Egr-1. Here, we show that pregnenolone sulfate stimulation leads to a significant elevation of activator protein-1 (AP-1) activity in insulinoma cells. Expression of the basic region leucine zipper (bZIP) transcription factors c-Jun and c-Fos is up-regulated in insulinoma cells and pancreatic β-cells in primary culture after pregnenolone sulfate stimulation. Up-regulation of a chromatin-embedded c-Jun promoter/luciferase reporter gene transcription in pregnenolone sulfate-stimulated insulinoma cells was impaired when the AP-1 binding sites were mutated, indicating that these motifs function as pregnenolone sulfate response elements. In addition, phosphorylation of cAMP response element (CRE)-binding protein is induced and transcription of a CRE-controlled reporter gene is stimulated after pregnenolone sulfate treatment, indicating that the CRE functions as a pregnenolone sulfate response element as well. Pharmacological and genetic experiments revealed that both L-type Ca(2+) channels and transient receptor potential melastatin 3 (TRPM3) channels are essential for connecting pregnenolone sulfate stimulation with enhanced AP-1 activity and bZIP-mediated transcription in insulinoma cells. In contrast, pregnenolone sulfate stimulation did not enhance AP-1 activity or c-Jun and c-Fos expression in pituitary corticotrophs that express functional L-type Ca(2+) channels but only trace amounts of TRPM3. We conclude that expression of L-type Ca(2+) channels is not sufficient to activate bZIP-mediated gene transcription by pregnenolone sulfate. Rather, additional expression of TRPM3 or depolarization of the cells is required to connect pregnenolone sulfate stimulation with enhanced gene transcription.

  17. Antioxidant effect of mogrosides against oxidative stress induced by palmitic acid in mouse insulinoma NIT-1 cells.

    PubMed

    Xu, Q; Chen, S Y; Deng, L D; Feng, L P; Huang, L Z; Yu, R R

    2013-11-18

    Excessive oxidative stress in pancreatic β cells, caused by glucose and fatty acids, is associated with the pathogenesis of type 2 diabetes. Mogrosides have shown antioxidant and antidiabetic activities in animal models of diabetes, but the underlying mechanisms remain unclear. This study evaluated the antioxidant effect of mogrosides on insulinoma cells under oxidative stress caused by palmitic acid, and investigated the underlying molecular mechanisms. Mouse insulinoma NIT-1 cells were cultured in medium containing 0.75 mM palmitic acid, mimicking oxidative stress. The effects of 1 mM mogrosides were determined with the dichlorodihydrofluorescein diacetate assay for intracellular reactive oxygen species (ROS) and FITC-Annexin V/PI assay for cell apoptosis. Expression of glucose transporter-2 (GLUT2) and pyruvate kinase was determined by semi-quantitative reverse-transcription polymerase chain reaction. Palmitic acid significantly increased intracellular ROS concentration 2-fold (P<0.05), and decreased expression of GLUT2 (by 60%, P<0.05) and pyruvate kinase (by 80%, P<0.05) mRNAs in NIT-1 cells. Compared with palmitic acid, co-treatment with 1 mM mogrosides for 48 h significantly reduced intracellular ROS concentration and restored mRNA expression levels of GLUT2 and pyruvate kinase. However, mogrosides did not reverse palmitic acid-induced apoptosis in NIT-1 cells. Our results indicate that mogrosides might exert their antioxidant effect by reducing intracellular ROS and regulating expression of genes involved in glucose metabolism. Further research is needed to achieve a better understanding of the signaling pathway involved in the antioxidant effect of mogrosides.

  18. Antioxidant effect of mogrosides against oxidative stress induced by palmitic acid in mouse insulinoma NIT-1 cells

    PubMed Central

    Xu, Q.; Chen, S.Y.; Deng, L.D.; Feng, L.P.; Huang, L.Z.; Yu, R.R.

    2013-01-01

    Excessive oxidative stress in pancreatic β cells, caused by glucose and fatty acids, is associated with the pathogenesis of type 2 diabetes. Mogrosides have shown antioxidant and antidiabetic activities in animal models of diabetes, but the underlying mechanisms remain unclear. This study evaluated the antioxidant effect of mogrosides on insulinoma cells under oxidative stress caused by palmitic acid, and investigated the underlying molecular mechanisms. Mouse insulinoma NIT-1 cells were cultured in medium containing 0.75 mM palmitic acid, mimicking oxidative stress. The effects of 1 mM mogrosides were determined with the dichlorodihydrofluorescein diacetate assay for intracellular reactive oxygen species (ROS) and FITC-Annexin V/PI assay for cell apoptosis. Expression of glucose transporter-2 (GLUT2) and pyruvate kinase was determined by semi-quantitative reverse-transcription polymerase chain reaction. Palmitic acid significantly increased intracellular ROS concentration 2-fold (P<0.05), and decreased expression of GLUT2 (by 60%, P<0.05) and pyruvate kinase (by 80%, P<0.05) mRNAs in NIT-1 cells. Compared with palmitic acid, co-treatment with 1 mM mogrosides for 48 h significantly reduced intracellular ROS concentration and restored mRNA expression levels of GLUT2 and pyruvate kinase. However, mogrosides did not reverse palmitic acid-induced apoptosis in NIT-1 cells. Our results indicate that mogrosides might exert their antioxidant effect by reducing intracellular ROS and regulating expression of genes involved in glucose metabolism. Further research is needed to achieve a better understanding of the signaling pathway involved in the antioxidant effect of mogrosides. PMID:24270904

  19. Glucose and GLP-1 Stimulate cAMP Production via Distinct Adenylyl Cyclases in INS-1E Insulinoma Cells

    PubMed Central

    Ramos, Lavoisier S.; Zippin, Jonathan Hale; Kamenetsky, Margarita; Buck, Jochen; Levin, Lonny R.

    2008-01-01

    In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades. PMID:18695009

  20. Giant malignant insulinoma

    PubMed Central

    Karavias, Dimitrios; Habeos, Ioannis; Maroulis, Ioannis; Kalogeropoulou, Christina; Tsamandas, Athanasios; Chaveles, Ioannis

    2015-01-01

    Insulinomas are the most common pancreatic neuroendocrine tumors. Most insulinomas are benign, small, intrapancreatic solid tumors and only large tumors have a tendency for malignancy. Most patients present with symptoms of hypoglycemia that are relieved with the administration of glucose. We herein present the case of a 75-year-old woman who presented with an acute hypoglycemic episode. Subsequent laboratory and radiological studies established the diagnosis of a 17-cm malignant insulinoma, with local invasion to the left kidney, lymph node metastasis, and hepatic metastases. Patient symptoms, diagnostic and imaging work-up and surgical management of both the primary and the metastatic disease are reviewed. PMID:25960993

  1. Signal transduction of pregnenolone sulfate in insulinoma cells: activation of Egr-1 expression involving TRPM3, voltage-gated calcium channels, ERK, and ternary complex factors.

    PubMed

    Mayer, Sabine I; Müller, Isabelle; Mannebach, Stefanie; Endo, Takeshi; Thiel, Gerald

    2011-03-25

    The neurosteroid pregnenolone sulfate acts on the nervous system by modifying neurotransmission and receptor functions, thus influencing synaptic strength, neuronal survival, and neurogenesis. Here we show that pregnenolone sulfate induces a signaling cascade in insulinoma cells leading to enhanced expression of the zinc finger transcription factor Egr-1 and Egr-1-responsive target genes. Pharmacological and genetic experiments revealed that influx of Ca(2+) ions via transient receptor potential M3 and voltage-gated Ca(2+) channels, elevation of the cytosolic Ca(2+) level, and activation of ERK are essential for connecting pregnenolone sulfate stimulation with enhanced Egr-1 biosynthesis. Expression of a dominant-negative mutant of Elk-1, a key regulator of gene transcription driven by a serum response element, attenuated Egr-1 expression following stimulation, indicating that Elk-1 or related ternary complex factors connect the transcription of the Egr-1 gene with the pregnenolone sulfate-induced intracellular signaling cascade elicited by the initial influx of Ca(2+). The newly synthesized Egr-1 was biologically active and bound under physiological conditions to the regulatory regions of the Pdx-1, Synapsin I, and Chromogranin B genes. Pdx-1 is a major regulator of insulin gene transcription. Accordingly, elevated insulin promoter activity and increased mRNA levels of insulin could be detected in pregnenolone sulfate-stimulated insulinoma cells. Likewise, the biosynthesis of synapsin I, a synaptic vesicle protein that is found at secretory granules in insulinoma cells, was stimulated in pregnenolone sulfate-treated INS-1 cells. Together, these data show that pregnenolone sulfate induces a signaling cascade in insulinoma cells that is very similar to the signaling cascade induced by glucose in β-cells.

  2. Extracts from Leonurus sibiricus L. increase insulin secretion and proliferation of rat INS-1E insulinoma cells.

    PubMed

    Schmidt, S; Jakab, M; Jav, S; Streif, D; Pitschmann, A; Zehl, M; Purevsuren, S; Glasl, S; Ritter, M

    2013-10-28

    Traditional Mongolian medicine (TMM) uses preparations from herbs as one form of medication for the treatment of a diversity of diseases including diabetes mellitus (DM). We evaluated the effect of extracts from the plant Leonurus sibiricus L. (LS), used in TMM to treat typical symptoms of type 2 DM, on insulin secretion, electrophysiological properties, intracellular calcium concentration and cell proliferation of INS-1E insulinoma cells under standard cell culture conditions (SCC; 11.1mM glucose). Insulin secretion was measured by ELISA, electrical properties were assessed by whole cell patch clamping, intracellular calcium concentration (Cai) by Fluo-4 time lapse imaging, insulin receptor expression was verified by RT-PCR and cell proliferation assessed by CellTiter-Glo® cell viability assay. Insulin released from INS-1E cells into the culture medium over 24h was significantly increased in presence of 500 mg/L aqueous LS extract (LS OWE) as well as methanolic LS extract (LS MeOH/H2O) but not in the presence of the butanol-soluble extract (LS MeOH/BuOH). Acute application of LS OWE resulted in a depolarization of the cell membrane potential paralleled by an initial increase and subsequent decline and silencing of action potential frequency, by KATP channel inhibition, persisting depolarization and an increase in Cai. The electrophysiological effects were comparable to those of 100 μM tolbutamide, which, however failed to elevate insulin secretion under SCC. Furthermore all LS extracts stimulated INS-1E cell proliferation. The finding that extracts from Leonurus sibiricus L. enhance insulin secretion and/or foster cell proliferation may provide possible explanations for the underlying therapeutic principles in the empirical use of LS-containing formulations in DM and DM-related disorders as applied in TMM. © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. HLA-DR-restricted T cell lines from newly diagnosed type 1 diabetic patients specific for insulinoma and normal islet beta cell proteins: lack of reactivity to glutamic acid decarboxylase.

    PubMed Central

    Huang, G C; Tremble, J; Bailyes, E; Arden, S D; Kaye, T; McGregor, A M; Banga, J P

    1995-01-01

    T cells reacting with pancreatic islet beta cell proteins play a pivotal role in the pathogenesis of type 1 diabetes in experimental animal models and man, although the islet cell autoantigens against which these T cells are directed remain to be characterized. We have previously shown the presence of disease-related antigens residing in the transplantable RIN insulinoma membranes which are recognized by T cells from diabetic NOD mice. We now report on the establishment of CD4+, T cell lines reacting with insulinoma membranes from six newly diagnosed type 1 diabetic patients. Detailed examination of T cell lines from two patients revealed that both the lines continued to react with normal islet cell proteins and, interestingly, were also stimulated by antigens present in brain microsomes. The two T cell lines showed reactivity with different molecular weight proteins of the insulinoma membranes and both the lines were histocompatibility-linked antigen (HLA)-DR restricted. Although the insulinoma membrane preparation is known to contain glutamic acid decarboxylase (GAD), none of the six T cell lines proliferates in response to purified GAD. These T cell lines will be valuable in characterizing novel islet beta cell antigens which are likely to be implicated in type 1 diabetes. PMID:7554382

  4. [Malignant insulinoma: recommendations for workup and treatment].

    PubMed

    Baudin, Eric; Caron, Philippe; Lombard-Bohas, Catherine; Tabarin, Antoine; Mitry, Emmanuel; Reznick, Yves; Taieb, David; Pattou, François; Goudet, Pierre; Vezzosi, Delphine; Scoazec, Jean-Yves; Cadiot, Guillaume; Borson-Chazot, Françoise; Do Cao, Christine

    2014-06-01

    Insulinoma are malignant in 4 to 14 % of cases. Their rarity and the sparse data available in the literature have limited publication of specific guidelines for their management. The following review aim to provide up-to-date recommendations on initial evaluation including pathologic grading, measures to control hypoglycemia, antitumor strategies and long term follow-up. Will be discussed in detail respective indications of surgery, diazoxide, somatostatin analogs, everolimus, sunitinib, liver directed treatments including arterial embolization, chemotherapy and radiometabolic therapy. A Medline search using terms "insulinoma", "neuroendocrine pancreatic tumors", "islet cell carcinoma", "malignant insulinoma" was performed limiting the selection to English language articles and adult age cases, along with cross referencing.

  5. [Hypoglycemia and insulinoma].

    PubMed

    Cazabat, L; Chanson, P

    2009-09-01

    Insulinomas are rare causes of hypoglycemia. After having ruled out non insulinomatous causes of hypoglycemia in a patient in whom Whipple's triad is documented, hyperinsulinism must be demonstrated biochemically, either during a spontaneous hypoglycemic episode or, more often, during a supervised fast which may be prolonged up to 72 h. A mixed-meal test may also help to diagnose the very rare cases of postprandial hypoglycemia related to non insulinoma pancreatogenic hypoglycemic syndrome (NIPHS) or to some rare insulinomas. Only when diagnosis of hypoglycemic hyperinsulinism is made, the tumor localization process may be initiated. This may be difficult due to the small size of insulinomas (generally < 1 cm). Multimodal approach is necessary. The association of endoscopic ultrasound and CT-scan or MRI seems optimal. Octreoscan will be also performed. First results with a very new technique, the GLP-1 receptor imaging, are promising for localizing very small tumors. This localization aims to allow a sparing surgery; enucleation of benign tumors, if possible, allows a pancreatic tissue preservation in patients with quite normal survival.

  6. Overexpression of Reg3alpha increases cell growth and the levels of cyclin D1 and CDK4 in insulinoma cells.

    PubMed

    Cui, Wei; De Jesus, Kristine; Zhao, Hong; Takasawa, Shin; Shi, Bingyin; Srikant, Coimbatore B; Liu, Jun-Li

    2009-06-01

    Regenerating gene (Reg) family protein Reg3alpha is normally expressed in pancreatic acinar and endocrine cells. In order to explore its effect on islet beta-cell replication, insulinoma MIN6 cells were stably transfected with murine Reg3alpha cDNA. Determined using real-time PCR and Western blots, the levels of Reg3alpha mRNA and protein in Reg3alpha-transfected clones were increased 10- and 6-fold, respectively. Western blots also revealed that the protein was released into the culture medium, consistent with an endocrine effect. In MTT cell proliferation assay, Reg3alpha-overexpressing cells exhibited a 2-fold increase in the rate of cell growth. In order to investigate the intracellular mechanism, we studied cell cycle regulatory proteins. In Reg3alpha-expressing cells, we detected 2.2- and 2.5-fold increased levels of cyclin D1 and CDK4, respectively, which paralleled a 1.8-fold increase in the rate of Akt phosphorylation. It is established that beta-cell replication is associated with increased cyclin D1 and CDK4 levels; deficiency in CDK4 or cyclin D2 results in reduced beta-cell mass and diabetes. Our results suggest that Reg3alpha stimulates beta-cell replication, by activating Akt kinase and increasing the levels of cyclin D1/CDK4.

  7. Baicalein protects rat insulinoma INS-1 cells from palmitate-induced lipotoxicity by inducing HO-1.

    PubMed

    Kwak, Hyun Jeong; Yang, Dongki; Hwang, Yongha; Jun, Hee-Sook; Cheon, Hyae Gyeong

    2017-01-01

    β-Cell dysfunction plays a central role in the pathogenesis of type 2 diabetes (T2D), and the identification of novel approaches to improve β-cell function is essential to treat this disease. Baicalein, a flavonoid originally isolated from the root of Scutellaria Baicalensis, has been shown to have beneficial effects on β-cell function. Here, the authors investigated the molecular mechanism responsible for the protective effects of baicalein against palmitate (PA)-induced impaired β-cell function, and placed focus on the role of heme oxygenase (HO)-1. Rat pancreatic β-cell line INS-1 cells or mouse pancreatic islets were cultured with PA (500 μM) to induce lipotoxicity in the presence or absence of baicalein (50 μM), and the expressions of the ER stress markers, ATF-3, CHOP and GRP78 were detected by Western blotting and/or qPCR. The involvement of HO-1 was evaluated by HO-1 siRNA transfection and using the HO-1 inhibitor ZnPP. Baicalein reduced PA-induced ER stress and inflammation and enhanced insulin secretion, and these effects were associated with the induction of HO-1. Furthermore, these protective effects were attenuated by ZnPP and by HO-1 siRNA. Pretreatment of PD98059 (an ERK inhibitor) significantly inhibited the protective effects of baicalein and blocked HO-1 induction. On the other hand, CO production by RuCO (a CO donor) ameliorated PA-induced ER stress, suggesting that CO production followed by HO-1 induction may contribute to the protective effects of baicalein against PA-induced β-cell dysfunction. Baicalein protects pancreatic β-cells from PA-induced ER stress and inflammation via an ERK-HO-1 dependent pathway. The authors suggest HO-1 induction in pancreatic β-cells appears to be a promising therapeutic strategy for T2D.

  8. Nutritional energy stimulates NAD+ production to promote tankyrase-mediated PARsylation in insulinoma cells.

    PubMed

    Zhong, Linlin; Yeh, Tsung-Yin J; Hao, Jun; Pourtabatabaei, Nasim; Mahata, Sushil K; Shao, Jianhua; Chessler, Steven D; Chi, Nai-Wen

    2015-01-01

    The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD+ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD+ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD+ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD+ since it is mirrored by the NAD+ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD+ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD+ production to expand NAD+ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD+ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD+ production by ATP suggests that nutritional energy may enhance the functions of other NAD+-driven enzymes including sirtuins.

  9. A comprehensive characterization of cell cultures and xenografts derived from a human verrucous penile carcinoma.

    PubMed

    Muñoz, Juan J; Drigo, Sandra A; Kuasne, Hellen; Villacis, Rolando A R; Marchi, Fabio A; Domingues, Maria A C; Lopes, Ademar; Santos, Tiago G; Rogatto, Silvia R

    2016-08-01

    This study aimed to establish and characterize primary cell cultures and xenografts derived from penile carcinoma (PeCa) in order to provide experimental models for cellular processes and efficacy of new treatments. A verrucous squamous cell carcinoma (VSCC) was macrodissected, dissociated, and cultivated in KSFM/DF12 medium. Cell cultures were evaluated at passage 5 (P5) using migration and invasion assays and were serially propagated, in vivo, in BALB/c nude mice until passage 3 (X1-X3). Immunophenotypic characterization of cultures and xenografts was performed. Genomic (CytoScan HD, Affymetrix) and transcriptomic profiles (HTA 2.0 platform, Affymetrix) for VSCC, cell cultures, and xenografts were assessed. P5 cells were able to migrate, invade the Matrigel, and produce tumors in immunodeficient mice, demonstrating their malignant potential. The xenografts unexpectedly presented a sarcomatoid-like carcinoma phenotype. Genomic analysis revealed a high similarity between the VSCC and tumor-derived xenograft, confirming its xenograft origin. Interestingly, a subpopulation of P5 cells presented stem cell-related markers (CD44(+)CD24(-) and ALDH1(high)) and sphere-forming capacity, suggesting their potential xenograft origin. Cell cultures and xenografts retained the genomic alterations present in the parental tumor. Compared to VSCC, differentially expressed transcripts detected in all experimental conditions were associated with cellular morphology, movement, and metabolism and organization pathways. Malignant cell cultures and xenografts derived from a verrucous penile carcinoma were established and fully characterized. Nevertheless, xenograft PeCa models must be used with caution, taking into consideration the selection of specific cell populations and anatomical sites for cell/tumor implantation.

  10. Gallbladder small cell carcinoma Xenograft established by serial transplantation in nude mice.

    PubMed

    Nishime, Chiyoko; Ohnishi, Yasuyuki; Suemizu, Hiroshi; Tamaoki, Norikazu; Suematsu, Makoto; Oida, Yasuhisa; Yamazaki, Hitoshi; Nakamura, Masato; Ueyama, Yoshito; Kijima, Hiroshi

    2006-01-01

    The GB-04-JCK xenograft line of human gallbladder small cell carcinoma was established in nude mice by serial transplantation. The xenotransplantability has been maintained for more than 20 years. The carcinoma cells grew in a solid-sheet pattern and were found to have hyperchromatic nuclei, finely dispersed chromatin and inconspicuous nucleoli in the primary gallbladder tumor, as well as in the established xenograft GB-04-JCK The carcinoma cells also had Grimelius argyrophil granules, electron-dense neuroendocrine granules bounded by a single membrane. The xenograft line retained histological and immunohistochemical characteristics of the primary gallbladder tumor and is the first reported xenotransplantable tumor of human gallbladder small cell carcinoma.

  11. Beware of contaminating mouse cells in human xenografts from nude mice.

    PubMed

    Yang, J; Liu, A; Dougherty, C; Chen, X; Guzman, R; Nandi, S

    2000-01-01

    Human tumor xenografts in nude mice are widely utilized model system for the transplantation of human surgical specimens and human established cell lines. Gene expression studies are often carried out in these model systems. With an increasing use of PCR based analyses, the extreme sensitivity of this technique poses a serious challenge with regards to the extent of contaminating host mouse cells in the human tumor xenografts. These xenografts are never free of host cell contamination. We detected mouse estrogen receptor expression in several human tumor xenografts using RT-PCR demonstrating that precaution is necessary when utilizing PCR based analyses in human tumor xenografts. A cytologically based methodology which distinguishes human versus mouse cells will be more suitable for ER expression studies using human xenograft models. Both (1) in situ hybridization using human probe and (2) immunocytochemistry using a monoclonal antibody directed against human cytokeratin have been used successfully to distinguish human cells versus host mouse cells in human xenografts in nude mice. Immunostaining of ER can then be utilized to determine the expression pattern of ER in the transplanted human cells.

  12. Intermittent everolimus administration for malignant insulinoma

    PubMed Central

    Brizzi, Maria Pia; Tampellini, Marco; Scagliotti, Giorgio Vittorio; Priola, Adriano; Terzolo, Massimo; Pia, Anna; Berruti, Alfredo

    2014-01-01

    Summary Insulinoma is a rare form of insulin-secreting pancreatic islet cell neuroendocrine (NE) tumor. The medical treatment of the malignant NE disease of the pancreas deeply changed in the last years, thanks to the introduction of new target molecules, as everolimus. Even if the exact mechanism is not actually known, one of the side effects of everolimus, hyperglycemia, has been demonstrated to be useful to contrast the typical hypoglycemia of the insulinoma. We report the case of a patient with a metastatic malignant insulinoma treated with intermittent everolimus, obtaining an important improvement in the quality of life; this suggests the necessity of preclinical studies to analyze the cellular pathways involved in insulin-independent gluconeogenesis. Learning points Effect of somatostatin analogs is long-lasting in the control of functioning NE tumors.Persistent everolimus control of hypoglycemia despite serum insulin levels and disease progression.Open issue: are disease progression and the increase in serum markers the only valid criteria to reject a treatment? PMID:25298880

  13. Hypoxia-regulated gene expression explains differences between melanoma cell line-derived xenografts and patient-derived xenografts.

    PubMed

    Bhadury, Joydeep; Einarsdottir, Berglind O; Podraza, Agnieszka; Bagge, Roger Olofsson; Stierner, Ulrika; Ny, Lars; Dávila López, Marcela; Nilsson, Jonas A

    2016-04-26

    Cell line-derived xenografts (CDXs) are an integral part of drug efficacy testing during development of new pharmaceuticals against cancer but their accuracy in predicting clinical responses in patients have been debated. Patient-derived xenografts (PDXs) are thought to be more useful for predictive biomarker identification for targeted therapies, including in metastatic melanoma, due to their similarities to human disease. Here, tumor biopsies from fifteen patients and ten widely-used melanoma cell lines were transplanted into immunocompromised mice to generate PDXs and CDXs, respectively. Gene expression profiles generated from the tumors of these PDXs and CDXs clustered into distinct groups, despite similar mutational signatures. Hypoxia-induced gene signatures and overexpression of the hypoxia-regulated miRNA hsa-miR-210 characterized CDXs. Inhibition of hsa-miR-210 with decoys had little phenotypic effect in vitro but reduced sensitivity to MEK1/2 inhibition in vivo, suggesting down-regulation of this miRNA could result in development of resistance to MEK inhibitors.

  14. Hypoxia-regulated gene expression explains differences between melanoma cell line-derived xenografts and patient-derived xenografts

    PubMed Central

    Bhadury, Joydeep; Einarsdottir, Berglind O.; Podraza, Agnieszka; Bagge, Roger Olofsson; Stierner, Ulrika; Ny, Lars; López, Marcela Dávila; Nilsson, Jonas A.

    2016-01-01

    Cell line-derived xenografts (CDXs) are an integral part of drug efficacy testing during development of new pharmaceuticals against cancer but their accuracy in predicting clinical responses in patients have been debated. Patient-derived xenografts (PDXs) are thought to be more useful for predictive biomarker identification for targeted therapies, including in metastatic melanoma, due to their similarities to human disease. Here, tumor biopsies from fifteen patients and ten widely-used melanoma cell lines were transplanted into immunocompromised mice to generate PDXs and CDXs, respectively. Gene expression profiles generated from the tumors of these PDXs and CDXs clustered into distinct groups, despite similar mutational signatures. Hypoxia-induced gene signatures and overexpression of the hypoxia-regulated miRNA hsa-miR-210 characterized CDXs. Inhibition of hsa-miR-210 with decoys had little phenotypic effect in vitro but reduced sensitivity to MEK1/2 inhibition in vivo, suggesting down-regulation of this miRNA could result in development of resistance to MEK inhibitors. PMID:27009863

  15. Coffee inhibits nuclear factor-kappa B in prostate cancer cells and xenografts.

    PubMed

    Kolberg, Marit; Pedersen, Sigrid; Mitake, Maiko; Holm, Kristine Lillebø; Bøhn, Siv Kjølsrud; Blomhoff, Heidi Kiil; Carlsen, Harald; Blomhoff, Rune; Paur, Ingvild

    2016-01-01

    Chronic inflammation contributes to prostate cancer and the transcription factor Nuclear Factor-kappa B (NF-κB) is constitutively active in most such cancers. We examine the effects of coffee on NF-κB and on the regulation of selected genes in human-derived prostate cancer cells (PC3) and in PC3 xenografts in athymic nude mice. PC3 cells stably transduced with an NF-κB-luciferase reporter were used both in vitro and for xenografts. NF-κB activity was measured by reporter assays, DNA binding and in vivo imaging. Gene expression was measured in PC3 cells, xenografts and tumor microenvironment by low-density arrays. Western blotting of activated caspases was used to quantify apoptosis. Coffee inhibited TNFα-induced NF-κB activity and DNA-binding in PC3 cells. Furthermore, coffee increased apoptosis and modulated expression of a number of inflammation- and cancer-related genes in TNFα-treated PC3 cells. In vivo imaging revealed a 31% lower NF-κB-luciferase activation in the xenografts of the mice receiving 5% coffee compared to control mice. Interestingly, we observed major changes in gene expression in the PC3 cells in xenografts as compared to PC3 cells in vitro. In PC3 xenografts, genes related to inflammation, apoptosis and cytoprotection were down-regulated in mice receiving coffee, and coffee also affected the gene expression in the xenograft microenvironment. Our data demonstrate that coffee inhibits NF-κB activity in PC3 cells in vitro and in xenografts. Furthermore, coffee modulates transcription of genes related to prostate cancer and inflammation. Our results are the first to suggest mechanistic links between coffee consumption and prostate cancer in an experimental mouse model. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Insulinoma in 2 guinea pigs (Cavia porcellus)

    PubMed Central

    2005-01-01

    Abstract This paper describes an insulinoma in 2 guinea pigs (Cavia porcellus). Both guinea pigs presented with neurologic signs and low blood glucose readings. The neurologic signs resolved with dextrose administration. Insulinoma was confirmed on postmortem examination. PMID:15943120

  17. Insulinoma-released exosomes or microparticles are immunostimulatory and can activate autoreactive T cells spontaneously developed in non-obese diabetes mice1

    PubMed Central

    Sheng, Huiming; Hassanali, Saleema; Nugent, Courtney; Wen, Li; Hamilton-Williams, Emma; Dias, Peter; Dai, Yang D.

    2011-01-01

    Exosomes (EXO) are secreted intracellular microparticles that can trigger inflammation and induce antigen-specific immune responses. To test possible roles of EXO in autoimmunity, we isolated small microparticles, mainly EXO, from mouse insulinoma and examined their activities to stimulate the autoimmune responses in non-obese diabetic (NOD) mice, a model for human type 1 diabetes. We demonstrate that the EXO contains strong innate stimuli and expresses candidate diabetes autoantigens. They can induce secretion of inflammatory cytokines through MyD88-dependent pathways, and activate purified APC and result in T cell proliferation. To address whether EXO or the secreted microparticles are possible autoimmune targets causing islet-specific inflammation, we monitored the T cell responses spontaneously developed in prediabetic NOD mice for their reactivity to the EXO, and compared this reactivity between diabetes-susceptible and -resistant congenic mouse strains. We found that older NOD females, which have advanced islet destruction, accumulated more EXO-reactive, IFN-gamma-producing lymphocytes than younger females or age-matched males, and that pancreatic lymph nodes from the prediabetic NOD, but not from the resistant mice, were also enriched with EXO-reactive Th1 cells. In vivo, immunization with the EXO accelerates insulitis development in diabetes-resistant NOR mice. Thus, EXO or small microparticles can be recognized by the diabetes-associated autoreactive T cells, supporting that EXO might be a possible autoimmune target and/or insulitis trigger in NOD or congenic mouse strains. PMID:21734072

  18. Role of aryl hydrocarbon receptor nuclear translocator in K{sub ATP} channel-mediated insulin secretion in INS-1 insulinoma cells

    SciTech Connect

    Kim, Ji-Seon; Zheng Haifeng; Kim, Sung Joon; Ho, Won-Kyung; Chun, Yang-Sook

    2009-02-20

    Aryl hydrocarbon receptor nuclear translocator (ARNT) has been known to participate in cellular responses to xenobiotic and hypoxic stresses, as a common partner of aryl hydrocarbon receptor and hypoxia inducible factor-1/2{alpha}. Recently, it was reported that ARNT is essential for adequate insulin secretion in response to glucose input and that its expression is downregulated in the pancreatic islets of diabetic patients. In the present study, the authors addressed the mechanism by which ARNT regulates insulin secretion in the INS-1 insulinoma cell line. In ARNT knock-down cells, basal insulin release was elevated, but insulin secretion was not further stimulated by a high-glucose challenge. Electrophysiological analyses revealed that glucose-dependent membrane depolarization was impaired in these cells. Furthermore, K{sub ATP} channel activity and expression were reduced. Of two K{sub ATP} channel subunits, Kir6.2 was found to be positively regulated by ARNT at the mRNA and protein levels. Based on these results, the authors suggest that ARNT expresses K{sub ATP} channel and by so doing regulates glucose-dependent insulin secretion.

  19. Rapid intraoperative insulin assay: a novel method to differentiate insulinoma from nesidioblastosis in the pediatric patient.

    PubMed

    Strong, Vivian E; Shifrin, Alexander; Inabnet, William B

    2007-10-24

    Hyperinsulinism is the most common cause of recurrent and persistent hypoglycemia in infancy and childhood. Causes can include nesidioblastosis, pancreatic islet cell tumors such as insulinoma, and associations with multiple endocrine neoplasia syndromes. Although new, improved imaging techniques have allowed for more precise preoperative localization of insulinomas, the differentiation of nesidioblastosis and insulinoma, particularly in children, can be challenging. To improve intraoperative localization and confirmation of successful resection of insulinoma, a novel hormonal assay, the rapid intraoperative insulin assay, is reported for the first time in a pediatric patient. This intraoperative radioimmunoassay for insulin yields results within several minutes and confirms complete resection of insulinoma. We present a case of pancreatic insulinoma in a child with symptoms of severe hypoglycemia, causing seizures. The insulinoma was enucleated laparoscopically, and rapid intra-operative insulin assay used to determine the success of the procedure. This rapid intra-operative test provides a valuable adjunct for determining complete excision in complicated cases of recurrent or questionable insulinoma. Although not a common problem, for pediatric patients in whom the diagnosis is not clear, this test may provide a novel approach to confirming disease. We propose the use of this assay in facilitating intra-operative resection and confirmation of complete excision in pediatric patients. This population may especially benefit from this novel assay to confirm complete resection and to differentiate multiple etiologies of hyperinsulinism.

  20. Paroxysmal Hypertension Induced by an Insulinoma

    PubMed Central

    Harada, Ko; Hanayama, Yoshihisa; Hasegawa, Kou; Iwamuro, Masaya; Hagiya, Hideharu; Yoshida, Ryuichi; Otsuka, Fumio

    2017-01-01

    Insulinoma is a rare, usually benign, pancreatic neuroendocrine tumor. The clinical features of an insulinoma are fasting hypoglycemia with neuroglycopenic symptoms including confusion and unusual behavior, while hypertension is usually not associated with the disease. We herein report a patient with insulinoma who manifested paroxysmal hypertension and neuroglycopenic symptoms. The possible etiology of hypertension induced by an insulinoma is catecholamine release in response to hypoglycemia, which may cause acute hypertension through activation of the sympatho-adrenal system. This case implies that sustained hyperinsulinemia due to insulinoma can be functionally linked to the induction of paroxysmal hypertension. PMID:28202863

  1. Clonal selection in xenografted human T cell acute lymphoblastic leukemia recapitulates gain of malignancy at relapse

    PubMed Central

    Clappier, Emmanuelle; Gerby, Bastien; Sigaux, François; Delord, Marc; Touzri, Farah; Hernandez, Lucie; Ballerini, Paola; Baruchel, André

    2011-01-01

    Genomic studies in human acute lymphoblastic leukemia (ALL) have revealed clonal heterogeneity at diagnosis and clonal evolution at relapse. In this study, we used genome-wide profiling to compare human T cell ALL samples at the time of diagnosis and after engraftment (xenograft) into immunodeficient recipient mice. Compared with paired diagnosis samples, the xenograft leukemia often contained additional genomic lesions in established human oncogenes and/or tumor suppressor genes. Mimicking such genomic lesions by short hairpin RNA–mediated knockdown in diagnosis samples conferred a selective advantage in competitive engraftment experiments, demonstrating that additional lesions can be drivers of increased leukemia-initiating activity. In addition, the xenograft leukemias appeared to arise from minor subclones existing in the patient at diagnosis. Comparison of paired diagnosis and relapse samples showed that, with regard to genetic lesions, xenograft leukemias more frequently more closely resembled relapse samples than bulk diagnosis samples. Moreover, a cell cycle– and mitosis-associated gene expression signature was present in xenograft and relapse samples, and xenograft leukemia exhibited diminished sensitivity to drugs. Thus, the establishment of human leukemia in immunodeficient mice selects and expands a more aggressive malignancy, recapitulating the process of relapse in patients. These findings may contribute to the design of novel strategies to prevent or treat relapse. PMID:21464223

  2. Clonal selection in xenografted human T cell acute lymphoblastic leukemia recapitulates gain of malignancy at relapse.

    PubMed

    Clappier, Emmanuelle; Gerby, Bastien; Sigaux, François; Delord, Marc; Touzri, Farah; Hernandez, Lucie; Ballerini, Paola; Baruchel, André; Pflumio, Françoise; Soulier, Jean

    2011-04-11

    Genomic studies in human acute lymphoblastic leukemia (ALL) have revealed clonal heterogeneity at diagnosis and clonal evolution at relapse. In this study, we used genome-wide profiling to compare human T cell ALL samples at the time of diagnosis and after engraftment (xenograft) into immunodeficient recipient mice. Compared with paired diagnosis samples, the xenograft leukemia often contained additional genomic lesions in established human oncogenes and/or tumor suppressor genes. Mimicking such genomic lesions by short hairpin RNA-mediated knockdown in diagnosis samples conferred a selective advantage in competitive engraftment experiments, demonstrating that additional lesions can be drivers of increased leukemia-initiating activity. In addition, the xenograft leukemias appeared to arise from minor subclones existing in the patient at diagnosis. Comparison of paired diagnosis and relapse samples showed that, with regard to genetic lesions, xenograft leukemias more frequently more closely resembled relapse samples than bulk diagnosis samples. Moreover, a cell cycle- and mitosis-associated gene expression signature was present in xenograft and relapse samples, and xenograft leukemia exhibited diminished sensitivity to drugs. Thus, the establishment of human leukemia in immunodeficient mice selects and expands a more aggressive malignancy, recapitulating the process of relapse in patients. These findings may contribute to the design of novel strategies to prevent or treat relapse.

  3. Andrographolide suppress tumor growth by inhibiting TLR4/NF-κB signaling activation in insulinoma.

    PubMed

    Zhang, Qian-Qian; Ding, Yi; Lei, Yan; Qi, Cui-Ling; He, Xiao-Dong; Lan, Tian; Li, Jiang-Chao; Gong, Ping; Yang, Xuesong; Geng, Jian-Guo; Wang, Li-Jing

    2014-01-01

    Insulinomas are rare tumors, and approximately 10% of insulinomas are malignant. Accumulating evidence has implicated that we still lack effective therapy to treat the patients who are diagnosed with rare malignant insulinoma. Previous studies have reported that Andrographolide (Andro) could inhibit cell cycle progression, reduce cell invasion and induce cell apoptosis in many common cancer cells. However, the effects of andro are cell type-dependent. So we emplored the β-TC-6 cells and the RIP1-Tag2 transgenic mouse model of endogenously growing insulinoma model to elucidate the possible anti-cancer effect of Andro on insulinoma, an uncommon type of malignant cancers in this study. Our experiments revealed that Andro significantly inhibited tumor growth at both the early-stage and the advanced-stage of insulinoma through targeting the TLR4/NF-κB signaling pathway. This work initially provides the evidence that the TLR4/NF-κB signaling pathway might be vital as a potential therapeutic target, and also indispensable in Andro-mediated anti-cancer effect in insulinoma.

  4. Pancreatic insulinoma. Case report and review of the literature

    PubMed Central

    NEGREAN, VASILE; TUDOR, ANDRA; AIOANEI, OANA; DOMSA, IACOB

    2013-01-01

    Insulinoma is a rare pancreatic islet cell tumor, the most common cause of hypoglycemia related to endogenous hyperinsulinism. We present the case of an adult patient with pancreatic insulinoma. The patient presented to our clinic after prior hospitalizations in diabetes and psychiatry hospital units for repeated episodes of loss of consciousness, sweating and tonic-clonic seizures. Early detection of the cause is important for prompt initiation of therapy and to reduce symptoms. We describe our diagnostic and therapeutic strategies with references to previously published reports. PMID:26527982

  5. Gonadal status of male recipient mice influences germ cell development in immature buffalo testis tissue xenograft.

    PubMed

    Reddy, Niranjan; Mahla, Ranjeet Singh; Thathi, Revanth; Suman, Sanjay Kumar; Jose, Jedy; Goel, Sandeep

    2012-01-01

    Growth and development of immature testis xenograft from various domestic mammals has been shown in mouse recipients; however, buffalo testis xenografts have not been reported to date. In this study, small fragments of testis tissue from 8-week-old buffalo calves were implanted subcutaneously onto the back of immunodeficient male mouse recipients, which were either castrated or left intact (non-castrated). The xenografts were retrieved and analyzed 12 and 24 weeks later. The grafted tissue survived and grew in both types of recipient with a significant increase in weight and seminiferous tubule diameter. Recovery of grafts from intact recipients 24 weeks post-grafting was significantly lower than that from the castrated recipients. Seminal vesicle indices and serum testosterone levels were lower in castrated recipients at both collection time points in comparison to the intact recipients and non-grafted intact mouse controls. Pachytene spermatocytes were the most advanced germ cells observed in grafts recovered from castrated recipients 24 weeks post-grafting. Complete spermatogenesis, as indicated by the presence of elongated spermatids, was present only in grafts from intact recipients collected 24 weeks post-grafting. However, significant number of germ cells with DNA damage was also detected in these grafts as indicated by TUNEL assay. The complete germ cell differentiation in xenografts from intact recipients may be attributed to efficient Sertoli cell maturation. These results suggest that germ cell differentiation in buffalo testis xenograft can be completed by altering the recipient gonadal status.

  6. Desmoplastic small round cell tumor (DSRCT) xenografts and tissue culture lines: Establishment and initial characterization

    PubMed Central

    MARKIDES, CONSTANTINE S.A.; COIL, DOUGLAS R.; LUONG, LINH H.; MENDOZA, JOHN; KOZIELSKI, TONY; VARDEMAN, DANA; GIOVANELLA, BEPPINO C.

    2013-01-01

    Desmoplastic small round cell tumor (DSRCT) is an extremely rare and aggressive neoplasm, which mainly affects young males and generally presents as a widely disseminated tumor within the peritoneal cavity. Due to the rarity of the tumor, its younger and overall healthier patient population (compared with other tumor types) and the fact that it lacks definitive histological and immunohistological features, the diagnosis of DSRCT may be frequently delayed or the tumor may be entirely misdiagnosed as a different type of abdominal sarcoma. The present study aimed to rectify the lack of models that exist for this rare neoplasm, through the development of several DSRCT tissue cultures and xenograft lines. Samples were received from surgeries and biopsies from patients worldwide and were immediately processed for xenograft development in nude mice. Tumor tissues were minced and fragments were injected into the dorsal flanks of nude mice. Of the 14 samples received, nine were established into xenograft lines and five into tissue culture lines. Xenografts displayed the microscopic histology of their parent tumors and demonstrated two different growth rates among the established xenograft lines. Overall, the establishment of these xenograft and tissue culture lines provides researchers with tools to evaluate DSRCT responses to chemotherapy and to investigate DSRCT-specific signaling pathways or mechanisms. PMID:23759995

  7. Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential.

    PubMed

    Spacek, Tomás; Santorová, Jitka; Zacharovová, Klára; Berková, Zuzana; Hlavatá, Lydie; Saudek, Frantisek; Jezek, Petr

    2008-01-01

    Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (DeltaPsi(m)) during GSIS are lacking. Using high resolution respirometry and parallel DeltaPsi(m) monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and DeltaPsi(m) during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the "toxic" effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, DeltaPsi(m) jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10-20mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.

  8. Ca(2+) clearance by plasmalemmal NCLX, Li(+)-permeable Na(+)/Ca(2+) exchanger, is required for the sustained exocytosis in rat insulinoma INS-1 cells.

    PubMed

    Han, Young-Eun; Ryu, Shin-Young; Park, Sun-Hyun; Lee, Kyu-Hee; Lee, Suk-Ho; Ho, Won-Kyung

    2015-12-01

    Na(+)/Ca(2+) exchangers are key players for Ca(2+) clearance in pancreatic β-cells, but their molecular determinants and roles in insulin secretion are not fully understood. In the present study, we newly discovered that the Li(+)-permeable Na(+)/Ca(2+) exchangers (NCLX), which were known as mitochondrial Na(+)/Ca(2+) exchangers, contributed to the Na(+)-dependent Ca(2+) movement across the plasma membrane in rat INS-1 insulinoma cells. Na(+)/Ca(2+) exchange activity by NCLX was comparable to that by the Na(+)/Ca(2+) exchanger, NCX. We also confirmed the presence of NCLX proteins on the plasma membrane using immunocytochemistry and cell surface biotinylation experiments. We further investigated the role of NCLX on exocytosis function by measuring the capacitance increase in response to repetitive depolarization. Small interfering (si)RNA-mediated downregulation of NCLX did not affect the initial exocytosis, but significantly suppressed sustained exocytosis and recovery of exocytosis. XIP (NCX inhibitory peptide) or Na(+) replacement for inhibiting Na(+)-dependent Ca(2+) clearance also selectively suppressed sustained exocytosis. Consistent with the idea that sustained exocytosis requires ATP-dependent vesicle recruitment, mitochondrial function, assessed by mitochondrial membrane potential (ΔΨ), was impaired by siNCLX or XIP. However, depolarization-induced exocytosis was hardly affected by changes in intracellular Na(+) concentration, suggesting a negligible contribution of mitochondrial Na(+)/Ca(2+) exchanger. Taken together, our data indicate that Na(+)/Ca(2+) exchanger-mediated Ca(2+) clearance mediated by NCLX and NCX is crucial for optimizing mitochondrial function, which in turn contributes to vesicle recruitment for sustained exocytosis in pancreatic β-cells.

  9. Anticancer effect of silibinin on the xenograft model using MDA-MB-468 breast cancer cells.

    PubMed

    Kil, Won Ho; Kim, Sang Min; Lee, Jeong Eon; Park, Kyoung Sik; Nam, Seok Jin

    2014-10-01

    The aim of this study is to know whether silibinin has an anticancer effect on triple negative breast cancer xenograft model using MDA-MB-468 cells. To establish the xenograft model, we injected the MDA-MB-468 cells into female Balb/c-nude mice. After establishing a xenograft model, oral silibinin was administered to the tested mice in the way of 200 mg/kg for 45 days. The difference of mean tumor volume between silibinin fed mice and control mice was analyzed. The epidermal growth factor receptor (EGFR) phosphorylation in MDA-MB-468 cells was analyzed by Western blotting. The expression of VEGF, COX-2, and MMP-9 genes in tumor tissue was analyzed by real-time polymerase chain reaction (PCR). In the xenograft model using MDA-MB-468 cells, we found that oral administration of silibinin significantly suppressed the tumor volume (silibinin treated mice vs. control mice; 230.3 ± 61.6 mm(3) vs. 435.7 ± 93.5 mm(3), P < 0.001). The phosphorylation of EGFR in MDA-MB-468 cells was inhibited by treatment with 50 µg/mL of silibinin. In real time-PCR analysis of tumor tissue obtained from sacrificed mice, the gene expression of MMP-9, VEGF, and COX-2 was 51.8%-80% smaller in silibinin group than that of control group and we can also verify the similar result using Western blotting analysis. We verified that silibinin had anticancer effect on xenograft model of MDA-MB-468 cells in the way of preventing the phosphorylation of EGFR and eventually suppressed the production of COX-2, VEGF, and MMP-9 expression. Finally, the tumor volume of xenograft models was decreased after administration of Silibinin.

  10. Anticancer effect of silibinin on the xenograft model using MDA-MB-468 breast cancer cells

    PubMed Central

    Kim, Sang Min; Lee, Jeong Eon; Park, Kyoung Sik; Nam, Seok Jin

    2014-01-01

    Purpose The aim of this study is to know whether silibinin has an anticancer effect on triple negative breast cancer xenograft model using MDA-MB-468 cells. Methods To establish the xenograft model, we injected the MDA-MB-468 cells into female Balb/c-nude mice. After establishing a xenograft model, oral silibinin was administered to the tested mice in the way of 200 mg/kg for 45 days. The difference of mean tumor volume between silibinin fed mice and control mice was analyzed. The epidermal growth factor receptor (EGFR) phosphorylation in MDA-MB-468 cells was analyzed by Western blotting. The expression of VEGF, COX-2, and MMP-9 genes in tumor tissue was analyzed by real-time polymerase chain reaction (PCR). Results In the xenograft model using MDA-MB-468 cells, we found that oral administration of silibinin significantly suppressed the tumor volume (silibinin treated mice vs. control mice; 230.3 ± 61.6 mm3 vs. 435.7 ± 93.5 mm3, P < 0.001). The phosphorylation of EGFR in MDA-MB-468 cells was inhibited by treatment with 50 µg/mL of silibinin. In real time-PCR analysis of tumor tissue obtained from sacrificed mice, the gene expression of MMP-9, VEGF, and COX-2 was 51.8%-80% smaller in silibinin group than that of control group and we can also verify the similar result using Western blotting analysis. Conclusion We verified that silibinin had anticancer effect on xenograft model of MDA-MB-468 cells in the way of preventing the phosphorylation of EGFR and eventually suppressed the production of COX-2, VEGF, and MMP-9 expression. Finally, the tumor volume of xenograft models was decreased after administration of Silibinin. PMID:25317410

  11. [Effects of baicalin on HL-60 cell xenografts in nude mice and its mechanism].

    PubMed

    Zheng, Jing; Hu, Jian-Da; Huang, Yi; Chen, Ying-Yu; Li, Jing; Chen, Bu-Yuan

    2012-10-01

    This study was aimed to investigate the effects of baicalin on HL-60 cell xenografts in nude mice in vivo and explore its mechanism. Xenograft tumor model of HL-60 cells in nude mice was established, which was divided randomly into 6 groups: negative control group (injection of 5% NaHCO(3)), 25, 50 and 100 mg/kg baicalin groups, combination group (50 mg/kg baicalin + 2 mg/kg VP16) and positive control group (VP16 4 mg/kg). The nude mice with HL-60 cell xenografts were treated with drugs via intraperitoneal injection daily. After treatment for 14 days average weigh and inhibitory rate of transplanted tumor stripped from 5 nude mice in each group were calculated, and the ultrastructure change of xenografts cells were tested by transmission electron microscopy. Histopathologic examination was used to observed the change of main organs in nude mice. The expression of signaling molecular PI3K/Akt proteins extracted from xenografts was detected by Western blot. The effects of baicalin on overall survival time in nude mice with HL-60 cell xenografts were evaluated. The results showed that baicalin could inhibit the growth of transplanted tumors in dose-dependent manner. There were more necrotic and apoptotic cells in mice of baicalin-treated groups and combination group than that in mice of negative control group. Baicalin could inhibit the proliferation of HL-60 cells in vivo by down-regulating the PI3K/Akt/mTOR signal pathway, where the expressions of p-Akt, mTOR and p-mTOR proteins decreased compared with negative control group, and no significant difference of Akt expression was found between different groups. Compared with negative control group, the median survival time of mice in combination group was more prolongated (P < 0.05). It is concluded that baicalin can inhibit growth and induce apoptosis of HL-60 cell xenografts in nude mice, and prolong median survival time of nude mice. The possible mechanisms may be related to inhibition of Akt activity and down

  12. Insulinoma--experience from 1950 to 1995.

    PubMed Central

    Boukhman, M P; Karam, J H; Shaver, J; Siperstein, A E; Duh, Q Y; Clark, O H

    1998-01-01

    Insulinomas are rare tumors that originate from the islet cells of the pancreas. The purpose of this study was to analyze our experience in patients with insulinoma and present our approach to these patients. Medical records of 67 patients treated at the University of California, San Francisco (UCSF) Medical Center, 56 surgically and 11 medically, from 1954 to 1995 were retrospectively reviewed. Presenting symptoms, physical findings, laboratory data, pre and intraoperative localization studies, operative management, operative success, and post-operative complications were analyzed. Among the entire cohort, there were 11 patients with Multiple Endocrine Neoplasia type I (MEN 1) and 7 patients with multiple tumors. 46 out of 48 patients (96%) having first operations for benign tumors and 5 out of 8 patients (63%) having reoperations for benign tumors were successful, as were 6 out of 12 patients (50%) having operations for islet cell carcinoma. Overall, preoperative localization studies were positive in only 46% of patients and therefore failed to improve our surgical outcome. Careful palpation with intraoperative ultrasonography gave the best localization results. Enucleation of solitary tumors is curative in sporadic cases and gives the lowest complication rate. In patients with MEN 1, subtotal pancreatectomy with enucleation of tumours from the pancreatic head and uncinate process is recommended over simple enucleation because of frequent multiple tumors. Images Figure 3. PMID:9735690

  13. Germ cell differentiation in cryopreserved, immature, Indian spotted mouse deer (Moschiola indica) testes xenografted onto mice.

    PubMed

    Pothana, Lavanya; Makala, Himesh; Devi, Lalitha; Varma, Vivek Phani; Goel, Sandeep

    2015-03-01

    Death of immature animals is one of the reasons for the loss of genetic diversity of rare and endangered species. Because sperm cannot be collected from immature males, cryobanking of testicular tissue combined with testis xenografting is a potential option for conservation. The objective of this study was to evaluate the establishment of spermatogenesis in cryopreserved immature testicular tissues from Indian spotted mouse deer (Moschiola indica) after ectopic xenografting onto immunodeficient nude mice. Results showed that testis tissues that were frozen in cryomedia containing either 10% DMSO with 80% fetal bovine serum (D10S80) or 20% DMSO with 20% fetal bovine serum (D20S20) had significantly more (P < 0.01) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled positive interstitial cells when compared with fresh testis tissues (46.3 ± 3.4 and 51.9 ± 4.0 vs. 22.8 ± 2.0). Xenografted testicular tissues showed degenerated seminiferous tubules 24 weeks after grafting in testes that had been cryopreserved in D20S20; alternatively, pachytene spermatocytes were the most advanced germ cells in testes that were cryopreserved in D10S80. Proliferating cell nuclear antigen staining confirmed the proliferative status of spermatocytes, and the increases in tubular and lumen diameters indicated testicular maturation in xenografts. However, persistent anti-Müllerian hormone staining in Sertoli cells of xenografts revealed incomplete testicular maturation. This study reports that cryopreserved testis tissue that had been xenografted from endangered animals onto mice resulted in the establishment of spermatogenesis with initiation of meiosis. These findings are encouraging for cryobanking of testicular tissues from immature endangered animals to conserve their germplasm. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b).

    PubMed

    Desai, Shruti S; Kharade, Sampada S; Parekh, Vaishali I; Iyer, Sucharitha; Agarwal, Sunita K

    2015-10-16

    Pancreatic islet β-cells that lack the MEN1-encoded protein menin develop into tumors. Such tumors express the phosphorylated isoform of the β-cell differentiation transcription factor HLXB9. It is not known how phospho-HLXB9 acts as an oncogenic factor in insulin-secreting β-cell tumors (insulinomas). In this study we investigated the binding partners and target genes of phospho-HLXB9 in mouse insulinoma MIN6 β-cells. Co-immunoprecipitation coupled with mass spectrometry showed a significant association of phospho-HLXB9 with the survival factor p54nrb/Nono (54-kDa nuclear RNA-binding protein, non-POU-domain-containing octamer). Endogenous phospho-HLXB9 co-localized with endogenous Nono in the nucleus. Overexpression of HLXB9 decreased the level of overexpressed Nono but not endogenous Nono. Anti-phospho-HLXB9 chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) identified the c-Met inhibitor, Cblb, as a direct phospho-HLXB9 target gene. Phospho-HLXB9 occupied the promoter of Cblb and reduced the expression of Cblb mRNA. Cblb overexpression or HLXB9 knockdown decreased c-Met protein and reduced cell migration. Also, increased phospho-HLXB9 coincided with reduced Cblb and increased c-Met in insulinomas of two mouse models of menin loss. These data provide mechanistic insights into the role of phospho-HLXB9 as a pro-oncogenic factor by interacting with a survival factor and by promoting the oncogenic c-Met pathway. These mechanisms have therapeutic implications for reducing β-cell proliferation in insulinomas by inhibiting phospho-HLXB9 or its interaction with Nono and modulating the expression of its direct (Cblb) or indirect (c-Met) targets. Our data also implicate the use of pro-oncogenic activities of phospho-HLXB9 in β-cell expansion strategies to alleviate β-cell loss in diabetes.

  15. Exogenous administration of recombinant human FSH does not improve germ cell survival in human prepubertal xenografts.

    PubMed

    Van Saen, Dorien; Goossens, Ellen; Haentjens, Patrick; Baert, Yoni; Tournaye, Herman

    2013-03-01

    In a previous study, meiotic activity was observed in human intratesticular xenografts from peripubertal patients. However, full spermatogenesis could not be established. The present study aimed to evaluate whether the administration of recombinant human FSH could improve the spermatogonial survival and the establishment of full spermatogenesis in intratesticular human xenografts. Human testicular tissue was obtained from six boys (aged 2.5-12.5years). The testicular biopsy was fragmented and one fragment of 1.5-3.0mm(3) was transplanted to the testis of immunodeficient nude mice. Transplanted mice were assigned to different experimental groups to enable evaluation of the effects of FSH administration and freezing. The structural integrity of the seminiferous tubules, the spermatogonial survival and the presence of differentiated cells were evaluated by histology and immunohistochemistry. Freezing or administration of FSH did not influence tubule integrity and germ cell survival in human xenografts. Meiotic germ cells were observed in the xenografts. More tubules containing only Sertoli cells were observed in frozen-thawed grafts, and more tubules with meiotic cells were present in fresh grafts. There was no clear influence of FSH treatment on meiotic differentiation. Administration of FSH did not improve the establishment of full spermatogenesis after intratesticular tissue grafting. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  16. Mesenchymal stem cell-based NK4 gene therapy in nude mice bearing gastric cancer xenografts.

    PubMed

    Zhu, Yin; Cheng, Ming; Yang, Zhen; Zeng, Chun-Yan; Chen, Jiang; Xie, Yong; Luo, Shi-Wen; Zhang, Kun-He; Zhou, Shu-Feng; Lu, Nong-Hua

    2014-01-01

    Mesenchymal stem cells (MSCs) have been recognized as promising delivery vehicles for gene therapy of tumors. Gastric cancer is the third leading cause of worldwide cancer mortality, and novel treatment modalities are urgently needed. NK4 is an antagonist of hepatocyte growth factor receptors (Met) which are often aberrantly activated in gastric cancer and thus represent a useful candidate for targeted therapies. This study investigated MSC-delivered NK4 gene therapy in nude mice bearing gastric cancer xenografts. MSCs were transduced with lentiviral vectors carrying NK4 complementary DNA or enhanced green fluorescent protein (GFP). Such transduction did not change the phenotype of MSCs. Gastric cancer xenografts were established in BALB/C nude mice, and the mice were treated with phosphate-buffered saline (PBS), MSCs-GFP, Lenti-NK4, or MSCs-NK4. The tropism of MSCs toward gastric cancer cells was determined by an in vitro migration assay using MKN45 cells, GES-1 cells and human fibroblasts and their presence in tumor xenografts. Tumor growth, tumor cell apoptosis and intratumoral microvessel density of tumor tissue were measured in nude mice bearing gastric cancer xenografts treated with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 via tail vein injection. The results showed that MSCs migrated preferably to gastric cancer cells in vitro. Systemic MSCs-NK4 injection significantly suppressed the growth of gastric cancer xenografts. MSCs-NK4 migrated and accumulated in tumor tissues after systemic injection. The microvessel density of tumor xenografts was decreased, and tumor cellular apoptosis was significantly induced in the mice treated with MSCs-NK4 compared to control mice. These findings demonstrate that MSC-based NK4 gene therapy can obviously inhibit the growth of gastric cancer xenografts, and MSCs are a better vehicle for NK4 gene therapy than lentiviral vectors. Further studies are warranted to explore the efficacy and safety of the MSC-based NK4 gene therapy in

  17. Immunomodulatory action of the DNA methyltransferase inhibitor SGI-110 in epithelial ovarian cancer cells and xenografts.

    PubMed

    Srivastava, Pragya; Paluch, Benjamin E; Matsuzaki, Junko; James, Smitha R; Collamat-Lai, Golda; Taverna, Pietro; Karpf, Adam R; Griffiths, Elizabeth A

    2015-01-01

    We aimed to determine the effect of SGI-110 on methylation and expression of the cancer testis antigens (CTAs) NY-ESO-1 and MAGE-A in epithelial ovarian cancer (EOC) cells in vitro and in vivo and to establish the impact of SGI-110 on expression of major histocompatibility (MHC) class I and Intracellular Adhesion Molecule 1 (ICAM-1) on EOC cells, and on recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. We also tested the impact of combined SGI-110 and NY-ESO-1-specific CD8+ T-cells on tumor growth and/or murine survival in a xenograft setting. EOC cells were treated with SGI-110 in vitro at various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1 and MAGE-A methylation, mRNA, and protein expression were determined and compared to controls. SGI-110 treated EOC cells were evaluated for expression of immune-modulatory genes using flow cytometry, and were co-cultured with NY-ESO-1 specific T-cell clones to determine immune recognition. In vivo administration of SGI-110 and CD8+ T-cells was performed to determine anti-tumor effects on EOC xenografts. SGI-110 treatment induced hypomethylation and CTA gene expression in a dose dependent manner both in vitro and in vivo, at levels generally superior to azacitidine or decitabine. SGI-110 enhanced the expression of MHC I and ICAM-1, and enhanced recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. Sequential SGI-110 and antigen-specific CD8+ cell treatment restricted EOC tumor growth and enhanced survival in a xenograft setting. SGI-110 is an effective hypomethylating agent and immune modulator and, thus, an attractive candidate for combination with CTA-directed vaccines in EOC.

  18. Immunomodulatory action of the DNA methyltransferase inhibitor SGI-110 in epithelial ovarian cancer cells and xenografts

    PubMed Central

    Srivastava, Pragya; Paluch, Benjamin E; Matsuzaki, Junko; James, Smitha R; Collamat-Lai, Golda; Taverna, Pietro; Karpf, Adam R; Griffiths, Elizabeth A

    2015-01-01

    We aimed to determine the effect of SGI-110 on methylation and expression of the cancer testis antigens (CTAs) NY-ESO-1 and MAGE-A in epithelial ovarian cancer (EOC) cells in vitro and in vivo and to establish the impact of SGI-110 on expression of major histocompatibility (MHC) class I and Intracellular Adhesion Molecule 1 (ICAM-1) on EOC cells, and on recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. We also tested the impact of combined SGI-110 and NY-ESO-1-specific CD8+ T-cells on tumor growth and/or murine survival in a xenograft setting. EOC cells were treated with SGI-110 in vitro at various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1 and MAGE-A methylation, mRNA, and protein expression were determined and compared to controls. SGI-110 treated EOC cells were evaluated for expression of immune-modulatory genes using flow cytometry, and were co-cultured with NY-ESO-1 specific T-cell clones to determine immune recognition. In vivo administration of SGI-110 and CD8+ T-cells was performed to determine anti-tumor effects on EOC xenografts. SGI-110 treatment induced hypomethylation and CTA gene expression in a dose dependent manner both in vitro and in vivo, at levels generally superior to azacitidine or decitabine. SGI-110 enhanced the expression of MHC I and ICAM-1, and enhanced recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. Sequential SGI-110 and antigen-specific CD8+ cell treatment restricted EOC tumor growth and enhanced survival in a xenograft setting. SGI-110 is an effective hypomethylating agent and immune modulator and, thus, an attractive candidate for combination with CTA-directed vaccines in EOC. PMID:25793777

  19. Scaffold architecture controls insulinoma clustering, viability, and insulin production.

    PubMed

    Blackstone, Britani N; Palmer, Andre F; Rilo, Horacio R; Powell, Heather M

    2014-07-01

    Recently, in vitro diagnostic tools have shifted focus toward personalized medicine by incorporating patient cells into traditional test beds. These cell-based platforms commonly utilize two-dimensional substrates that lack the ability to support three-dimensional cell structures seen in vivo. As monolayer cell cultures have previously been shown to function differently than cells in vivo, the results of such in vitro tests may not accurately reflect cell response in vivo. It is therefore of interest to determine the relationships between substrate architecture, cell structure, and cell function in 3D cell-based platforms. To investigate the effect of substrate architecture on insulinoma organization and function, insulinomas were seeded onto 2D gelatin substrates and 3D fibrous gelatin scaffolds with three distinct fiber diameters and fiber densities. Cell viability and clustering was assessed at culture days 3, 5, and 7 with baseline insulin secretion and glucose-stimulated insulin production measured at day 7. Small, closely spaced gelatin fibers promoted the formation of large, rounded insulinoma clusters, whereas monolayer organization and large fibers prevented cell clustering and reduced glucose-stimulated insulin production. Taken together, these data show that scaffold properties can be used to control the organization and function of insulin-producing cells and may be useful as a 3D test bed for diabetes drug development.

  20. Speed of leukemia development and genetic diversity in xenograft models of T cell acute lymphoblastic leukemia

    PubMed Central

    Poglio, Sandrine; Lewandowski, Daniel; Calvo, Julien; Caye, Aurélie; Gros, Audrey; Laharanne, Elodie; Leblanc, Thierry; Landman-Parker, Judith; Baruchel, André; Soulier, Jean; Ballerini, Paola; Clappier, Emmanuelle; Pflumio, Françoise

    2016-01-01

    T cell acute lymphoblastic leukemia (T-ALL) develops through accumulation of multiple genomic alterations within T-cell progenitors resulting in clonal heterogeneity among leukemic cells. Human T-ALL xeno-transplantation in immunodeficient mice is a gold standard approach to study leukemia biology and we recently uncovered that the leukemia development is more or less rapid depending on T-ALL sample. The resulting human leukemia may arise through genetic selection and we previously showed that human T-ALL development in immune-deficient mice is significantly enhanced upon CD7+/CD34+ leukemic cell transplantations. Here we investigated the genetic characteristics of CD7+/CD34+ and CD7+/CD34− cells from newly diagnosed human T-ALL and correlated it to the speed of leukemia development. We observed that CD7+/CD34+ or CD7+/CD34− T-ALL cells that promote leukemia within a short-time period are genetically similar, as well as xenograft-derived leukemia resulting from both cell fractions. In the case of delayed T-ALL growth CD7+/CD34+ or CD7+/CD34− cells were either genetically diverse, the resulting xenograft leukemia arising from different but branched subclones present in the original sample, or similar, indicating decreased fitness to mouse micro-environment. Altogether, our work provides new information relating the speed of leukemia development in xenografts to the genetic diversity of T-ALL cell compartments. PMID:27191650

  1. Biological Analysis of Human CML Stem Cells; Xenograft Model of Chronic Phase Human Chronic Myeloid Leukemia.

    PubMed

    Abraham, Sheela A

    2016-01-01

    Xenograft mouse models have been instrumental in expanding our knowledge of hematopoiesis and can provide a functional description of stem cells that possess engrafting potential. Here we describe methodology outlining one way of analyzing human malignant cells that are able to engraft immune compromised mice. Using models such as these will allow researchers to gain valuable insight into the primitive leukemic subtypes that evade current therapy regimes and are critical to understand, in order to eradicate malignancy.

  2. T-cell number and subtype influence the disease course of primary chronic lymphocytic leukaemia xenografts in alymphoid mice

    PubMed Central

    Oldreive, Ceri E.; Skowronska, Anna; Davies, Nicholas J.; Parry, Helen; Agathanggelou, Angelo; Krysov, Sergey; Packham, Graham; Rudzki, Zbigniew; Cronin, Laura; Vrzalikova, Katerina; Murray, Paul; Odintsova, Elena; Pratt, Guy; Taylor, A. Malcolm R.; Moss, Paul; Stankovic, Tatjana

    2015-01-01

    ABSTRACT Chronic lymphocytic leukaemia (CLL) cells require microenvironmental support for their proliferation. This can be recapitulated in highly immunocompromised hosts in the presence of T cells and other supporting cells. Current primary CLL xenograft models suffer from limited duration of tumour cell engraftment coupled with gradual T-cell outgrowth. Thus, a greater understanding of the interaction between CLL and T cells could improve their utility. In this study, using two distinct mouse xenograft models, we investigated whether xenografts recapitulate CLL biology, including natural environmental interactions with B-cell receptors and T cells, and whether manipulation of autologous T cells can expand the duration of CLL engraftment. We observed that primary CLL xenografts recapitulated both the tumour phenotype and T-cell repertoire observed in patients and that engraftment was significantly shorter for progressive tumours. A reduction in the number of patient T cells that were injected into the mice to 2-5% of the initial number or specific depletion of CD8+ cells extended the limited xenograft duration of progressive cases to that characteristic of indolent disease. We conclude that manipulation of T cells can enhance current CLL xenograft models and thus expand their utility for investigation of tumour biology and pre-clinical drug assessment. PMID:26398941

  3. Curcumin inhibits human non-small cell lung cancer xenografts by targeting STAT3 pathway

    PubMed Central

    Xu, Xiaofang; Zhu, Yuping

    2017-01-01

    Human non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in men. Signal transducers and activators of transcription 3 (STAT3) is a potential molecular target in angiogenesis-mediated cancer therapy. In this study, we subcutaneously injected athymic nude mice with NCI-H460 cells to induce ectopic xenograft model, and treated the animals with curcumin (100 mg/kg) or vehicle by oral gavage. Tumor size and tumor weight were significantly reduced by curcumin treatment. Besides, curcumin significantly decreased hemoglobin content and mRNA expression of CD31 and CD105 in tumor tissue, suggesting that curcumin could inhibit angiogenesis in NSCLC xenograft. Similarly, we intrathoracally injected athymic nude mice with H1975 cells to induce orthotopic xenograft model, in which curcumin significantly reduced tumor weight as well as improved the survival rate of mice. STAT3 pathway was involved in curcumin-induced tumor inhibition, in which phosphorylation of STAT3 and JAK in ectopic xenograft were both declined after curcumin treatment, and the STAT3-regulated promoter activation of VEGF, Bcl-xL, Cyclin D1 was also significantly reduced after treatment. In in vitro assays, curcumin significantly inhibited cell migration and tube formation of NCI-H460 cells, but transfection with pMXs-Stat3C, a dominant active mutant, could abolish the inhibitory effects of curcumin on the cells, suggesting curcumin inhibited tumor angiogenesis of NCI-H460 cells through the inactivation of STAT3. All data showed that curcumin could be a potential drug targeting STAT3 to treat NSCLC. PMID:28861154

  4. Mapping of homozygous deletions in verified esophageal adenocarcinoma cell lines and xenografts.

    PubMed

    Boonstra, Jurjen J; van Marion, Ronald; Douben, Hannie J C W; Lanchbury, Jerry S; Timms, Kirsten M; Abkevich, Victor; Tilanus, Hugo W; de Klein, Annelies; Dinjens, Winand N M

    2012-03-01

    Human esophageal adenocarcinoma (EAC) cell lines and xenografts are powerful tools in the search for genetic alterations because these models are composed of pure human cancer cell populations without admixture of normal human cells. In particular detection of homozygous deletions (HDs) is easier using these pure populations of cancer cells. Identification of HDs could potentially lead to the subsequent identification of new tumor suppressor genes (TSGs) involved in esophageal adenocarcinogenesis. Genome wide single nucleotide polymorphism (SNP) arrays were used to identify HDs in 10 verified EAC cell lines and nine EAC xenografts. In total, 61 HDs (range 1-6 per sample) were detected and confirmed by polymerase chain reaction. Besides HDs observed in common fragile genomic regions (n = 26), and gene deserts (n = 8), 27 HDs were located in gene-containing regions. HDs were noted for known TSGs, including CDKN2A, SMAD4 and CDH3/CDH1. Twenty-two new chromosomal regions were detected harboring potentially new TSGs involved in EAC carcinogenesis. Two of these regions of homozygous loss, encompassing the ITGAV and RUNX1 gene, were detected in multiple samples indicating a potential role in the carcinogenesis of EAC. To exclude culturing artifacts, these last two deletions were confirmed by fluorescent in situ hybridization in the primary tumors of which the involved cell lines and xenografts were derived. In summary, in this report we describe the identification of HDs in a series of verified EAC cell lines and xenografts. The deletions documented here are a step forward identifying the key genes involved in EAC development.

  5. The emerging technologies of neural xenografting and stem cell transplantation for treating neurodegenerative disorders.

    PubMed

    Harrower, Timothy P; Barker, Roger A

    2004-02-01

    Neural transplantation has normally been considered in the context of the neurodegenerative disorders, Parkinson's and Huntington's disease, which are characterized pathologically by the predominant loss of specific cells in the basal ganglia. This approach has now emerged from the experimental arena into the level of clinical trial, at least with respect to fetal human allografts. However the ethical and practical problems with using such tissue has led to the search for alternative sources of cells of which two of the most promising are cells from another species, such as the pig (xenografts), and stem cells. Neural transplantation using cells derived from the developing pig brain offers many advantages. Firstly, time-mated litters will overcome the issue of donor tissue supply. Secondly, advances in genetic technology have led to the development of pigs which have a reduced rejection potential. Thirdly, xenografted neural fiber outgrowth may be superior to that from neural grafts derived from the same species (allografts) which may increase the potential for circuit reconstruction. Disadvantages with this tissue source include concerns about transmission of zoonotic infections and the immunological rejection of the xenograft. Stem cells are defined as cells capable of division (self-renewal) and differentiation into a range of different cell types (differentiation). A variety of such cells exist including embryonic stem cells, neural stem cells derived from the developing fetal brain (neural progenitor cells), adult neural stem cells and adult stem cells originating from outside of the central nervous system. Each of these different types of stem cell have their own unique benefits but also disadvantages, and access to each type is constrained by a number of limiting factors. All of this means that the translation of these cell therapies into practice is not straightforward and must be done at a pace dictated by laboratory-based research rather than corporate

  6. Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells.

    PubMed

    Epstein, Sharon; Kirkpatrick, Clare L; Castillon, Guillaume A; Muñiz, Manuel; Riezman, Isabelle; David, Fabrice P A; Wollheim, Claes B; Riezman, Howard

    2012-03-01

    Sphingolipids are not only important components of membranes but also have functions in protein trafficking and intracellular signaling. The LCB1 gene encodes a subunit of the serine palmitoyltransferase, which is responsible for the first step of sphingolipid synthesis. Here, we show that activation of the unfolded protein response (UPR) can restore normal ceramide levels and viability in yeast cells with a conditional defect in LCB1. Dependence on UPR was demonstrated by showing the HAC1-dependence of the suppression. A similar induction of ceramides by UPR seems to take place in mammalian cells. In rat pancreatic INS-1E cells, UPR activation induces the transcription of the CerS6 gene, which encodes a ceramide synthase. This correlates with the specific accumulation of ceramide with a C16 fatty acyl chain upon UPR activation. Therefore, our study reveals a novel connection between UPR induction and ceramide synthesis that seems to be conserved between yeast and mammalian cells.

  7. Mitochondrial malic enzyme (ME2) in pancreatic islets of the human, rat and mouse and clonal insulinoma cells.

    PubMed

    MacDonald, Michael J; Longacre, Melissa J; Kendrick, Mindy A

    2009-08-15

    Despite interest in malic enzyme(ME)s in insulin cells, mitochondrial malic enzyme (ME2) has only been studied with estimates of mRNA or with mRNA knockdown. Because an mRNA's level does not necessarily reflect the level of its cognate enzyme, we designed a simple spectrophotometric enzyme assay to measure ME2 activity of insulin cells by utilizing the distinct kinetic properties of ME2. Mitochondrial ME2 uses either NAD or NADP as a cofactor, has a high Km for malate and is allosterically activated by fumarate and inhibited by ATP. Cytosolic ME (ME1) and the other mitochondrial ME (ME3) use only NADP as a cofactor and have lower Kms for malate. The assay easily showed for the first time that substantial ME2 activity is present in pancreatic islets of humans, rats and mice and INS-1 832/13 cells. ME2's presence was confirmed with immunoblotting. There was no evidence that ME3 is present in these tissues.

  8. Insulinoma presenting as idiopathic hypersomnia.

    PubMed

    Maestri, Michelangelo; Monzani, Fabio; Bonanni, Enrica; Di Coscio, Elisa; Cignoni, Fabio; Dardano, Angela; Iudice, Alfonso; Murri, Luigi

    2010-06-01

    We report the case of a 32-year-old woman with a history of increased sleep need and difficulty waking up; the diagnosis of idiopathic hypersomnia was hypothesized. During ambulatory polysomnography (PSG), the patient presented an episode characterized by loss of consciousness and jerking of the four limbs. A video-PSG monitoring was performed and the patient showed unresponsiveness and drowsiness at 7 a.m. During the episode, EEG showed theta-delta diffuse activity, and blood glucose level was 32 mg dl(-1). The diagnosis of insulinoma was then assumed; CT scan showed a hypodense mass into the pancreatic tail, and a partial pancreasectomy was performed. The described symptoms disappeared, and 5 years later the findings of a complete clinical and neurophysiological examination were negative. The clinical picture of insulinoma presenting with paroxysmal disorders has been previously described; however, whereas hypersomnia is uncommon, in the current case it represents the main symptom. Clinicians should keep in mind that neuroglycopenia should be considered in the differential diagnosis of patients with hypersomnia, particularly if the clinical scenario does not conform to standard criteria.

  9. Cyclophilin A Enhances Cell Proliferation and Xenografted Tumor Growth of Early Gastric Cancer.

    PubMed

    Feng, Wenhua; Xin, Yan; Xiao, Yuping; Li, Wenhui; Sun, Dan

    2015-09-01

    Recently Cyclophilin A (CypA) was identified as a candidate target protein in gastric carcinoma. However, the role of CypA in gastric cancer (GC) has not been investigated extensively so far. The purpose of this study was to determine the expression pattern of CypA in human GC, and to explore the effects of suppressed CypA expression on cell proliferation and xenografted tumor growth of gastric cancer. In the present study, we detected the expression pattern of CypA in human GC by immunohistochemistry analysis. Further, the RNAi method was used to silence CypA, and colony formation assay, growth curves, cell cycle and mouse xenograft were analysed. An elevated expression of CypA in GC tissues compared with normal gastric mucosa was observed, especially in TNM stage-I and intestinal type of tumor. CypA was overexpressed in most GC cell lines and endogenous expression of CypA correlated with cell growth phenotypes. Transient suppression of CypA reduced the proliferation of BGC-823 and SGC-7901 GC cell lines. Exogenous CypA promoted the proliferation of NCI-N87 GC cells in a concentration dependent manner. Further study revealed that stable CypA silencing inhibited the proliferation, prevented cell cycle and reduced autophagy of BGC-823 GC cells in vitro through suppressing the ERK1/2 signal pathway. Stable CypA silencing also inhibited the growth of xenografted tumor of BGC-823 GC cell in nude mice. These results indicate a special function mode for CypA of playing more important roles in the early stage of gastric tumorigenesis and suggest CypA as a new molecular target of diagnosis and treatment for GC patients.

  10. Raman spectroscopy identifies radiation response in human non-small cell lung cancer xenografts

    NASA Astrophysics Data System (ADS)

    Harder, Samantha J.; Isabelle, Martin; Devorkin, Lindsay; Smazynski, Julian; Beckham, Wayne; Brolo, Alexandre G.; Lum, Julian J.; Jirasek, Andrew

    2016-02-01

    External beam radiation therapy is a standard form of treatment for numerous cancers. Despite this, there are no approved methods to account for patient specific radiation sensitivity. In this report, Raman spectroscopy (RS) was used to identify radiation-induced biochemical changes in human non-small cell lung cancer xenografts. Chemometric analysis revealed unique radiation-related Raman signatures that were specific to nucleic acid, lipid, protein and carbohydrate spectral features. Among these changes was a dramatic shift in the accumulation of glycogen spectral bands for doses of 5 or 15 Gy when compared to unirradiated tumours. When spatial mapping was applied in this analysis there was considerable variability as we found substantial intra- and inter-tumour heterogeneity in the distribution of glycogen and other RS spectral features. Collectively, these data provide unique insight into the biochemical response of tumours, irradiated in vivo, and demonstrate the utility of RS for detecting distinct radiobiological responses in human tumour xenografts.

  11. Midazolam Induces Cellular Apoptosis in Human Cancer Cells and Inhibits Tumor Growth in Xenograft Mice

    PubMed Central

    Mishra, Siddhartha Kumar; Kang, Ju-Hee; Lee, Chang Woo; Oh, Seung Hyun; Ryu, Jun Sun; Bae, Yun Soo; Kim, Hwan Mook

    2013-01-01

    Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosis-inducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-XL and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties. PMID:24008365

  12. Low dose carboplatin combined with angiostatic agents prevents metastasis in human testicular germ cell tumor xenografts.

    PubMed

    Abraham, Dietmar; Abri, Samad; Hofmann, Michael; Höltl, Wolfgang; Aharinejad, Seyedhossein

    2003-10-01

    Low dose chemotherapy combined with angiogenesis inhibitors has been shown to be more effective for experimental tumor treatment than chemotherapy alone. To our knowledge whether germ cell tumors could benefit from this treatment strategy remains to be evaluated. We examined the efficacy of angiostatic thrombospondin-1 (TSP-1), endostatin and combined angiostatic/low dose carboplatin in mice xenografted with human nonseminomatous germ cell tumor. We monitored tumor progression and angiogenesis in the established model of human nonseminomatous germ cell tumor xenograft in 120 SCID mice using intravital video microscopy, immunocytochemistry and real-time polymerase chain reaction. Mice received TSP-1 (20 mg/kg daily) or endostatin (10 mg/kg daily) subcutaneously (via osmotic mini pumps) for 2 weeks starting 15 days after cancer cell grafting, carboplatin cycled twice (30 mg/kg intraperitoneally days 14 and 21 after cancer cell grafting), or a combination of carboplatin with TSP-1 or endostatin. Untreated, sham and tumor bearing mice treated with Ringer's solution served as controls. Primary tumor development was not affected in mice treated with TSP-1, endostatin or carboplatin alone. All animals had metastases at 6 months, while metastasis did not develop following the combination of carboplatin with TSP-1 or endostatin. This combined therapy suppressed tumor angiogenesis, enhanced apoptosis in tumor cells and decreased vascular endothelial growth factor-A tissue mRNA expression vs controls (p <0.05). These data indicate that angiostatic agents added to low dose carboplatin have the ability to suppress the progression of human germ cell tumor xenografts toward a metastatic phenotype. Therefore, this treatment strategy might be beneficial to prevent metastasis in germ cell tumors.

  13. Occult sporadic insulinoma: localization and surgical strategy.

    PubMed

    Abboud, Bassam; Boujaoude, Joe

    2008-02-07

    Insulinomas continue to pose a diagnostic challenge to physicians, surgeons and radiologists alike. Most are intrapancreatic, benign and solitary. Biochemical diagnosis is obtained and imaging techniques to localize lesions continue to evolve. Surgical resection is the treatment of choice. Despite all efforts, an occult insulinoma (occult insulinoma refers to a biochemically proven tumor with indeterminate anatomical site before operation) may still be encountered. New localization preoperative techniques decreases occult cases and the knowledge of the site of the mass before surgery allows to determine whether enucleation of the tumor or pancreatic resection is likely to be required and whether the tumor is amenable to removal via a laparoscopic approach. In absence of preoperative localization and intraoperative detection of an insulinoma, blind pancreatic resection is not recommended.

  14. Teratomas produced from human pluripotent stem cells xenografted into immunodeficient mice - a histopathology atlas.

    PubMed

    Damjanov, Ivan; Andrews, Peter W

    2016-01-01

    This atlas illustrates the microscopic features of tumors produced from human pluripotent stem cells (hPSCs) xenografted into immunosuppressed mice, according to the generally accepted protocols for performing this teratoma assay of stem cell pluripotency. Microphotographs depict various hematoxylin and eosin (H&E) stained tissues derived from all three embryonic germ layers (ectoderm, mesoderm and endoderm). The appearance of persistent hPSC in teratomas is also described with special emphasis on the morphogenesis of embryoid bodies and yolk sac components surrounding them. The use of immunohistochemistry for analyzing hPSC-derived teratomas is also illustrated.

  15. Teratomas produced from human pluripotent stem cells xenografted into immunodeficient mice - a histopathology atlas

    PubMed Central

    Damjanov, Ivan; Andrews, Peter W.

    2017-01-01

    This atlas illustrates the microscopic features of tumors produced from human pluripotent stem cells (hPSCs) xenografted into immunosuppressed mice, according to the generally accepted protocols for performing this teratoma assay of stem cell pluripotency. Microphotographs depict various hematoxylin and eosin (H&E) stained tissues derived from all three embryonic germ layers (ectoderm, mesoderm and endoderm). The appearance of persistent hPSC in teratomas is also described with special emphasis on the morphogenesis of embryoid bodies and yolk sac components surrounding them. The use of immunohistochemistry for analyzing hPSC-derived teratomas is also illustrated. PMID:28000905

  16. EUS-Guided Ethanol Ablation of Insulinomas

    PubMed Central

    Qin, Shan-yu; Lu, Xiu-ping; Jiang, Hai-xing

    2014-01-01

    Abstract Surgical resection is a standard treatment for insulinomas; however, it is associated with a high risk of complications and limited to specific suitable candidates. In recent years, endoscopic ultrasound (EUS)-guided ethanol ablation of insulinomas has emerged as a new therapeutic option, especially for elderly patients and candidates unfit for surgery. We aimed to evaluate the feasibility and safety of this technique for insulinomas. Four patients diagnosed with insulinomas based on EUS–fine-needle aspiration and immunohistochemistry results underwent EUS-guided 95% ethanol ablation. A comprehensive literature review was performed to understand the current status of the feasibility, safety, and effects of EUS-guided ethanol ablation of insulinomas. EUS-guided ethanol ablation of insulinomas was successfully completed in all the 4 patients. There were no perioperative or postoperative complications. The patients were discharged at 3 days after the procedure. No recurrence of hypoglycemia or tumors was noted during follow-up (range, 3–6 months). Literature review showed 8 patients with insulinomas who underwent EUS-guided ethanol ablation. All the procedures were successful, with no need for further surgical treatment. Among these reviewed cases, 6 patients had no post-procedural complications, while other 2 patients showed a mild increase in the serum levels of lipase and/or pancreatic enzymes within 48 h post-procedure; furthermore, 1 of these 2 patients presented at a later date with medically controllable hematoma and ulceration. During follow-up, 6 patients remained asymptomatic and normoglycemic, while the 2 patients who presented post-procedural complications developed occasional mild confusion. EUS-guided ethanol ablation of insulinomas is an effective and safe modality, with an acceptable level of post-procedural complications. However, the long-term effects of this new therapeutic option need to be validated in a large randomized controlled

  17. Videolaparoscopic resection of insulinomas: experience in two institutions.

    PubMed

    Gramática, Luis; Herrera, Miguel F; Mercado-Luna, Andrés; Sierra, Mauricio; Verasay, Guillermo; Brunner, Noemí

    2002-10-01

    Laparoscopic resection of islet cell tumors has been performed in some selected cases. The aim of the study was to analyze the experience of two institutions in the laparoscopic management of insulinomas. In a 4-year period, videolaparoscopic resection of sporadic insulinomas was performed in 9 patients. All patients had hypoglycemia/hyperinsulinism and a solitary tumor demonstrated by image studies. Demographics, surgical findings, results, and complications were analyzed. Mean age of the patients was 43 years. One patient was male and eight were females. One tumor was located in the head of the pancreas, 4 in the body, and 4 in the tail. Laparoscopic resection was completed in all patients. Procedures included 4 enucleations and 5 distal pancreatectomies. Pancreatic resection with splenic preservation was achieved in 4 cases. Intraoperative ultrasound was used in 7 patients. Mean size of the tumors was 1.6 cm. All patients became normoglycemic after surgery. Complications included one pancreatic fistula, one pleural effusion, and one peripancreatic fluid collection. All resolved spontaneously. In a follow-up period between 3 and 48 months no evidence of recurrence has been observed. This series supports laparoscopic resection of preoperatively localized benign solitary insulinomas. The operation provides the advantages of minimally invasive surgery and can be safely performed in most cases.

  18. Erlotinib pretreatment improves photodynamic therapy of non-small cell lung carcinoma xenografts via multiple mechanisms

    PubMed Central

    Gallagher-Colombo, Shannon M.; Miller, Joann; Cengel, Keith A.; Putt, Mary E.; Vinogradov, Sergei A.; Busch, Theresa M.

    2015-01-01

    Aberrant expression of the epidermal growth factor receptor (EGFR) is a common characteristic of many cancers including non-small cell lung carcinoma (NSCLC), head and neck squamous cell carcinoma, and ovarian cancer. While EGFR is currently a favorite molecular target for treatment of these cancers, inhibition of the receptor with small molecule inhibitors (i.e.- erlotinib) or monoclonal antibodies (i.e.- cetuximab) does not provide long-term therapeutic benefit as standalone treatment. Interestingly, we have found that addition of erlotinib to photodynamic therapy (PDT) can improve treatment response in typically erlotinib-resistant NSCLC tumor xenografts. Ninety-day complete response rates of 63% are achieved when erlotinib is administered in three doses before PDT of H460 human tumor xenografts, compared to 16% after PDT-alone. Similar benefit is found when erlotinib is added to PDT of A549 NCSLC xenografts. Improved response is accompanied by increased vascular shutdown, and erlotinib increases the in vitro cytotoxicity of PDT to endothelial cells. Tumor uptake of the photosensitizer (benzoporphyrin derivative monoacid ring A; BPD) is increased by the in vivo administration of erlotinib; nevertheless, this elevation of BPD levels only partially accounts for the benefit of erlotinib to PDT. Thus, pretreatment with erlotinib augments multiple mechanisms of PDT effect that collectively lead to large improvements in therapeutic efficacy. These data demonstrate that short-duration administration of erlotinib before PDT can greatly improve the responsiveness of even erlotinib-resistant tumors to treatment. Results will inform clinical investigation of EGFR-targeting therapeutics in conjunction with PDT. PMID:26054596

  19. Xenografting of sheep testis tissue and isolated cells as a model for preservation of genetic material from endangered ungulates.

    PubMed

    Arregui, Lucía; Rathi, Rahul; Megee, Susan O; Honaramooz, Ali; Gomendio, Montserrat; Roldan, Eduardo R S; Dobrinski, Ina

    2008-07-01

    Recovery of germ cells could be an option for preservation of the genetic pool of endangered animals. In immature males, xenografting of testis tissue provides the opportunity to recover sperm from these animals. In adult animals, xenografting has been less successful, but de novo morphogenesis of functional testis tissue from dissociated testis cells could be an alternative. To assess the potential use of these techniques in endangered bovid species, the domestic sheep was used as a model. Testes from 2-week-old lambs were grafted as tissue fragments or cell suspensions into nude mice. Grafts were recovered at 4, 8, 12 and 16 weeks post grafting. For isolated cells, two additional time points at 35 and 40 weeks after grafting were added. In addition, to analyse the possible effect of social stress among mice within a group on the development of the grafts, testis tissue grafts were recovered 13 weeks post grafting from mice housed individually and in groups. Complete spermatogenesis occurred in sheep testis xenografts at 12 weeks, similar to the situation in situ. Isolated sheep testis cells were able to reorganize and form functional testicular tissue de novo. Housing mice individually or in groups did not have any effect on the development of xenografts. Xenografting of testis tissue might be useful to obtain sperm from immature endangered ungulates that die prematurely. Testis tissue de novo morphogenesis from isolated cells could open interesting options to recover germ cells from mature males with impaired spermatogenesis.

  20. ABCG2-overexpressing H460/MX20 cell xenografts in athymic nude mice maintained original biochemical and cytological characteristics

    PubMed Central

    Zhang, Wei; Chen, Zhen; Chen, Likun; Wang, Fang; Li, Furong; Wang, Xiaokun; Fu, Liwu

    2017-01-01

    H460/MX20 are derived from large cell lung cancer H460 cell line and then transformed into ABCG2-overexpressing cells by mitoxantrone’s induction, which are widely used in study of multidrug resistance (MDR) in vitro. To establish and spread the model of H460/MX20 cell xenografts, we investigated whether cell biological characteristics and the MDR phenotype were maintained in vivo model. Our results demonstrated that the cell proliferation, cell cycle, and ABCG2 expression level in xH460/MX20 cells isolated from H460/MX20 cell xenografts were similar to H460/MX20 cells in vitro. Importantly, xH460/MX20 cells exhibited high levels of resistance to ABCG2 substrates such as mitoxantrone and topotecan as H460/MX20 cells did. Furthermore, lapatinib, the inhibitor of ABCG2, potently reversed mitoxantrone- and topotecan-resistance of xH460/MX20 cells. Taken together, these results suggest that H460/MX20 cell xenografts in athymic nude mice still retain their original cytological characteristics and MDR phenotype. Thus, the H460/MX20 cell xenografts model could serve as a sound model in vivo for study on reversal MDR. PMID:28059154

  1. ABCG2-overexpressing H460/MX20 cell xenografts in athymic nude mice maintained original biochemical and cytological characteristics.

    PubMed

    Zhang, Wei; Chen, Zhen; Chen, Likun; Wang, Fang; Li, Furong; Wang, Xiaokun; Fu, Liwu

    2017-01-06

    H460/MX20 are derived from large cell lung cancer H460 cell line and then transformed into ABCG2-overexpressing cells by mitoxantrone's induction, which are widely used in study of multidrug resistance (MDR) in vitro. To establish and spread the model of H460/MX20 cell xenografts, we investigated whether cell biological characteristics and the MDR phenotype were maintained in vivo model. Our results demonstrated that the cell proliferation, cell cycle, and ABCG2 expression level in xH460/MX20 cells isolated from H460/MX20 cell xenografts were similar to H460/MX20 cells in vitro. Importantly, xH460/MX20 cells exhibited high levels of resistance to ABCG2 substrates such as mitoxantrone and topotecan as H460/MX20 cells did. Furthermore, lapatinib, the inhibitor of ABCG2, potently reversed mitoxantrone- and topotecan-resistance of xH460/MX20 cells. Taken together, these results suggest that H460/MX20 cell xenografts in athymic nude mice still retain their original cytological characteristics and MDR phenotype. Thus, the H460/MX20 cell xenografts model could serve as a sound model in vivo for study on reversal MDR.

  2. Recombinant leukemia inhibitory factor suppresses human medullary thyroid carcinoma cell line xenografts in mice.

    PubMed

    Starenki, Dmytro; Singh, Nishant K; Jensen, Davin R; Peterson, Francis C; Park, Jong-In

    2013-10-01

    Medullary thyroid carcinoma (MTC) is a neoplasm of the endocrine system, which originates from parafollicular C-cells of the thyroid gland. For MTC therapy, the Food and Drug Administration recently approved vandetanib and cabozantinib, multi-kinase inhibitors targeting RET and other tyrosine kinase receptors of vascular endothelial growth factor, epidermal growth factor, or hepatocyte growth factor. Nevertheless, not all patients with the progressive MTC respond to these drugs, requiring the development of additional therapeutic modalities that have distinct activity. Previously, we reported that expression of activated Ras or Raf in the human MTC cell lines, TT and MZ-CRC-1, can induce growth arrest and RET downregulation via a leukemia inhibitory factor (LIF)-mediated autocrine/paracrine loop. In this study, we aimed to evaluate bacterially-produced recombinant human LIF for its efficacy to suppress human MTC xenografts in mice. Here, we report that, consistent with its effects in vitro, locally or systemically administered recombinant LIF effectively suppressed growth of TT and MZ-CRC-1 xenografts in mice. Further, as predicted from its effects in TT and MZ-CRC-1 cell cultures in vitro, recombinant LIF activated the JAK/STAT pathway and downregulated RET and E2F1 expression in tumors in mice. These results suggest that LIF is a potent cytostatic agent for MTC cells, which regulates unique mechanisms that are not targeted by currently available therapeutic agents. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. Monitoring Serial Changes in Circulating Human Breast Cancer Cells in Mruine Xenograft Models

    PubMed Central

    Eliane, Jean-Pierre; Repollet, Madeline; Luker, Kathryn E.; Brown, Martha; Rae, James M.; Dontu, Gabriela; Schott, Anne F.; Wicha, Max; Doyle, Gerald V.; Hayes, Daniel F.; Luker, Gary D.

    2009-01-01

    Circulating tumor cells (CTC) are emerging as a powerful prognostic and predictive biomarker in several types of cancer, including breast, colon, and prostate. Studies of CTC in metastasis and further development of CTC as a biomarker in cancer have been limited by the inability to repetitively monitor CTC in mouse models of cancer. We have validated a method to enumerate CTC in blood samples obtained from living mice using a modified version of an in vitro diagnostic system for quantifying CTC in patients. Different routes of blood collection were tested to identify a method to reproducibly recover CTC from tumor-bearing mice without interference from contaminating normal murine epithelial cells. CTC are present in blood samples from mice bearing orthotopic xenografts of several different breast cancer cell lines and primary breast cancer cells from patient biopsies. We also show that this technology can be used for serial monitoring of CTC in mouse xenograft models of human breast cancer. These results establish a new method for studying CTC in mouse models of epithelial cancer, providing the foundation for studies of molecular regulation of CTC in cancer and CTC as biomarker for therapeutic efficacy. PMID:18632603

  4. Clonal dominance between subpopulations of mixed small cell lung cancer xenografts implanted ectopically in nude mice.

    PubMed

    Aabo, K; Vindeløv, L L; Spang-Thomsen, M

    1995-01-01

    Clonal evolution of neoplastic cells during solid tumour growth leads to the emergence of new tumour cell subpopulations with diverging phenotypic characteristics which may alter the behaviour of a malignant disease. Cellular interaction was studied in mixed xenografts in nude mice and during in vitro growth of two sets of small cell lung cancer (SCLC) subpopulations (54A, 54B and NYH, NYH2). The tumour cell lines differed in cellular DNA content enabling flow cytometric DNA analysis (FCM) to be used to monitor changes in the fractional composition of the mixed cell populations. The progeny clone 54B was found to dominate the parent 54A clone when grown as mixed subcutaneous xenografts in nude mice, whereas no dominance was exerted during in vitro growth. The in vivo dominance could not be explained by differences in growth kinetics between the two tumour cell lines, and the interaction was not dependent on 54B being in excess in mixed tumours. The dominance was dependent on close in vivo contact as no remote effect on the growth of 54A was found when the dominating 54B cells were growing in the opposite flank of tumour-bearing mice. Irradiation inactivated 54B cells were unable to exert the dominating effect, indicating that the interaction required viable and proliferating cells. Clonal dominance was not found in mixed NYH-NYH2 tumours indicating that the dominance mechanism(s) may not always be operational between subpopulations in heterogeneous tumours. Recognition of interaction between tumour cell populations may result in a better understanding of the behaviour of heterogeneous human malignancies.

  5. A zebrafish xenograft model for studying human cancer stem cells in distant metastasis and therapy response.

    PubMed

    Chen, L; Groenewoud, A; Tulotta, C; Zoni, E; Kruithof-de Julio, M; van der Horst, G; van der Pluijm, G; Ewa Snaar-Jagalska, B

    2017-01-01

    Lethal and incurable bone metastasis is one of the main causes of death in multiple types of cancer. A small subpopulation of cancer stem/progenitor-like cells (CSCs), also known as tumor-initiating cells from heterogenetic cancer is considered to mediate bone metastasis. Although over the past decades numerous studies have been performed in different types of cancer, it is still difficult to track small numbers of CSCs during the onset of metastasis. With use of noninvasive high-resolution imaging, transparent zebrafish embryos can be employed to dynamically visualize cancer progression and reciprocal interaction with stroma in a living organism. Recently we established a zebrafish CSC-xenograft model to visually and functionally analyze the role of CSCs and their interactions with the microenvironment at the onset of metastasis. Given the highly conserved human and zebrafish genome, transplanted human cancer cells are able to respond to zebrafish cytokines, modulate the zebrafish microenvironment, and take advantage of the zebrafish stroma during cancer progression. This chapter delineates the zebrafish CSC-xenograft model as a useful tool for both CSC biological study and anticancer drug screening. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Patient-derived xenograft models of squamous cell carcinoma of the uterine cervix.

    PubMed

    Rofstad, Einar K; Simonsen, Trude G; Huang, Ruixia; Andersen, Lise Mari K; Galappathi, Kanthi; Ellingsen, Christine; Wegner, Catherine S; Hauge, Anette; Gaustad, Jon-Vidar

    2016-04-10

    Patient-derived xenograft (PDX) models of cancer are considered to reflect the biology and treatment response of human tumors to a larger extent than xenograft models initiated from established cell lines. The characterization of a panel of four novel PDX models of cervical carcinoma of the uterine cervix is described in this communication. The outcome of treatment differed substantially among the donor patients, and the PDX models were found to mirror the histology, aggressiveness, and metastatic propensity of the donor patients' tumors. Two of the models (BK-12 and LA-19) were highly metastatic, one model (ED-15) was poorly metastatic, and one model (HL-16) was non-metastatic. The primary tumors of the two highly metastatic models showed high density of intratumoral lymphatics, whereas the other two models did not develop intratumoral lymphatics. The potential of the models to metastasize to lymph nodes was associated with high expression of both angiogenesis-related genes and cancer stem cell-related genes. The models may be highly valuable for studying mechanisms linking lymph node metastasis to lymphangiogenesis, hemangiogenesis, and the presence of cancer stem cells.

  7. T Cell-Mediated Rejection of Human CD34(+) Cells Is Prevented by Costimulatory Blockade in a Xenograft Model.

    PubMed

    Oh, Annie L; Mahmud, Dolores; Nicolini, Benedetta; Mahmud, Nadim; Senyuk, Vitalyi; Patel, Pritesh R; Bonetti, Elisa; Arpinati, Mario; Ferrara, James L M; Rondelli, Damiano

    2017-08-14

    A xenograft model of stem cell rejection was developed by co-transplantating human CD34(+) and allogeneic CD3(+) T cells into NOD-scid ɣ-chain(null) mice. T cells caused graft failure when transplanted at any CD34/CD3 ratio between 1:50 and 1:.1. Kinetics experiments showed that 2 weeks after transplantation CD34(+) cells engrafted the marrow and T cells expanded in the spleen. Then, at 4 weeks only memory T cells populated both sites and rejected CD34(+) cells. Blockade of T cell costimulation was tested by injecting the mice with abatacept (CTLA4-IgG1) from day -1 to +27 (group A), from day -1 to +13 (group B), or from day +14 to +28 (group C). On day +56 groups B and C had rejected the graft, whereas in group A graft failure was completely prevented, although with lower stem cell engraftment than in controls (P = .03). Retransplantation of group A mice with same CD34(+) cells obtained a complete reconstitution of human myeloid and B cell lineages and excluded latent alloreactivity. In this first xenograft model of stem cell rejection we showed that transplantation of HLA mismatched CD34(+) cells may be facilitated by treatment with abatacept and late stem cell boost. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  8. Bevacizumab enhances the therapeutic efficacy of Irinotecan against human head and neck squamous cell carcinoma xenografts.

    PubMed

    Cao, Shousong; Durrani, Farukh A; Toth, Karoly; Rustum, Youcef M; Seshadri, Mukund

    2011-06-01

    Combining antiangiogenic agents with traditional cytotoxic chemotherapy offers the potential to target both vascular and cellular components of a growing tumor mass. Here, we examined the antitumor activity of the vascular endothelial growth factor antibody, Bevacizumab (Avastin®) in combination with the topoisomerase I inhibitor, Irinotecan (CPT-11) against human head and neck squamous cell carcinoma (HNSCC) xenografts. Bevacizumab was administered daily (at 5 or 20mg/kg) to nude mice bearing FaDu HNSCC xenografts for 28days with the first dose beginning seven days prior to Irinotecan (100mg/kg, weekly × 4). Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and immunohistochemical (IHC) methods were employed to study the antiangiogenic effects of Bevacizumab in vivo. Kinetics of tumor response to treatment was studied by monitoring tumor volume over a 60-day period. DCE-MRI detected a significant reduction in vascular permeability following treatment with Bevacizumab (5mg/kg) while high dose Bevacizumab (20mg/kg) induced significant microvascular damage and tumor necrosis, confirmed by immunohistochemistry (IHC). Irinotecan alone resulted in complete tumor regression (cures) in ∼40% of animals while Bevacizumab alone did not result in any cures. Treatment with Bevacizumab (5mg/kg/day×28days) in combination with Irinotecan (100mg/kg, weekly × 4) was highly effective in inhibiting FaDu tumor growth and resulted in complete tumor regression in 80% of animals. These results demonstrate that long term administration of Bevacizumab effectively modulates chemotherapeutic efficacy against HNSCC xenografts. Further investigation into the therapeutic potential of this combination strategy against HNSCC is warranted.

  9. Increased expression of Beige/Brown adipose markers from host and breast cancer cells influence xenograft formation in mice

    PubMed Central

    Singh, Rajan; Parveen, Meher; Basgen, John M.; Fazel, Sayeda; Meshesha, Meron F.; Thames, Easter C.; Moore, Brandis; Martinez, Luis; Howard, Carolyn B.; Vergnes, Laurent; Reue, Karen; Pervin, Shehla

    2016-01-01

    The initiation and progression of breast cancer is a complex process that is influenced by heterogeneous cell populations within the tumor microenvironment (TME). Although adipocytes have been shown to promote breast cancer development, adipocyte characteristics involved in this process remain poorly understood. In this study, we demonstrate enrichment of beige/brown adipose markers, contributed from the host as well as tumor cells, in the xenografts from breast cancer cell lines. In addition to uncoupling protein-1 (UCP1) that is exclusively expressed in beige/brown adipocytes, gene expression for classical brown (MYF5, EVA1 and OPLAH), as well as beige (CD137/TNFRSF9 and TBX1) adipocyte markers, were also elevated in the xenografts. Enrichment of beige/brown characteristics in the xenografts was independent of the site of implantation of the breast tumor cells. Early stages of xenografts showed an expansion of a subset of mammary cancer stem cells (MCSCs) that expressed PRDM16, a master regulator of brown adipocyte differentiation. Depletion of UCP1+ or Myf5+ cells significantly reduced tumor development. There was increased COX-2 (MT-CO2) expression, which is known to stimulate formation of beige adipocytes in early xenografts and treatment with a COX-2 inhibitor (SC236) reduced tumor growth. By contrast, treatment with factors that induce brown adipocyte differentiation in vitro led to larger tumors in vivo. A panel of xenografts derived from established breast tumor cells as well as patient-tumor tissues were generated that expressed key brown adipose tissue (BAT)-related markers and contained cells that morphologically resembled brown adipocytes. Implications This is the first report demonstrating that beige /brown adipocyte characteristics could play an important role in breast tumor development and suggest a potential target for therapeutic drug design. PMID:26464213

  10. Insulinoma in a patient with NIDDM.

    PubMed

    Kane, L A; Grant, C S; Nippoldt, T B; Service, F J

    1993-09-01

    To confirm insulinoma as the cause of hypoglycemia in a patient with NIDDM and determine the frequency of the co-occurrence of these two conditions. The patient underwent an in-hospital prolonged fast (< or = 72 h), according to standard protocol, and an ultrasound examination of the pancreas. All cases of histologically confirmed insulinoma at this institution over the period of 1927-1992 were reviewed to determine the prevalence of pre-existent diabetes mellitus. After 10 h of fasting, plasma glucose was low (1.89 mM); plasma insulin (258 pM) and C-peptide (1.39 nM) were elevated in the absence of sulfonylurea in the plasma. An insulinoma detected by ultrasonography was removed surgically with subsequent reoccurrence of insulin-requiring diabetes. Among 313 cases of insulinoma confirmed at this institution, this patient is the only one with pre-existent diabetes mellitus. Insulinoma occurs extraordinarily rarely in patients with pre-existing NIDDM.

  11. New model of in-situ xenograft lymphangiogenesis by a human colonic adenocarcinoma cell line in nude mice.

    PubMed

    Sun, Jian-Jun; Jing, Wei; Ni, Yan-Yan; Yuan, Xiao-Jian; Zhou, Hai-Hua; Fan, Yue-Zu

    2012-01-01

    To explore a new model of in-situ xenograft lymphangiogenesis of human colonic adenocarcinomas in nude mice. On the basis of establishing subcutaneous xenograft lymphangiogenesis model of human colonic adenocarcinoms, in-situ xenografts were established through the in situ growth of the HT-29 human colonic adenocarcinoma cell line in nude mice. The numbers of lymphangiogenic microvessels, the expression of lymphatic endothelial cell markers lymphatic vessel endothelial hyaloronic acid receptor-1 (LYVE-1), D2-40 and the lymphatic endothelial growth factors vascular endothelial growth factor-C (VEGF-C), -D (VEGF-D) and receptor-3 (VEGFR-3) were compared by immunohistochemical staining, Western bolt and quantitative RT-PCR in xenograft in-situ models. Some microlymphatics with thin walls, large and irregular or collapsed cavities and increased LMVD, with strong positive of LYVE-1, D2-40 in immunohistochemistry, were observed, identical with the morphological characteristics of lymphatic vessels and capillaries. Expression of LYVE-1 and D2-40 proteins and mRNAs were significantly higher in xenografts in-situ than in the negative control group (both P<0.01). Moreover, the expression of VEGF-C, VEGF-D and VEGFR-3 proteins and mRNAs were significantly higher in xenografts in-situ (both P<0.01), in conformity with the signal regulation of the VEGF-C,-D/VEGFR-3 axis of tumor lymphangiogenesis. In-situ xenografts of a human colonic adenocarcinoma cell line demonstrate tumor lymphangiogenesis. This novel in-situ animal model should be useful for further studying mechanisms of lymph node metastasis, drug intervention and anti-metastasis therapy in colorectal cancer.

  12. Crizotinib Fails to Enhance the Effect of Radiation in Head and Neck Squamous Cell Carcinoma Xenografts.

    PubMed

    Baschnagel, Andrew M; Galoforo, Sandra; Thibodeau, Bryan J; Ahmed, Samreen; Nirmal, Sonali; Akervall, Jan; Wilson, George D

    2015-11-01

    Mesenchymal-epithelial transition factor (MET), a receptor tyrosine kinase, is expressed in head and neck squamous cell carcinomas (HNSCC) and is involved in tumor progression and associated with poor prognosis. MET can be inhibited by crizotinib, a potent ATP-competitive kinase inhibitor. We examined the effects of combining crizotinib and radiation in a pre-clinical HNSCC model. Nine HNSCC cell lines were screened for MET expression, copy-number amplification and mutational status. The in vitro effects of crizotinib and radiation were assessed with clonogenic survival assays. MET signaling proteins were assessed with western blot and receptor tyrosine kinase array. Tumor growth-delay experiments with UT-SCC-14 and UT-SCC-15 oral tongue xenografts were used to assess in vivo tumor radiosensitivity. All nine HNSCC cell lines showed a varying degree of MET protein and RNA expression. Increased MET copy number was not present. MET was expressed after irradiation both in vitro and in vivo. Crizotinib alone inhibited phosphorylation of MET and inhibited cell growth in vitro but did not inhibit phosphorylation of downstream signaling proteins: MAPK, AKT or c-SRC. When combined with radiation in vitro, crizotinib demonstrated radiation enhancement in only one cell line. Crizotinib did not enhance the effect of radiation in either UT-SCC-14 or UT-SCC-15 tumors grown as xenografts. MET is overexpressed in HNSCC cell lines, however, crizotinib failed to enhance the radiation response and failed to inhibit MET downstream signaling proteins in this HNSCC model. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  13. Survivin Antisense Oligonucleotides Effectively Radiosensitize Colorectal Cancer Cells in Both Tissue Culture and Murine Xenograft Models

    SciTech Connect

    Roedel, Franz; Capalbo, Gianni; Weiss, Christian; Roedel, Claus

    2008-05-01

    Purpose: Survivin shows a radiation resistance factor in colorectal cancer. In the present study, we determined whether survivin messenger RNA levels in patients with rectal cancer predict tumor response after neoadjuvant radiochemotherapy and whether inhibition of survivin by the use of antisense oligonucleotides (ASOs) enhances radiation responses. Methods and Materials: SW480 colorectal carcinoma cells were transfected with survivin ASO (LY2181308) and irradiated with doses ranging from 0-8 Gy. Survivin expression, cell-cycle distribution, {gamma}H2AX fluorescence, and induction of apoptosis were monitored by means of immunoblotting, flow cytometry, and caspase 3/7 activity. Clonogenic survival was determined by using a colony-forming assay. An SW480 xenograft model was used to investigate the effect of survivin attenuation and irradiation on tumor growth. Furthermore, survivin messenger RNA levels were studied in patient biopsy specimens by using Affymetrix microarray analysis. Results: In the translational study of 20 patients with rectal cancer, increased survivin levels were associated with significantly greater risk of local tumor recurrence (p = 0.009). Treatment of SW480 cells with survivin ASOs and irradiation resulted in an increased percentage of apoptotic cells, caspase 3/7 activity, fraction of cells in the G{sub 2}/M phase, and H2AX phosphorylation. Clonogenic survival decreased compared with control-treated cells. Furthermore, treatment of SW480 xenografts with survivin ASOs and irradiation resulted in a significant delay in tumor growth. Conclusion: Survivin appears to be a molecular biomarker in patients with rectal cancer. Furthermore, in vitro and in vivo data suggest a potential role of survivin as a molecular target to improve treatment response to radiotherapy in patients with rectal cancer.

  14. Bevacizumab radiosensitizes non-small cell lung cancer xenografts by inhibiting DNA double-strand break repair in endothelial cells.

    PubMed

    Gao, Hui; Xue, Jianxin; Zhou, Lin; Lan, Jie; He, Jiazhuo; Na, Feifei; Yang, Lifei; Deng, Lei; Lu, You

    2015-08-28

    The aims of this study were to evaluate the effects of biweekly bevacizumab administration on a tumor microenvironment and to investigate the mechanisms of radiosensitization that were induced by it. Briefly, bevacizumab was administered intravenously to Balb/c nude mice bearing non-small cell lung cancer (NSCLC) H1975 xenografts; in addition, bevacizumab was added to NSCLC or endothelial cells (ECs) in vitro, followed by irradiation (IR). The anti-tumor efficacy, anti-angiogenic efficacy and repair of DNA double-strand breaks (DSBs) were evaluated. The activation of signaling pathways was determined using immunoprecipitation (IP) and WB analyses. Finally, biweekly bevacizumab administration inhibited the growth of H1975 xenografts and induced vascular normalization periodically. Bevacizumab more significantly increased cellular DSB and EC apoptosis when administered 1 h prior to 12 Gy/1f IR than when administered 5 days prior to IR, thereby inhibiting tumor angiogenesis and growth. In vitro, bevacizumab more effectively increased DSBs and apoptosis prior to IR and inhibited the clonogenic survival of ECs but not NSCLC cells. Using IP and WB analyses, we confirmed that bevacizumab can directly inhibit the phosphorylation of components of the VEGR2/PI3K/Akt/DNA-PKcs signaling pathway that are induced by IR in ECs. In conclusion, bevacizumab radiosensitizes NSCLC xenografts mainly by inhibiting DSB repair in ECs rather than by inducing vascular normalization.

  15. DHEA increases epithelial markers and decreases mesenchymal proteins in breast cancer cells and reduces xenograft growth.

    PubMed

    Colín-Val, Zaira; González-Puertos, Viridiana Yazmín; Mendoza-Milla, Criselda; Gómez, Erika Olivia; Huesca-Gómez, Claudia; López-Marure, Rebeca

    2017-10-15

    Breast cancer is one of the most common neoplasias and the leading cause of cancer death in women worldwide. Its high mortality rate is linked to a great metastatic capacity associated with the epithelial-mesenchymal transition (EMT). During this process, a decrease in epithelial proteins expression and an increase of mesenchymal proteins are observed. On the other hand, it has been shown that dehydroepiandrosterone (DHEA), the most abundant steroid in human plasma, inhibits migration of breast cancer cells; however, the underlying mechanisms have not been elucidated. In this study, the in vitro effect of DHEA on the expression pattern of some EMT-related proteins, such as E-cadherin (epithelial), N-cadherin, vimentin and Snail (mesenchymal) was measured by Western blot and immunofluorescence in MDA-MB-231 breast cancer cells with invasive, metastatic and mesenchymal phenotype. Also, the in vivo effect of DHEA on xenograft tumor growth in nude mice (nu(-)/nu(-)) and on expression of the same epithelial and mesenchymal proteins in generated tumors was evaluated. We found that DHEA increased expression of E-cadherin and decreased N-cadherin, vimentin and Snail expression both in MD-MB-231 cells and in the formed tumors, possibly by DHEA-induced reversion of mesenchymal phenotype. These results were correlated with a tumor size reduction in mouse xenografts following DHEA administration either a week earlier or concurrent with breast cancer cells inoculation. In conclusion, DHEA could be useful in the treatment of breast cancer with mesenchymal phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Insulinoma: A Comprehensive Summary of Two Cases

    PubMed Central

    Sharma, Pankaj; Sharma, Sonia; Singh, Bhanu Pratap; Sharma, Sonam

    2014-01-01

    Pancreatic endocrine tumours are rare in occurrence. They may present with a baffling range of symptoms which pose a diagnostic dilemma. Two cases of pancreatic insulinoma are presented herewith. Both presented with neuro-psychiatric complaints and were treated symptomatically for depression, anxiety, seizures etc. The diagnosis in these patients was missed for years. Insulinoma being a great diagnostic challenge, requires reasonably high clinical suspicion, accurate biochemical diagnosis and radiological localization to avoid extensive surgery. The aim is to highlight the occurrence of this rare tumour at a tertiary care hospital. PMID:25386444

  17. Inhibition of telomerase in the endothelial cells disrupts tumor angiogenesis in glioblastoma xenografts.

    PubMed

    Falchetti, Maria Laura; Mongiardi, Maria Patrizia; Fiorenzo, Paolo; Petrucci, Giovanna; Pierconti, Francesco; D'Agnano, Igea; D'Alessandris, Giorgio; Alessandri, Giulio; Gelati, Maurizio; Ricci-Vitiani, Lucia; Maira, Giulio; Larocca, Luigi Maria; Levi, Andrea; Pallini, Roberto

    2008-03-15

    Tumor angiogenesis is a complex process that involves a series of interactions between tumor cells and endothelial cells (ECs). In vitro, glioblastoma multiforme (GBM) cells are known to induce an increase in proliferation, migration and tube formation by the ECs. We have previously shown that in human GBM specimens the proliferating ECs of the tumor vasculature express the catalytic component of telomerase, hTERT, and that telomerase can be upregulated in human ECs by exposing these cells to GBM in vitro. Here, we developed a controlled in vivo assay of tumor angiogenesis in which primary human umbilical vascular endothelial cells (HUVECs) were subcutaneously grafted with or without human GBM cells in immunocompromised mice as Matrigel implants. We found that primary HUVECs did not survive in Matrigel implants, and that telomerase upregulation had little effect on HUVEC survival. In the presence of GBM cells, however, the grafted HUVECs not only survived in Matrigel implants but developed tubule structures that integrated with murine microvessels. Telomerase upregulation in HUVECs enhanced such effect. More importantly, inhibition of telomerase in HUVECs completely abolished tubule formation and greatly reduced survival of these cells in the tumor xenografts. Our data demonstrate that telomerase upregulation by the ECs is a key requisite for GBM tumor angiogenesis. (c) 2007 Wiley-Liss, Inc.

  18. Growth suppression of MCF-7 cancer cell-derived xenografts in nude mice by caveolin-1

    SciTech Connect

    Wu Ping; Wang Xiaohui; Li Fei; Qi Baoju; Zhu Hua; Liu Shuang; Cui Yeqing; Chen Jianwen

    2008-11-07

    Caveolin-1 is an essential structural constituent of caveolae membrane domains that has been implicated in mitogenic signaling and oncogenesis. However, the exact functional role of caveolin-1 still remains controversial. In this report, utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/cav-1 cells), we demonstrate that caveolin-1 expression dramatically inhibits invasion and migration of these cells. Importantly, in vivo experiments employing xenograft tumor models demonstrated that expression of caveolin-1 results in significant growth inhibition of breast tumors. Moreover, a dramatic delay in tumor progression was observed in MCF-7/cav-1 cells as compared with MCF-7 cells. Histological analysis of tumor sections demonstrated a marked decrease in the percentage of proliferating tumor cells (Ki-67 assay) along with an increase in apoptotic tumor cells (TUNEL assay) in MCF-7/cav-1-treated animals. Our current findings provide for the first time in vivo evidence that caveolin-1 can indeed function as a tumor suppressor in human breast adenocarcinoma derived from MCF-7 cells rather than as a tumor promoter.

  19. Topographic abnormalities of proinsulin to insulin conversion in functioning human insulinomas. Comparison of immunoelectron microscopic and clinical data.

    PubMed Central

    Roth, J.; Komminoth, P.; Heitz, P. U.

    1995-01-01

    It has been proposed that the major defect in human insulinomas is a decreased hormone storage capacity resulting in uncontrolled release of proinsulin and insulin. By immunoelectron microscopy with monoclonal antibodies we studied the subcellular distribution of proinsulin and insulin in benign and malignant functioning insulinomas of different histology and compared the findings with various clinical and pathohistological parameters. We found that, in contrast to normal B cells, the proinsulin to insulin conversion in insulinomas occurs already in the trans Golgi apparatus but remains incomplete, resulting in the formation of secretory granules containing both proinsulin and insulin. Thus, in functioning insulinomas, sorting into secretory granules is not a prerequisite for hormone conversion. Furthermore, proinsulin and insulin storage and most probably subsequent secretion occurs through the secretory granules via the regulated pathway. A substantial variability for both proinsulin and insulin immunolabeling in secretory granules was found not only in individual tumor cells but also among the insulinomas studied. This observed variability may account for the lack of correlation between pathohistological, immunohistochemical, and clinical parameters in functioning insulinomas. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7639339

  20. Comparison of the Gene Expression Profiles of Human Hematopoietic Stem Cells between Humans and a Humanized Xenograft Model.

    PubMed

    Matsuzawa, Hideyuki; Matsushita, Hiromichi; Yahata, Takashi; Tanaka, Masayuki; Ando, Kiyoshi

    2017-04-20

    The aim of this study is to evaluate the feasibility of NOD/Shi-scid-IL2Rγ(null)(NOG) mice transplanted with human CD34(+)/CD38(-)/Lin(-/low) hematopoietic cells from cord blood (CB) as an experimental model of the gene expression in human hematopoiesis. We compared the gene expressions of human CD34(+)/CD38(-)/Lin(-/low) cells from human bone marrow (BM) and in xenograft models. The microarray data revealed that 25 KEGG pathways were extracted from the comparison of human CD34(+)/CD38(-)/Lin(-/low) HSCs between CB and BM, and that 17 of them--which were mostly related to cellular survival, RNA metabolism and lymphoid development--were shared with the xenograft model. When the probes that were commonly altered in CD34(+)/CD38(-)/Lin(-/low) cells from both human and xenograft BM were analyzed, most of them, including the genes related hypoxia, hematopoietic differentiation, epigenetic modification, translation initiation, and RNA degradation, were downregulated. These alterations of gene expression suggest a reduced differentiation capacity and likely include key alterations of gene expression for settlement of CB CD34(+)/CD38(-)/Lin(-/low) cells in BM. Our findings demonstrate that the xenograft model of human CB CD34(+)/CD38(-)/Lin(-/low) cells using NOG mice was useful, at least in part, for the evaluation of the gene expression profile of human hematopoietic stem cells.

  1. Chondrocytic differentiation of peripheral neuroectodermal tumor cell line in nude mouse xenograft.

    PubMed

    Goji, J; Sano, K; Nakamura, H; Ito, H

    1992-08-01

    We have established a cell line (KU-SN) from a peripheral neuroectodermal tumor originating in the left scapula of a 4-year-old girl. The original tumor was immunoreactive with antibodies for neurofilament proteins, neuron-specific enolase, vimentin, S100 protein, and beta 2-microglobulin. Dense core granules, 50-150 nm in diameter, were identified by electron microscopy. The cell line was established from tumor cells in metastatic lung fluid. KU-SN cells were immunoreactive with the antibodies for neurofilament proteins, vimentin, neuron-specific enolase, S100 protein, glial fibrillary acidic protein, cytokeratin, and carcinoembryonic antigen. Besides these neuronal features, KU-SN cells express type 2 collagen and insulin-like growth factor 1 receptor. The addition of insulin-like growth factor 1 (100 ng/ml) increased the growth rate of KU-SN cells 2.1-fold over control. Some cells were positive for Alcian blue and alkaline phosphatase staining. Cytogenetic analysis of KU-SN cells disclosed a reciprocal chromosomal translocation [t(11,22)]. Northern blot analysis of KU-SN cells demonstrated amplified expression of the c-myc gene but not the N-myc gene. When tumor cells were transplanted into nude mice, cartilage was formed. The cartilage was immunoreactive with the antibody for HLA-ABC, indicating that it was derived from the tumor cells, not from mouse tissue. Chondrocytic differentiation was not observed in xenografts of Ewing's sarcoma cell lines SK-ES or RD-ES or the peripheral neuroectodermal tumor cell line SK-N-MC. These results indicate that KU-SN cells represent primitive neural crest cells having the potential for chondrocytic differentiation.

  2. In vivo cell cycle profiling in xenograft tumors by quantitative intravital microscopy

    PubMed Central

    Chittajallu, Deepak R; Florian, Stefan; Kohler, Rainer H; Iwamoto, Yoshiko; Orth, James D; Weissleder, Ralph; Danuser, Gaudenz; Mitchison, Timothy J

    2015-01-01

    Quantification of cell-cycle state at a single-cell level is essential to understand fundamental three-dimensional biological processes such as tissue development and cancer. Analysis of 3D in vivo images, however, is very challenging. Today’s best practice, manual annotation of select image events, generates arbitrarily sampled data distributions, unsuitable for reliable mechanistic inferences. Here, we present an integrated workflow for quantitative in vivo cell-cycle profiling. It combines image analysis and machine learning methods for automated 3D segmentation and cell-cycle state identification of individual cell-nuclei with widely varying morphologies embedded in complex tumor environments. We applied our workflow to quantify cell-cycle effects of three antimitotic cancer drugs over 8 days in HT-1080 fibrosarcoma xenografts in living mice using a dataset of 38,000 cells and compared the induced phenotypes. In contrast to 2D culture, observed mitotic arrest was relatively low, suggesting involvement of additional mechanisms in their antitumor effect in vivo. PMID:25867850

  3. [Detecting doxorubicin concentration in KBv200 and KB cell xenografts in nude mice by high-performance liquid chromatography].

    PubMed

    Deng, Wen-Jing; Zeng, Zhao-Lei; Liang, Yong-Ju; Dai, Chun-Ling; Zhang, Jian-Ye; Fu, Li-Wu

    2008-04-01

    Doxorubicin (Dox) is one of the most commonly used chemotherapeutic drugs. Drug concentrations in tumor tissue can predict the drugos efficacy better than that in serum do. This study was to detect and compare the concentration of Dox in KBv200 and KB cell xenografts in nude mice by high-performance liquid chromatography (HPLC). Tumor models in nude mice were established with KB cells (drug-sensitive) and KBv200 cells (multidrug-resistant). Dox was injected via the tail vein. The concentration of Dox in tumor tissue was detected by HPLC at 1, 3 and 5 h after injection. A Hypersil reversed-phase BDS C18 column (250 mm x 4.6 mm, ID 5 microm) and mobile phases that were composed of acetonitrile and 0.02 mol/L KH2PO4 (1/2.4, V/V, pH 3.9) at a flow rate of 1.0 mL/min were used for setting a fluorescence detector (excitation wave length was 480 nm; emission wave length was 580 nm). Under the condition of HPLC, the calibration curve of Dox concentration in tumor tissue was linear within a range of 29.3-7 500 ng/g (r=0.9998). The limit of detection in tumor tissue was 14 ng/g. At the concentration of 3 750, 468.8 and 117.2 ng/g, extraction recovery were (99.35+/-7.65)%, (99.79+/-5.73)% and (103.67+/-6.76)%, respectively, method recovery were (91.89+/-7.03)%, (94.94+/-5.18)% and (100.83+/-5.32)%, respectively. The relative standard deviation (RSD) of the intra-day and inter-day precision were less than 4.2%. At 1, 3, 5 h after Dox injection, the concentrations of Dox were (139.32+/-54.68), (260.00+/-126.11) and (173.26+/-13.88) ng/g in KBv cell xenografts, respectively, and (385.13+/-42.55), (523.38+/-138.84) and (460.75+/-86.85) ng/g in KB cell xenografts, respectively. The Dox concentration was significantly higher in KB cell xenografts than in KBv200 cell xenografts at the same time point (P<0.05). Detected by HPLC, the concentration of Dox is much lower in multidrug-resistant cell xenografts than in sensitive cell xenografts.

  4. Autologous bone marrow Th cells can support multiple myeloma cell proliferation in vitro and in xenografted mice.

    PubMed

    Wang, D; Fløisand, Y; Myklebust, C V; Bürgler, S; Parente-Ribes, A; Hofgaard, P O; Bogen, B; Tasken, K; Tjønnfjord, G E; Schjesvold, F; Dalgaard, J; Tveita, A; Munthe, L A

    2017-02-24

    Multiple myeloma (MM) is a plasma cell malignancy where MM cell growth is supported by the bone marrow (BM) microenvironment with poorly defined cellular and molecular mechanisms. MM cells express CD40, a receptor known to activate autocrine secretion of cytokines and elicit proliferation. Activated T helper (Th) cells express CD40 ligand (CD40L) and BM Th cells are significantly increased in MM patients. We hypothesized that activated BM Th cells could support MM cell growth. We here found that activated autologous BM Th cells supported MM cell growth in a contact- and CD40L-dependent manner in vitro. MM cells had retained the ability to activate Th cells that reciprocated and stimulated MM cell proliferation. Autologous BM Th cells supported MM cell growth in xenografted mice and were found in close contact with MM cells. MM cells secreted chemokines that attracted Th cells, secretion was augmented by CD40-stimulation. Within 14 days of culture of whole bone marrow aspirates in autologous serum, MM cells and Th cells mutually stimulated each other, and MM cells required Th cells for further expansion in vitro and in mice. The results suggest that Th cells may support the expansion of MM cells in patients.Leukemia accepted article preview online, 24 February 2017. doi:10.1038/leu.2017.69.

  5. Cancer initiating-cells are enriched in the CA9 positive fraction of primary cervix cancer xenografts

    PubMed Central

    Marie-Egyptienne, Delphine Tamara; Chaudary, Naz; Kalliomäki, Tuula; Hedley, David William; Hill, Richard Peter

    2017-01-01

    Numerous studies have suggested that Cancer Initiating Cells (CIC) can be identified/enriched in cell populations obtained from solid tumors based on the expression of cell surface marker proteins. We used early passage primary cervix cancer xenografts to sort cells based on the expression of the intrinsic hypoxia marker Carbonic Anhydrase 9 (CA9) and tested their cancer initiation potential by limiting dilution assay. We demonstrated that CICs are significantly enriched in the CA9+ fraction in 5/6 models studied. Analyses of the expression of the stem cell markers Oct4, Notch1, Sca-1 & Bmi1 showed a trend toward an increase in the CA9+ populations, albeit not significant. We present evidence that enhanced autophagy does not play a role in the enhanced growth of the CA9+ cells. Our study suggests a direct in vivo functional link between hypoxic cells and CICs in primary cervix cancer xenografts. PMID:27901496

  6. mtDNA depletion confers specific gene expression profiles in human cells grown in culture and in xenograft

    PubMed Central

    Magda, Darren; Lecane, Philip; Prescott, Julia; Thiemann, Patricia; Ma, Xuan; Dranchak, Patricia K; Toleno, Donna M; Ramaswamy, Krishna; Siegmund, Kimberly D; Hacia, Joseph G

    2008-01-01

    Background Interactions between the gene products encoded by the mitochondrial and nuclear genomes play critical roles in eukaryotic cellular function. However, the effects mitochondrial DNA (mtDNA) levels have on the nuclear transcriptome have not been defined under physiological conditions. In order to address this issue, we characterized the gene expression profiles of A549 lung cancer cells and their mtDNA-depleted ρ0 counterparts grown in culture and as tumor xenografts in immune-deficient mice. Results Cultured A549 ρ0 cells were respiration-deficient and showed enhanced levels of transcripts relevant to metal homeostasis, initiation of the epithelial-mesenchymal transition, and glucuronidation pathways. Several well-established HIF-regulated transcripts showed increased or decreased abundance relative to the parental cell line. Furthermore, growth in culture versus xenograft has a significantly greater influence on expression profiles, including transcripts involved in mitochondrial structure and both aerobic and anaerobic energy metabolism. However, both in vitro and in vivo, mtDNA levels explained the majority of the variance observed in the expression of transcripts in glucuronidation, tRNA synthetase, and immune surveillance related pathways. mtDNA levels in A549 xenografts also affected the expression of genes, such as AMACR and PHYH, involved in peroxisomal lipid metabolic pathways. Conclusion We have identified mtDNA-dependent gene expression profiles that are shared in cultured cells and in xenografts. These profiles indicate that mtDNA-depleted cells could provide informative model systems for the testing the efficacy of select classes of therapeutics, such as anti-angiogenesis agents. Furthermore, mtDNA-depleted cells grown culture and in xenografts provide a powerful means to investigate possible relationships between mitochondrial activity and gene expression profiles in normal and pathological cells. PMID:18980691

  7. STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis

    PubMed Central

    Qiu, Qing-chao; Hu, Bo; He, Xiu-pei; Luo, Qiao; Tang, Guo-hua; Long, Zhi-feng; Chen, Zhu-chu; He, Xiu-sheng

    2012-01-01

    STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors. PMID:22481869

  8. Tumorigenesis and spontaneous metastasis by luciferase-labeled human xenograft osteosarcoma cells in nude mice.

    PubMed

    Du, Lin; Xu, Wen-ting; Fan, Qi-ming; Tu, Bing; Shen, Yang; Yan, Wei; Tang, Ting-ting; Wang, You

    2012-11-01

    Osteosarcoma (OS) is the most common primary malignant tumor of bone. Mouse models of human OS can invariably provide greater insight into the complex mechanisms that underlie the development and pathogenesis of this aggressive tumor. Bioluminescence technology favored tracing cancer cells in vivo. In this study, an OS model was described and evaluated using human OS cell line, Saos2, labeled with luciferase (Saos2-luc). Saos2 cells were infected by lentivirus loading a firefly luciferase gene. Luciferase expression of Saos2-luc cells was characterized both in vitro and in vivo. Specific biologic and oncologic features of Saos2-luc cells were analyzed. The OS was established as orthotopic xenografts in nude mice. Both orthotopic tumors and spontaneous lung metastasis were analyzed. Tumorigenesis and spontaneous lung metastasis in nude mice could be monitored in vivo through in vivo imaging system. The enhancement in proliferation, migration and invasion abilities and the attenuation in adhesion ability were observed in Saos2-luc cells compared with Saos2 cells. Furthermore, there were the up-regulation of Osteocalcin, CCR10, CXCR1 and ID1 and the down-regulation of ALP, collagen I, CCR1, CCR3, CXCR3, NID and N-cadherin in Saos2-luc cells compare to Saos2 cells. The rate of spontaneous lung metastasis in Saos2-luc cells was higher than that in Saos2 cells, although without significant difference. Lentivirus transfection may cause alteration of gene expression profiles and further biological functions. This model can be used in the elucidation of molecular mechanisms of tumorigenesis and the screening of new therapeutic agents.

  9. Suppression of T cells results in long-term survival of mouse heart xenografts in C6-deficient rats.

    PubMed

    Wu, G; Korsgren, O; van Rooijen, N; Tibell, A

    2001-11-01

    The present study aimed to investigate the role of cellular immune response in the absence of membrane attack complex (MAC) formation in the concordant mouse-to-rat heart xenografting. Hearts from BALB/c mice were transplanted into the neck vessels of C6-competent (C6(+)) and C6-deficient (C6(-)) PVG rats. Liposome-encapsulated dichloro-methylene diphosphonate (Lip-Cl2MDP) was administered at a dose of 10 ml/kg 2 days before transplantation and every 5 days thereafter. Cyclosporine (CsA) was administered intramuscularly (i.m.) at a dose of 15 mg/kg per day. The heart xenografts were harvested for immuno-histological analysis at the time of rejection and the functioning grafts were removed at 70 days after transplantation. In untreated C6(+) rats, xeno-grafts survived for 2.3 +/- 0.5 days. Treatment with CsA or Lip-Cl(2)MDP in C6(+) rats did not significantly affect graft survival (2.5 +/- 0.6 and 2.3 +/- 0.4 days, respectively). In untreated C6(-) rats, xenografts survived for 5.0 +/- 0.6 days. However, Lip-Cl(2)MDP in C6(-) rats resulted in a prolongation of graft survival to 11 +/- 2.3 days (P < 0.05 vs. untreated C6(-) rats), while treatment with CsA alone in these rats led to more than 70 days' survival in four out of six grafts (61 +/- 16 days). In untreated C6(+) rats, immunohistology showed a severe myocardial necrosis and thrombosis with a scarce cellular infiltrate in the rejected xenografts. By contrast, in untreated C6(-) rats, xenografts were heavily infiltrated by macrophages and T cells. The number of macrophages, but not T cells, was markedly reduced in Lip-Cl(2)MDP-treated rats. In CsA-treated C6(-) rats, the grafts harvested at 70 days after transplantation had a normal morphology, with a minimal cellular infiltrate. Our data indicate that MAC-mediated injury plays an essential role in concordant xenograft rejection. Once this mechanism has been prevented, suppression of T cells allows for long-term xenograft survival.

  10. Inhibition of leukemic cells by valproic acid, an HDAC inhibitor, in xenograft tumors

    PubMed Central

    Zhang, Zhihua; Hao, Changlai; Wang, Lihong; Liu, Peng; Zhao, Lei; Zhu, Cuimin; Tian, Xia

    2013-01-01

    The chimeric fusion protein, AML1-ETO, generated by translocation of t(8;21), abnormally recruits histone deacetylase (HDAC) to the promoters of AML1 target genes, resulting in transcriptional repression of the target genes and development of t(8;21) acute myeloid leukemia. Abnormal expression of cyclin-dependent kinase inhibitors, especially p21, is considered a possible mechanism of the arrested maturation and differentiation seen in leukemia cells. A new generation of HDAC inhibitors is becoming an increasing focus of attention for their ability to induce differentiation and apoptosis in tumor cells and to block the cell cycle. Our previous research had demonstrated that valproic acid induces G0/G1 arrest of Kasumi-1 cells in t(8;21) acute myeloid leukemia. In this study, we further confirmed that valproic acid inhibits the growth of Kasumi-1 cells in a murine xenograft tumor model, and that this occurs via upregulation of histone acetylation in the p21 promoter region, enhancement of p21 expression, suppression of phosphorylation of retinoblastoma protein, blocking of transcription activated by E2F, and induction of G0/G1 arrest. PMID:23836985

  11. Improved Method for Ex Ovo-Cultivation of Developing Chicken Embryos for Human Stem Cell Xenografts

    PubMed Central

    Schomann, Timo; Qunneis, Firas; Widera, Darius; Kaltschmidt, Christian; Kaltschmidt, Barbara

    2013-01-01

    The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26–HH28/E5—E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages. PMID:23554818

  12. Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo

    PubMed Central

    Tchoghandjian, Aurélie; Carré, Manon; Colin, Carole; Jiglaire, Carine Jiguet; Mercurio, Sandy; Beclin, Christophe; Figarella-Branger, Dominique

    2014-01-01

    Glioblastoma is the most frequent primary brain tumor in adults. Because of molecular and cellular heterogeneity, high proliferation rate and significant invasive ability, prognosis of patients is poor. Recent therapeutic advances increased median overall survival but tumor recurrence remains inevitable. In this context, we used a high throughput screening approach to bring out novel compounds with anti-proliferative and anti-migratory properties for glioblastoma treatment. Screening of the Prestwick chemical library® of 1120 molecules identified proscillaridin A, a cardiac glycoside inhibitor of the Na+/K+ ATPase pump, with most significant effects on glioblastoma cell lines. In vitro effects of proscillaridin A were evaluated on GBM6 and GBM9 stem-like cell lines and on U87-MG and U251-MG cell lines. We showed that proscillaridin A displayed cytotoxic properties, triggered cell death, induced G2/M phase blockade in all the glioblastoma cell lines and impaired GBM stem self-renewal capacity even at low concentrations. Heterotopic and orthotopic xenotransplantations were used to confirm in vivo anticancer effects of proscillaridin A that both controls xenograft growth and improves mice survival. Altogether, results suggest that proscillaridin A is a promising candidate as cancer therapies in glioblastoma. This sustains previous reports showing that cardiac glycosides act as anticancer drugs in other cancers. PMID:25400117

  13. Improved method for ex ovo-cultivation of developing chicken embryos for human stem cell xenografts.

    PubMed

    Schomann, Timo; Qunneis, Firas; Widera, Darius; Kaltschmidt, Christian; Kaltschmidt, Barbara

    2013-01-01

    The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26-HH28/E5-E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages.

  14. Dissection of stromal and cancer cell-derived signals in melanoma xenografts before and after treatment with DMXAA

    PubMed Central

    Henare, K; Wang, L; Wang, L-Cs; Thomsen, L; Tijono, S; Chen, C-Jj; Winkler, S; Dunbar, P R; Print, C; Ching, L-M

    2012-01-01

    Background: The non-malignant cells of the tumour stroma have a critical role in tumour biology. Studies dissecting the interplay between cancer cells and stromal cells are required to further our understanding of tumour progression and methods of intervention. For proof-of-principle of a multi-modal approach to dissect the differential effects of treatment on cancer cells and stromal cells, we analysed the effects of the stromal-targeting agent 5,6-dimethylxanthenone-4-acetic acid on melanoma xenografts. Methods: Flow cytometry and multi-colour immunofluorescence staining was used to analyse leukocyte numbers in xenografts. Murine-specific and human-specific multiplex cytokine panels were used to quantitate cytokines produced by stromal and melanoma cells, respectively. Human and mouse Affymetrix microarrays were used to separately identify melanoma cell-specific and stromal cell-specific gene expression. Results: 5,6-Dimethylxanthenone-4-acetic acid activated pro-inflammatory signalling pathways and cytokine expression from both stromal and cancer cells, leading to neutrophil accumulation and haemorrhagic necrosis and a delay in tumour re-growth of 26 days in A375 melanoma xenografts. Conclusion: 5,6-Dimethylxanthenone-4-acetic acid and related analogues may potentially have utility in the treatment of melanoma. The experimental platform used allowed distinction between cancer cells and stromal cells and can be applied to investigate other tumour models and anti-cancer agents. PMID:22415295

  15. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    PubMed Central

    Cordeiro, Ingrid R.; Lopes, Daiana V.; Abreu, José G.; Carneiro, Katia; Rossi, Maria I. D.; Brito, José M.

    2015-01-01

    ABSTRACT Human adipose-derived stromal cells (hADSC) are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1) regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues. PMID:26319582

  16. Comparative oncological studies of feline bronchioloalveolar lung carcinoma, its derived cell line and xenograft.

    PubMed

    Grossman, Deborah A; Hiti, Alan L; McNiel, Elizabeth A; Ye, Yin; Alpaugh, Mary L; Barsky, Sanford H

    2002-07-01

    Although certain neoplasms are unique to man, others occur across species. One such neoplasm is bronchioloalveolar lung carcinoma (BAC), a neoplasm of the Type II pneumocyte that affects humans, sheep, and small animals (dogs and cats). Human BAC occurs largely in nonsmokers. Sheep BAC is caused by the jaagsiekte retrovirus and is endemic and contagious. Feline BAC is neither endemic nor contagious and occurs sporadically and spontaneously in older purebred cats. In these respects, feline BAC is more closely similar to human BAC than sheep BAC (jaagsiekte) is. To study feline BAC further, we established the first immortal cell line (SPARKY) and transplantable scid mouse xenograft (Sparky-X) from a malignant pleural effusion of a 12-year-old Persian male with autopsy-confirmed BAC. SPARKY exhibited a Type II pneumocyte phenotype characterized by surfactant and thyroid-transcription factor-1 immunoreactivities and lamellar bodies. SPARKY's karyotype was aneuploid (66 chromosomes: 38, normal cat) and showed evidence of genomic instability analogous to human lung cancers. p53 showed a homozygous G to T transversion at codon 167, the feline equivalent of human codon 175, one of the many hot spots mutated in the lung cancers of smokers. H-ras and K-ras were not altered. By reverse transcription-PCR, SPARKY lacked expression of retroviral JSRVgag transcripts that were present in the lungs of sheep BAC (jaagsiekte). Unlike human BAC xenografts, SPARKY-X retained its unique lepidic BAC growth pattern even though it was grown in murine s.c. tissues. This property may be related to the ability of SPARKY-X to up-regulate its surfactant genes (SP-A, SP-B, and SP-D). These studies of feline BAC may allow insights into the human disease that are not possible by studying human BAC directly.

  17. Angiotensin-(1-7) Decreases Cell Growth and Angiogenesis of Human Nasopharyngeal Carcinoma Xenografts.

    PubMed

    Pei, Nana; Wan, Renqiang; Chen, Xinglu; Li, Andrew; Zhang, Yanling; Li, Jinlong; Du, Hongyan; Chen, Baihong; Wei, Wenjin; Qi, Yanfei; Zhang, Yi; Katovich, Michael J; Sumners, Colin; Zheng, Haifa; Li, Hongwei

    2016-01-01

    Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone acting through the Mas receptor (MasR), with antiproliferative and antiangiogenic properties. Recent studies have shown that Ang-(1-7) has an antiproliferative action on lung adenocarcinoma cells and prostate cancer cells. In this study, we report that MasR levels were significantly upregulated in nasopharyngeal carcinoma (NPC) specimens and NPC cell lines. Viral vector-mediated expression of Ang-(1-7) dramatically suppressed NPC cell proliferation and migration in vitro. These effects were completely blocked by the specific Ang-(1-7) receptor antagonist A-779, suggesting that they are mediated by the Ang-(1-7) receptor Mas. In this study, Ang-(1-7) not only caused a significant reduction in the growth of human nasopharyngeal xenografts, but also markedly decreased vessel density, suggesting that the heptapeptide inhibits angiogenesis to reduce tumor size. Mechanistic investigations revealed that Ang-(1-7) inhibited the expression of the proangiogenic factors VEGF and PlGF. Taken together, the data suggest that upregulation of MasR could be used as a diagnostic marker of NPC and Ang-(1-7) may be a novel therapeutic agent for nasopharyngeal cancer therapy because it exerts significant antiangiogenic activity.

  18. Antitumor Activity of Garcinol in Human Prostate Cancer Cells and Xenograft Mice.

    PubMed

    Wang, Yu; Tsai, Mei-Ling; Chiou, Li-Yu; Ho, Chi-Tang; Pan, Min-Hsiung

    2015-10-21

    Garcinol, which is isolated from fruit rinds of Garcinia indica, is a polyisoprenylated benzophenone. It has been studied for its antitumor activity by inducing apoptosis and inhibiting autophagy in human prostate cancer cells. The Bax/Bcl-2 ratio increased when garcinol was applied to PC-3 cells indicating a presence of apoptosis. Meanwhile, procaspases-9 and -3 were suppressed with attenuating PARP and DFF-45. Autophagy was inhibited through activating p-mTOR and p-PI3 Kinase/AKT by garcinol, which as a result induced the cells to apoptosis directly. In addition, the apoptosis effect of garcinol in a xenograft mouse model was also tested, suggesting a consistent result with PC-3 cell model. The tumor size was reduced more than 80 percent after the mouse accepted the garcinol treatment. Garcinol was demonstrated to have a strong antitumor activity through inhibiting autophagy and inducing apoptosis, which was discovered for the first time. Based on these findings, our data suggests that garcinol deserves further investigation as a potent chemopreventive agent.

  19. Pancratistatin selectively targets cancer cell mitochondria and reduces growth of human colon tumor xenografts.

    PubMed

    Griffin, Carly; Karnik, Aditya; McNulty, James; Pandey, Siyaram

    2011-01-01

    The naturally occurring Amaryllidaceae alkaloid pancratistatin exhibits potent apoptotic activity against a large panel of cancer cells lines and has an insignificant effect on noncancerous cell lines, although with an elusive cellular target. Many current chemotherapeutics induce apoptosis via genotoxic mechanisms and thus have low selectivity. The observed selectivity of pancratistatin for cancer cells promoted us to consider the hypothesis that this alkaloid targets cancer cell mitochondria rather than DNA or its replicative machinery. In this study, we report that pancratistatin decreased mitochondrial membrane potential and induced apoptotic nuclear morphology in p53-mutant (HT-29) and wild-type p53 (HCT116) colorectal carcinoma cell lines, but not in noncancerous colon fibroblast (CCD-18Co) cells. Interestingly, pancratistatin was found to be ineffective against mtDNA-depleted (ρ(0)) cancer cells. Moreover, pancratistatin induced cell death in a manner independent of Bax and caspase activation, and did not alter β-tubulin polymerization rate nor cause double-stranded DNA breaks. For the first time we report the efficacy of pancratistatin in vivo against human colorectal adenocarcinoma xenografts. Intratumor administration of pancratistatin (3 mg/kg) caused significant reduction in the growth of subcutaneous HT-29 tumors in Nu/Nu mice (n = 6), with no apparent toxicity to the liver or kidneys as indicated by histopathologic analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Altogether, this work suggests that pancratistatin may be a novel mitochondria-targeting compound that selectively induces apoptosis in cancer cells and significantly reduces tumor growth.

  20. An orthotopic xenograft model of intraneural NF1 MPNST suggests a potential association between steroid hormones and tumor cell proliferation.

    PubMed

    Perrin, George Q; Li, Hua; Fishbein, Lauren; Thomson, Susanne A; Hwang, Min S; Scarborough, Mark T; Yachnis, Anthony T; Wallace, Margaret R; Mareci, Thomas H; Muir, David

    2007-11-01

    Malignant peripheral nerve sheath tumors (MPNST) are the most aggressive cancers associated with neurofibromatosis type 1 (NF1). Here we report a practical and reproducible model of intraneural NF1 MPNST, by orthotopic xenograft of an immortal human NF1 tumor-derived Schwann cell line into the sciatic nerves of female scid mice. Intraneural injection of the cell line sNF96.2 consistently produced MPNST-like tumors that were highly cellular and showed extensive intraneural growth. These xenografts had a high proliferative index, were angiogenic, had significant mast cell infiltration and rapidly dominated the host nerve. The histopathology of engrafted intraneural tumors was consistent with that of human NF1 MPNST. Xenograft tumors were readily examined by magnetic resonance imaging, which also was used to assess tumor vascularity. In addition, the intraneural proliferation of sNF96.2 cell tumors was decreased in ovariectomized mice, while replacement of estrogen or progesterone restored tumor cell proliferation. This suggests a potential role for steroid hormones in supporting tumor cell growth of this MPNST cell line in vivo. The controlled orthotopic implantation of sNF96.2 cells provides for the precise initiation of intraneural MPNST-like tumors in a model system suitable for therapeutic interventions, including inhibitors of angiogenesis and further study of steroid hormone effects on tumor cell growth.

  1. Immunomodulatory action of SGI-110, a hypomethylating agent, in acute myeloid leukemia cells and xenografts.

    PubMed

    Srivastava, Pragya; Paluch, Benjamin E; Matsuzaki, Junko; James, Smitha R; Collamat-Lai, Golda; Karbach, Julia; Nemeth, Michael J; Taverna, Pietro; Karpf, Adam R; Griffiths, Elizabeth A

    2014-11-01

    The mechanism of clinical action for the FDA approved hypomethylating drugs azacitidine and decitabine remains unresolved and in this context the potential immunomodulatory effect of these agents on leukemic cells is an area of active investigation. Induced expression of methylated Cancer Testis Antigen (CTA) genes has been demonstrated in leukemic cell lines following exposure to hypomethylating drugs in vitro. SGI-110 is a novel hypomethylating dinucleotide with prolonged in vivo exposure and clinical activity in patients with MDS and AML. We demonstrate that this agent, like decitabine, produces robust re-expression of the CTAs NY-ESO-1 and MAGE-A, both in vitro and in leukemia-bearing AML xenografts. Upregulation of these genes in vitro was sufficient to induce cytotoxicity by HLA-compatible CD8+ T-cells specific for NY-ESO-1, a well-recognized and immunogenic CTA. Additionally, exposure to SGI-110 enhances MHC class I and co-stimulatory molecule expression, potentially contributing to recognition of CTAs. SGI-110, like the parent compound decitabine, induces expression of CTAs and might modulate immune recognition of myeloid malignancy.

  2. Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma.

    PubMed

    Shankavaram, Uma T; Bredel, Markus; Burgan, William E; Carter, Donna; Tofilon, Philip; Camphausen, Kevin

    2012-03-01

    Cell line models have been widely used to investigate glioblastoma multiforme (GBM) pathobiology and in the development of targeted therapies. However, GBM tumours are molecularly heterogeneous and how cell lines can best model that diversity is unknown. In this report, we investigated gene expression profiles of three preclinical growth models of glioma cell lines, in vitro and in vivo as subcutaneous and intracerebral xenografts to examine which cell line model most resembles the clinical samples. Whole genome DNA microarrays were used to profile gene expression in a collection of 25 high-grade glioblastomas, and comparisons were made to profiles of cell lines under three different growth models. Hierarchical clustering revealed three molecular subtypes of the glioblastoma patient samples. Supervised learning algorithm, trained on glioma subtypes predicted the intracerebral cell line model with one glioma subtype (r = 0.68; 95% bootstrap CI -0.41, 0.46). Survival analysis of enriched gene sets (P < 0.05) revealed 19 biological categories (146 genes) belonging to neuronal, signal transduction, apoptosis- and glutamate-mediated neurotransmitter activation signals that are associated with poor prognosis in this glioma subclass. We validated the expression profiles of these gene categories in an independent cohort of patients from 'The Cancer Genome Atlas' project (r = 0.62, 95% bootstrap CI: -0.42, 0.43). We then used these data to select and inhibit a novel target (glutamate receptor) and showed that LY341595, a glutamate receptor specific antagonist, could prolong survival in intracerebral tumour-implanted mice in combination with irradiation, providing an in vivo cell line system of preclinical studies. © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  3. Targeting Tumor Vasculature Endothelial Cells and Tumor Cells for Immunotherapy of Human Melanoma in a Mouse Xenograft Model

    NASA Astrophysics Data System (ADS)

    Hu, Zhiwei; Sun, Ying; Garen, Alan

    1999-07-01

    An immunotherapy treatment for cancer that targets both the tumor vasculature and tumor cells has shown promising results in a severe combined immunodeficient mouse xenograft model of human melanoma. The treatment involves systemic delivery of an immunoconjugate molecule composed of a tumor-targeting domain conjugated to the Fc effector domain of human IgG1. The effector domain induces a cytolytic immune response against the targeted cells by natural killer cells and complement. Two types of targeting domains were used. One targeting domain is a human single-chain Fv molecule that binds to a chondroitin sulfate proteoglycan expressed on the surface of most human melanoma cells. Another targeting domain is factor VII (fVII), a zymogen that binds with high specificity and affinity to the transmembrane receptor tissue factor (TF) to initiate the blood coagulation cascade. TF is expressed by endothelial cells lining the tumor vasculature but not the normal vasculature, and also by many types of tumor cells including melanoma. Because the binding of a fVII immunoconjugate to TF might cause disseminated intravascular coagulation, the active site of fVII was mutated to inhibit coagulation without affecting the affinity for TF. The immunoconjugates were encoded as secreted molecules in a replication-defective adenovirus vector, which was injected into the tail vein of severe combined immunodeficient mice. The results demonstrate that a mutated fVII immunoconjugate, administered separately or together with a single-chain Fv immunoconjugate that binds to the tumor cells, can inhibit the growth or cause regression of an established human tumor xenograft. This procedure could be effective in treating a broad spectrum of human solid tumors that express TF on vascular endothelial cells and tumor cells.

  4. Enantiomeric CopA3 dimer peptide suppresses cell viability and tumor xenograft growth of human gastric cancer cells.

    PubMed

    Lee, Joon Ha; Kim, In-Woo; Shin, Yong Pyo; Park, Ho Jin; Lee, Young Shin; Lee, In Hee; Kim, Mi-Ae; Yun, Eun-Young; Nam, Sung-Hee; Ahn, Mi-Young; Kang, Dongchul; Hwang, Jae Sam

    2016-03-01

    The CopA3 dimer peptide is a coprisin analog that has an anticancer effect against human cancer cells in vitro. In this study, we investigated the anticancer activity of the enantiomeric CopA3 dimer peptide in human gastric cancer cell lines as well as in an in vivo tumor xenograft model. Enantiomeric CopA3 reduced gastric cancer cell viability and exhibited cytotoxicity against cancer cells. Enantiomeric CopA3-induced cell death was mediated by specific interactions with phosphatidylserine and phosphatidylcholine, membrane components that are enriched in cancer cells, in a calcein leakage assay. Moreover, acridine orange/ethidium bromide staining, flow cytometric analysis, and Western blot analysis showed that enantiomeric CopA3 induced apoptotic and necrotic gastric cancer cell death. The antitumor effect was also observed in a mouse tumor xenograft model in which intratumoral inoculation of the peptide resulted in a significant decrease in the SNU-668 gastric cancer tumor volume. In addition, periodic acid-Schiff and hematoxylin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed apoptotic and necrotic cell death in tumor masses treated with greater than 150 μg CopA3. Collectively, these results indicate that the enantiomeric CopA3 dimer peptide induces apoptosis and necrosis of gastric cancer cells in vitro and in vivo, indicating that the peptide is a potential candidate for the treatment of gastric cancer, which is a common cause of cancer and cancer deaths worldwide.

  5. BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity.

    PubMed

    Zheng, Lu; Liang, Ping; Zhou, JianBo; Huang, XiaoBing; Wen, Yu; Wang, Zheng; Li, Jing

    2012-02-01

    The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG(2) cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG(2) cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG(2) cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G(1) phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.

  6. Cisplatin and photodynamic therapy exert synergistic inhibitory effects on small-cell lung cancer cell viability and xenograft tumor growth.

    PubMed

    Chen, You-Shuang; Peng, Yin-Bo; Yao, Min; Teng, Ji-Ping; Ni, Da; Zhu, Zhi-Jun; Zhuang, Bu-Feng; Yang, Zhi-Yin

    2017-06-03

    Lung cancer is the leading cause of cancer death worldwide. Small-cell lung cancer (SCLC) is an aggressive type of lung cancer that shows an overall 5-year survival rate below 10%. Although chemotherapy using cisplatin has been proven effective in SCLC treatment, conventional dose of cisplatin causes adverse side effects. Photodynamic therapy, a form of non-ionizing radiation therapy, is increasingly used alone or in combination with other therapeutics in cancer treatment. Herein, we aimed to address whether low dose cisplatin combination with PDT can effectively induce SCLC cell death by using in vitro cultured human SCLC NCI-H446 cells and in vivo tumor xenograft model. We found that both cisplatin and PDT showed dose-dependent cytotoxic effects in NCI-H446 cells. Importantly, co-treatment with low dose cisplatin (1 μM) and PDT (1.25 J/cm(2)) synergistically inhibited cell viability and cell migration. We further showed that the combined therapy induced a higher level of intracellular ROS in cultured NCI-H446 cells. Moreover, the synergistic effect by cisplatin and PDT was recapitulated in tumor xenograft as revealed by a more robust increase in the staining of TUNEL (a marker of cell death) and decrease in tumor volume. Taken together, our findings suggest that low dose cisplatin combination with PDT can be an effective therapeutic modality in the treatment of SCLC patients. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. CpG oligodeoxynucleotide stimulates protective innate immunity against human renal cell carcinoma xenografted in nude mice.

    PubMed

    Zhou, Pei-jun; Ma, Bin-bin; He, Wei; Xu, Da; Wang, Xiang-hui

    2011-09-01

    Renal cell carcinoma easily develops metastasis, which is highly resistant to a variety of therapies. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are potent activators of innate and adaptive immunity. CpG ODN is inclined to be used as vaccine adjuvant or in combination with other therapies to exert antitumor effect mediated by the adaptive immunity. Herein, we examined the antitumor effect of CpG ODN monotherapy and the role of innate immunity on human RCC Caki-1 cells xenografted in nude mice. Our results indicated that the peritumoral subcutaneous injections of CpG ODN1826 once a week resulted in significant inhibition of the growth of Caki-1 xenografts compared with the control groups, and the survival of tumor-bearing mice were also prolonged significantly. When cytotoxicity of splenic cells from host mice was assessed, it was found that CpG ODN1826 significantly promoted the cytotoxicities of splenocytes targeting primary Caki-1 cells or YAC-1 cells, indicating that the activity of natural killer cells in tumor-bearing nude mice was enhanced by CpG ODN1826 monotherapy. The serum concentrations of interleukin-12 were increased in mice treated with CpG ODN1826. Thus, CpG ODN1826 monotherapy induces significant inhibitory effects on the growth of human RCC xenografted in athymic immunodeficient mice, and the tumor-bearing mice achieves long-term survival, which might be attributed to enhanced innate immunity.

  8. † THE GROUP VIA CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 (iPLA2β)1 PARTICIPATES IN ER STRESS-INDUCED INS-1 INSULINOMA CELL APOPTOSIS BY PROMOTING CERAMIDE GENERATION VIA HYDROLYSIS OF SPHINGOMYELINS BY NEUTRAL SPHINGOMYELINASE

    PubMed Central

    Lei, Xiao-Yong; Zhang, Sheng; Bohrer, Alan; Bao, Shunzhong; Song, Haowei; Ramanadham, Sasanka

    2008-01-01

    β-cell mass is regulated by a balance between β-cell growth and β-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the Group VIA Ca2+-independent phospholipase A2 (iPLA2β), associated with increased ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2β was over expressed (OE INS-1 cells). These findings suggested that iPLA2β and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we addressed this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2β-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and BiP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Ceramide accumulation during ER stress was not associated with changes in mRNA levels of serine palmitoyl-transferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, temporally increased in the INS-1 cells. The increases in NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2β protein and catalytic activity. Pretreatment with BEL inactivated iPLA2β and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2β or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress

  9. The isolation and characterization of renal cancer initiating cells from human Wilms' tumour xenografts unveils new therapeutic targets†

    PubMed Central

    Pode-Shakked, Naomi; Shukrun, Rachel; Mark-Danieli, Michal; Tsvetkov, Peter; Bahar, Sarit; Pri-Chen, Sara; Goldstein, Ronald S; Rom-Gross, Eithan; Mor, Yoram; Fridman, Edward; Meir, Karen; Simon, Amos; Magister, Marcus; Kaminski, Naftali; Goldmacher, Victor S; Harari-Steinberg, Orit; Dekel, Benjamin

    2013-01-01

    There are considerable differences in tumour biology between adult and paediatric cancers. The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear. Here, we show the successful propagation of primary human Wilms' tumour (WT), a common paediatric renal malignancy, in immunodeficient mice, demonstrating the presence of a population of highly proliferative CIC/CSCs capable of serial xenograft initiation. Cell sorting and limiting dilution transplantation analysis of xenograft cells identified WT CSCs that harbour a primitive undifferentiated – NCAM1 expressing – “blastema” phenotype, including a capacity to expand and differentiate into the mature renal-like cell types observed in the primary tumour. WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family. Complete eradication of WT in multiple xenograft models was achieved with a human NCAM antibody drug conjugate. The existence of CIC/CSCs in WT provides new therapeutic targets. PMID:23239665

  10. The isolation and characterization of renal cancer initiating cells from human Wilms' tumour xenografts unveils new therapeutic targets.

    PubMed

    Pode-Shakked, Naomi; Shukrun, Rachel; Mark-Danieli, Michal; Tsvetkov, Peter; Bahar, Sarit; Pri-Chen, Sara; Goldstein, Ronald S; Rom-Gross, Eithan; Mor, Yoram; Fridman, Edward; Meir, Karen; Simon, Amos; Magister, Marcus; Kaminski, Naftali; Goldmacher, Victor S; Harari-Steinberg, Orit; Dekel, Benjamin

    2013-01-01

    There are considerable differences in tumour biology between adult and paediatric cancers. The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear. Here, we show the successful propagation of primary human Wilms' tumour (WT), a common paediatric renal malignancy, in immunodeficient mice, demonstrating the presence of a population of highly proliferative CIC/CSCs capable of serial xenograft initiation. Cell sorting and limiting dilution transplantation analysis of xenograft cells identified WT CSCs that harbour a primitive undifferentiated-NCAM1 expressing-"blastema" phenotype, including a capacity to expand and differentiate into the mature renal-like cell types observed in the primary tumour. WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family. Complete eradication of WT in multiple xenograft models was achieved with a human NCAM antibody drug conjugate. The existence of CIC/CSCs in WT provides new therapeutic targets.

  11. An insulinoma presenting with reactive hypoglycaemia.

    PubMed Central

    Connor, H.; Scarpello, J. H.

    1979-01-01

    A 57-year-old woman presented with symptoms which were cured by the removal of an insulinoma. The case was atypical in that symptomatic hypoglycaemia occurred only after meals or glucose administration but never during fasting, and thus if plasma insulin activity had not been measured an incorrect diagnosis of reactive hypoglycaemia might have been made on the basis of symptoms and oral glucose-tolerance test. Reactive hypoglycaemia resulted from an increased rate of glucose assimilation and possibly also from a decreased rate of gluconeogenesis due to the immense insulin secretion provoked by glucose or food. The findings suggest that a diagnosis of hypoglycaemia should not be made until the possibility of an insulinoma has been excluded by measurement of plasma insulin activity during a period of hypoglycaemia. PMID:231776

  12. Differential modulation of nicotine-induced gemcitabine resistance by GABA receptor agonists in pancreatic cancer cell xenografts and in vitro.

    PubMed

    Banerjee, Jheelam; Al-Wadei, Hussein An; Al-Wadei, Mohammed H; Dagnon, Koami; Schuller, Hildegard M

    2014-09-27

    Pancreatic cancer is frequently resistant to cancer therapeutics. Smoking and alcoholism are risk factors and pancreatic cancer patients often undergo nicotine replacement therapy (NRT) and treatment for alcohol dependence. Based on our report that low dose nicotine within the range of NRT causes gemcitabine resistance in pancreatic cancer, our current study has tested the hypothesis that GABA or the selective GABA-B-R agonist baclofen used to treat alcohol dependence reverse nicotine-induced gemcitabine resistance in pancreatic cancer. Using mouse xenografts from the gemcitabine--sensitive pancreatic cancer cell line BXPC-3, we tested the effects of GABA and baclofen on nicotine-induced gemcitabine resistance. The levels of cAMP, p-SRC, p-ERK, p-AKT, p-CREB and cleaved caspase-3 in xenograft tissues were determined by ELISA assays. Expression of the two GABA-B receptors, metalloproteinase-2 and 9 and EGR-1 in xenograft tissues was monitored by Western blotting. Mechanistic studies were conducted in vitro, using cell lines BXPC-3 and PANC-1 and included analyses of cAMP production by ELISA assay and Western blots to determine protein expression of GABA-B receptors, metalloproteinase-2 and 9 and EGR-1. Our data show that GABA was as effective as gemcitabine and significantly reversed gemcitabine resistance induced by low dose nicotine in xenografts whereas baclofen did not. These effects of GABA were accompanied by decreases in cAMP, p-CREB, p-AKT, p-Src, p-ERK metalloproteinases-9 and -2 and EGR-1 and increases in cleaved caspase-3 in xenografts whereas baclofen had the opposite effects. In vitro exposure of cells to single doses or seven days of nicotine induced the protein expression of MMP-2, MMP-9 and EGR-1 and these responses were blocked by GABA. Baclofen downregulated the protein expression of GABA-B-Rs in xenograft tissues and in cells exposed to baclofen for seven days in vitro. This response was accompanied by inversed baclofen effects from inhibition of

  13. Discovery of a drug targeting microenvironmental support for lymphoma cells by screening using patient-derived xenograft cells

    PubMed Central

    Sugimoto, Keiki; Hayakawa, Fumihiko; Shimada, Satoko; Morishita, Takanobu; Shimada, Kazuyuki; Katakai, Tomoya; Tomita, Akihiro; Kiyoi, Hitoshi; Naoe, Tomoki

    2015-01-01

    Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from oxidative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development. PMID:26278963

  14. Discovery of a drug targeting microenvironmental support for lymphoma cells by screening using patient-derived xenograft cells.

    PubMed

    Sugimoto, Keiki; Hayakawa, Fumihiko; Shimada, Satoko; Morishita, Takanobu; Shimada, Kazuyuki; Katakai, Tomoya; Tomita, Akihiro; Kiyoi, Hitoshi; Naoe, Tomoki

    2015-08-17

    Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from oxidative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development.

  15. [Effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expression of membrane transport proteins in K562/A02 cell xenografts].

    PubMed

    Li, Dong-Yun; Zheng, Zhi; Hou, Li; Jiang, Miao; Dong, Qing; Tian, Shao-Dan; Ma, Wei; Chen, Ju; Wang, Jing; Chen, Xin-Yi

    2010-02-01

    This study was purposed to investigate the effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expressions of P-gp, MRP, LRP in K562/A02 cell xenografts. Tumor xenograft model were established by injecting the multidrug resistant cell line K562/A02 in the axillary flank of BALB/c-nu-nu mice. CZBG-intragastric administration and doxorubicin-intraperitoneal injection in combination were given to the BALB/c-nu nude mice. The tumor xenografts were made into slice after the dissection, and the expression of P-gp, MRP, LRP in K562/A02 tumor xenografts in mice were investigated by immunohistochemical technique. The integral optical density (IOD) of P-gp, MRP, LRP in K562/A02 tumor xenografts were measured by Image Pro Plus 6.0. The results showed that as compared with the doxorubicin alone, the combination of the doxorubicin and CZBG with high, middle and low dosage could decrease IOD of P-gp, MRP in K562/A02 tumor xenografts with statistical significance (p < 0.05). The LRP expression in K562/A02 tumor xenografts was not observed in five groups. It is concluded that the combination of CZBG with doxorubicin decreases the expressions of P-gp, MRP in K562/A02 tumor xenografts of mice.

  16. 68Ga-labelled exendin-3, a new agent for the detection of insulinomas with PET

    PubMed Central

    Oyen, Wim J. G.; Joosten, Lieke; Gotthardt, Martin; Boerman, Otto C.

    2010-01-01

    Purpose Insulinomas are neuroendocrine tumours derived from pancreatic β-cells. The glucagon-like peptide 1 receptor (GLP-1R) is expressed with a high incidence (>90%) and high density in insulinomas. Glucagon-like peptide 1 (GLP-1), the natural ligand of GLP-1R, is rapidly degraded in vivo. A more stable agonist of GLP-1R is exendin-3. We investigated imaging of insulinomas with DOTA-conjugated exendin-3 labelled with 68Ga. Methods Targeting of insulinomas with [Lys40(DOTA)]exendin-3 labelled with either 111In or 68Ga was investigated in vitro using insulinoma tumour cells (INS-1). [Lys40(111In-DTPA)]Exendin-3 was used as a reference in this study. In vivo targeting was investigated in BALB/c nude mice with subcutaneous INS-1 tumours. PET imaging was performed using a preclinical PET/CT scanner. Results In vitro exendin-3 specifically bound and was internalized by GLP-1R-positive cells. In BALB/c nude mice with subcutaneous INS-1 tumours a high uptake of [Lys40(111In-DTPA)]exendin-3 in the tumour was observed (33.5 ± 11.6%ID/g at 4 h after injection). Uptake was specific, as determined by coinjection of an excess of unlabelled [Lys40]exendin-3 (1.8 ± 0.1%ID/g). The pancreas also exhibited high and specific uptake (11.3 ± 1.0%ID/g). High uptake was also found in the kidneys (144 ± 24%ID/g) and this uptake was not receptor-mediated. In this murine tumour model optimal targeting of the GLP-1R expressing tumour was obtained at exendin doses ≤0.1 µg. Remarkably, tumour uptake of 68Ga-labelled [Lys40(DOTA)]exendin-3 was lower (8.9 ± 3.1%ID/g) than tumour uptake of 111In-labelled [Lys40(DTPA)]exendin-3 (25.4 ± 7.2%ID/g). The subcutaneous tumours were clearly visualized by small-animal PET imaging after injection of 3 MBq of [Lys40(68Ga-DOTA)]exendin-3. Conclusion [Lys40(68Ga-DOTA)]Exendin-3 specifically accumulates in insulinomas, although the uptake is lower than that of [Lys40(111In-DTPA)]exendin-3. Therefore, [Lys40(68Ga

  17. Insulinoma--a deceptive endocrine tumour.

    PubMed

    Rehman, Abdul

    2011-09-01

    Insulinoma is a deceptive endocrine tumour that can easily mislead even an astute clinician because of its bizarre and nonspecific symptom complex. A 45 year old woman presented with altered behaviour, seizures and spells of coma and was being treated as a case of hysterical neurosis. Biochemical and radiological investigations revealed fasting hypoglycaemia, endogenous hyperinsulinism, and a pancreatic parenchymal lesion. Removal of the pancreatic lesion resulted in abrupt restoration of euglycaemia and complete disappearance of patients' symptoms.

  18. Diagnostic Difficulties in a Pediatric Insulinoma

    PubMed Central

    Miron, Ingrith; Diaconescu, Smaranda; Aprodu, Gabriel; Ioniuc, Ileana; Diaconescu, Mihai Radu; Miron, Lucian

    2016-01-01

    Abstract Insulinomas are functional neuroendocrine pancreatic tumors rarely encountered in pediatric pathology. Insulinomas are usually solitary and sporadic, but may occur in association with multiple endocrine neoplasia type 1. Whipple's triad—hypoglycemia, simultaneous compatible adrenergic and/or neurological signs, and relief of symptoms upon the administration of glucose—remains the fundamental diagnostic tool. We report a case of insulinoma in an 11-year-old boy with malnutrition and mild psychic retardation. History revealed neuroglycopenic symptoms associated with hypoglycemia that returned to normal values after glucose intravenous infusion; before admission in our unit, the levels of circulating insulin, as well as the abdominal ultrasound and abdominal computed tomography scan, were reported within normal range. During hospitalization in our service, the glycemic curves showed recurring low values associated with low glycated hemoglobin, positive fasting test, and elevated C-peptide. The pancreatic ultrasound was inconclusive, but the magnetic resonance imaging revealed a high signal focal area with a diameter of 1 cm, located in the tail of pancreas. Conventional enucleation of the lesion prompted a spectacular normalization of glucose metabolism and the alleviation of the main clinical symptoms. The child had a favorable evolution in the clinical follow-up, presenting with weight gain and progressive remission to complete disappearance of most symptoms—except for the mental impairments. Although in our case Whipple's triad was apparent from the beginning, the diagnosis was delayed due to the failure of conventional imaging methods in locating the tumor. Weight loss and mental impairment contributed to the diagnosis pitfalls. Pediatricians should be aware of confusing and nonspecific symptoms, especially when children with insulinoma present mental or neurological retardation. Despite the existence of medical regimens, surgery remains the gold

  19. Case report: a glucose responsive insulinoma--implication for the diagnosis of insulin secreting tumors.

    PubMed

    Sjoberg, R J; Kidd, G S

    1992-09-01

    Normal insulin secretagogues, including glucose, usually have little influence on insulin secretion from insulinomas. Therefore, insulinomas typically cause fasting hypoglycemia with relative hyperinsulinemia. This report describes a patient with hyperinsulinemia due to an islet cell adenoma with microadenomatosis, which, upon provocative in vivo testing, was found to be profoundly responsive to hypoglycemic and hyperglycemic stimuli. A 72 hr fast followed by brisk exercise resulted in a gradual reduction of serum glucose and insulin concentrations, but did not provoke symptomatic hypoglycemia. Oral glucose tolerance testing resulted in a prompt 10-fold increase in serum insulin accompanied by a mildly symptomatic and gradual fall in serum glucose to 30 mg/dl 90 minutes after glucose ingestion. An intravenous glucose challenge caused an acute increase in serum insulin to more than 1200 microU/ml with a resulting serum glucose of 11 mg/dl 25 minutes later, associated with loss of consciousness. Although a prolonged fast has proven to be the best diagnostic test for insulin secreting tumors, many other provocative tests that use normal insulin secretagogues have been somewhat useful in this regard. The patient in this report supports the concept that insulinomas vary widely in their response to a number of normal physiologic regulators of insulin secretion, including the serum glucose concentration. A variety of provocative tests may be needed to fully evaluate the rare patient in whom there is a strong clinical suspicion of insulinoma but who has a nondiagnostic prolonged fast.

  20. Enhancement of glucose uptake in skeletal muscle L6 cells and insulin secretion in pancreatic hamster-insulinoma-transfected cells by application of non-thermal plasma jet

    NASA Astrophysics Data System (ADS)

    Kumar, Naresh; Kaushik, Nagendra K.; Park, Gyungsoon; Choi, Eun H.; Uhm, Han S.

    2013-11-01

    Type-II diabetes Mellitus is characterized by defects in insulin action on peripheral tissues, such as skeletal muscle, adipose tissue, and liver and pancreatic beta cells. Since the skeletal muscle accounts for approximately 75% of insulin-stimulated glucose-uptake in our body, impaired insulin secretion from defected beta cell plays a major role in the afflicted glucose homoeostasis. It was shown that the intracellular reactive oxygen species and nitric oxide level was increased by non-thermal-plasma treatment in ambient air. These increased intracellular reactive species may enhance glucose uptake and insulin secretion through the activation of intracellular calcium (Ca+) and cAMP production.

  1. Dynamics of genomic clones in breast cancer patient xenografts at single cell resolution

    PubMed Central

    Eirew, Peter; Steif, Adi; Khattra, Jaswinder; Ha, Gavin; Yap, Damian; Farahani, Hossein; Gelmon, Karen; Chia, Stephen; Mar, Colin; Wan, Adrian; Laks, Emma; Biele, Justina; Shumansky, Karey; Rosner, Jamie; McPherson, Andrew; Nielsen, Cydney; Roth, Andrew J. L.; Lefebvre, Calvin; Bashashati, Ali; de Souza, Camila; Siu, Celia; Aniba, Radhouane; Brimhall, Jazmine; Oloumi, Arusha; Osako, Tomo; Bruna, Alejandra; Sandoval, Jose; Algara, Teresa; Greenwood, Wendy; Leung, Kaston; Cheng, Hongwei; Xue, Hui; Wang, Yuzhuo; Lin, Dong; Mungall, Andrew J.; Moore, Richard; Zhao, Yongjun; Lorette, Julie; Nguyen, Long; Huntsman, David; Eaves, Connie J.; Hansen, Carl; Marra, Marco A.; Caldas, Carlos; Shah, Sohrab P.; Aparicio, Samuel

    2016-01-01

    Human cancers, including breast cancers, are comprised of clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution1,2, underpinning important emergent features such as drug resistance and metastasis3–7. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours8–10. However the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours has not been systematically examined at single cell resolution. Here we show by both deep genome and single cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (<5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies utilizing patient-derived breast cancer xenoengraftment. PMID:25470049

  2. Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution.

    PubMed

    Eirew, Peter; Steif, Adi; Khattra, Jaswinder; Ha, Gavin; Yap, Damian; Farahani, Hossein; Gelmon, Karen; Chia, Stephen; Mar, Colin; Wan, Adrian; Laks, Emma; Biele, Justina; Shumansky, Karey; Rosner, Jamie; McPherson, Andrew; Nielsen, Cydney; Roth, Andrew J L; Lefebvre, Calvin; Bashashati, Ali; de Souza, Camila; Siu, Celia; Aniba, Radhouane; Brimhall, Jazmine; Oloumi, Arusha; Osako, Tomo; Bruna, Alejandra; Sandoval, Jose L; Algara, Teresa; Greenwood, Wendy; Leung, Kaston; Cheng, Hongwei; Xue, Hui; Wang, Yuzhuo; Lin, Dong; Mungall, Andrew J; Moore, Richard; Zhao, Yongjun; Lorette, Julie; Nguyen, Long; Huntsman, David; Eaves, Connie J; Hansen, Carl; Marra, Marco A; Caldas, Carlos; Shah, Sohrab P; Aparicio, Samuel

    2015-02-19

    Human cancers, including breast cancers, comprise clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution, underpinning important emergent features such as drug resistance and metastasis. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours. However, the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours have not been systematically examined at single-cell resolution. Here we show, using deep-genome and single-cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (<5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single-cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies using patient-derived breast cancer xenoengraftment.

  3. Monitoring PAI-1 and VEGF Levels in 6 Human Squamous Cell Carcinoma Xenografts During Fractionated Irradiation

    SciTech Connect

    Bayer, Christine; Kielow, Achim; Schilling, Daniela; Maftei, Constantin-Alin; Zips, Daniel; Yaromina, Ala; Baumann, Michael; Molls, Michael; Multhoff, Gabriele

    2012-11-01

    Purpose: Previous studies have shown that the plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are regulated by hypoxia and irradiation and are involved in neoangiogenesis. The aim of this study was to determine in vivo whether changes in PAI-1 and VEGF during fractionated irradiation could predict for radiation resistance. Methods and Materials: Six xenografted tumor lines from human squamous cell carcinomas (HSCC) of the head and neck were irradiated with 0, 3, 5, 10, and 15 daily fractions of 2 Gy. The PAI-1 and VEGF antigen levels in tumor lysates were determined by enzyme-linked immunosorbent assay kits. The amounts of PAI-1 and VEGF were compared with the dose to cure 50% of tumors (TCD{sub 50}). Colocalization of PAI-1, pimonidazole (hypoxia), CD31 (endothelium), and Hoechst 33342 (perfusion) was examined by immunofluorescence. Results: Human PAI-1 and VEGF (hVEGF) expression levels were induced by fractionated irradiation in UT-SCC-15, UT-SCC-14, and UT-SCC-5 tumors, and mouse VEGF (msVEGF) was induced only in UT-SCC-5 tumors. High hVEGF levels were significantly associated with radiation sensitivity after 5 fractions (P=.021), and high msVEGF levels were significantly associated with radiation resistance after 10 fractions (P=.007). PAI-1 staining was observed in the extracellular matrix, the cytoplasm of fibroblast-like stroma cells, and individual tumor cells at all doses of irradiation. Colocalization studies showed PAI-1 staining close to microvessels. Conclusions: These results indicate that the concentration of tumor-specific and host-specific VEGF during fractionated irradiation could provide considerably divergent information for the outcome of radiation therapy.

  4. Molecular characterization of chordoma xenografts generated from a novel primary chordoma cell source and two chordoma cell lines.

    PubMed

    Karikari, Isaac O; Gilchrist, Christopher L; Jing, Liufang; Alcorta, David A; Chen, Jun; Richardson, William J; Gabr, Mostafa A; Bell, Richard D; Kelley, Michael J; Bagley, Carlos A; Setton, Lori A

    2014-09-01

    Chordoma cells can generate solid-like tumors in xenograft models that express some molecular characteristics of the parent tumor, including positivity for brachyury and cytokeratins. However, there is a dearth of molecular markers that relate to chordoma tumor growth, as well as the cell lines needed to advance treatment. The objective in this study was to isolate a novel primary chordoma cell source and analyze the characteristics of tumor growth in a mouse xenograft model for comparison with the established U-CH1 and U-CH2b cell lines. Primary cells from a sacral chordoma, called "DVC-4," were cultured alongside U-CH1 and U-CH2b cells for more than 20 passages and characterized for expression of CD24 and brachyury. While brachyury is believed essential for driving tumor formation, CD24 is associated with healthy nucleus pulposus cells. Each cell type was subcutaneously implanted in NOD/SCID/IL2Rγ(null) mice. The percentage of solid tumors formed, time to maximum tumor size, and immunostaining scores for CD24 and brachyury (intensity scores of 0-3, heterogeneity scores of 0-1) were reported and evaluated to test differences across groups. The DVC-4 cells retained chordoma-like morphology in culture and exhibited CD24 and brachyury expression profiles in vitro that were similar to those for U-CH1 and U-CH2b. Both U-CH1 and DVC-4 cells grew tumors at rates that were faster than those for U-CH2b cells. Gross tumor developed at nearly every site (95%) injected with U-CH1 and at most sites (75%) injected with DVC-4. In contrast, U-CH2b cells produced grossly visible tumors in less than 50% of injected sites. Brachyury staining was similar among tumors derived from all 3 cell types and was intensely positive (scores of 2-3) in a majority of tissue sections. In contrast, differences in the pattern and intensity of staining for CD24 were noted among the 3 types of cell-derived tumors (p < 0.05, chi-square test), with evidence of intense and uniform staining in a

  5. Effect of dietary selenium and cancer cell xenograft on peripheral T and B lymphocytes in adult nude mice.

    PubMed

    Cheng, Wen-Hsing; Holmstrom, Alexandra; Li, Xiangdong; Wu, Ryan T Y; Zeng, Huawei; Xiao, Zhengguo

    2012-05-01

    Selenium (Se) is known to regulate tumorigenesis and immunity at the nutritional and supranutritional levels. Because the immune system provides critical defenses against cancer and the athymic, immune-deficient NU/J nude mice are known to gradually develop CD8(+) and CD4(+) T cells, we investigated whether B and T cell maturation could be modulated by dietary Se and by tumorigenesis in nude mice. Fifteen homozygous nude mice were fed a Se-deficient, Torula yeast basal diet alone (Se-) or supplemented with 0.15 (Se+) or 1.0 (Se++) mg Se/kg (as Na(2)SeO(4)) for 6 months, followed by a 7-week time course of PC-3 prostate cancer cell xenograft (2 × 10(6) cells/site, 2 sites/mouse). Here, we show that peripheral B cell levels decreased in nude mice fed the Se -  or Se++ diet and the CD4(+) T cell levels increased in mice fed the Se++ diet. During the PC-3 cell tumorigenesis, dietary Se status did not affect peripheral CD4(+) or CD8(+) T cells in nude mice whereas mice fed with the Se++ diet appeared to exhibit greater peripheral CD25(+)CD4(+) T cells on day 9. Dietary Se status did not affect spleen weight in nude mice 7 weeks after the xenograft. Spleen weight was associated with frequency of peripheral CD4(+), but not CD8(+) T cells. Taken together, dietary Se at the nutritional and supranutritional levels regulates peripheral B and T cells in adult nude mice before and after xenograft with PC-3 prostate cancer cells.

  6. Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis

    SciTech Connect

    Santamaria-Martinez, Albert; Barquinero, Jordi; Barbosa-Desongles, Anna; Hurtado, Antoni; Pinos, Tomas; Seoane, Joan; Poupon, Marie-France; Morote, Joan; Reventos, Jaume; Munell, Francina

    2009-10-15

    Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45{sup -}, CD81{sup +} and Sca-1{sup +}). We also demonstrated that SP clonal cells secrete transforming growth factor {beta}1 (TGF-{beta}1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-{beta}1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

  7. Anti-GPC3-CAR T Cells Suppress the Growth of Tumor Cells in Patient-Derived Xenografts of Hepatocellular Carcinoma.

    PubMed

    Jiang, Zhiwu; Jiang, Xiaofeng; Chen, Suimin; Lai, Yunxin; Wei, Xinru; Li, Baiheng; Lin, Simiao; Wang, Suna; Wu, Qiting; Liang, Qiubin; Liu, Qifa; Peng, Muyun; Yu, Fenglei; Weng, Jianyu; Du, Xin; Pei, Duanqing; Liu, Pentao; Yao, Yao; Xue, Ping; Li, Peng

    2016-01-01

    The lack of a general clinic-relevant model for human cancer is a major impediment to the acceleration of novel therapeutic approaches for clinical use. We propose to establish and characterize primary human hepatocellular carcinoma (HCC) xenografts that can be used to evaluate the cytotoxicity of adoptive chimeric antigen receptor (CAR) T cells and accelerate the clinical translation of CAR T cells used in HCC. Primary HCCs were used to establish the xenografts. The morphology, immunological markers, and gene expression characteristics of xenografts were detected and compared to those of the corresponding primary tumors. CAR T cells were adoptively transplanted into patient-derived xenograft (PDX) models of HCC. The cytotoxicity of CAR T cells in vivo was evaluated. PDX1, PDX2, and PDX3 were established using primary tumors from three individual HCC patients. All three PDXs maintained original tumor characteristics in their morphology, immunological markers, and gene expression. Tumors in PDX1 grew relatively slower than that in PDX2 and PDX3. Glypican 3 (GPC3)-CAR T cells efficiently suppressed tumor growth in PDX3 and impressively eradicated tumor cells from PDX1 and PDX2, in which GPC3 proteins were highly expressed. GPC3-CAR T cells were capable of effectively eliminating tumors in PDX model of HCC. Therefore, GPC3-CAR T cell therapy is a promising candidate for HCC treatment.

  8. Metabolic studies in a child with a pancreatic insulinoma.

    PubMed

    Ginsberg-Fellner, F; Rayfield, E J

    1980-01-01

    An 8-year-old boy with a convulsive disorder for 3 1/2 years remined seizure free for 20 months while being treated with phenytoin (diphenylhydantoin) sodium, and then he had a relapse. He first demonstrated hypoglycemia when he fasted prior to being placed on a ketogenic diet. An oral glucose tolerance test indicated fasting and postglucose hypoglycemia and substantial hyperinsulinemia. Somatostatin infusion resulted in a modest increase in plasma glucose levels and a decrease in serum insulin concentrations. A discrete pancreatic mass was demonstrated preoperatively by celiac angiography that on surgical extirpation, proved to be a benign intrapancreatic insulinoma. Evaluation for islet cell tumors is of importance in children with seizure disorders unresponsive to anticonvulsant medication. Furthermore, somatostatin may be useful preoperatively in maintaining normal blood glucose concentrations in patients with islet cell adenomas.

  9. Soma-to-germline transmission of RNA in mice xenografted with human tumour cells: possible transport by exosomes.

    PubMed

    Cossetti, Cristina; Lugini, Luana; Astrologo, Letizia; Saggio, Isabella; Fais, Stefano; Spadafora, Corrado

    2014-01-01

    Mendelian laws provide the universal founding paradigm for the mechanism of genetic inheritance through which characters are segregated and assorted. In recent years, however, parallel with the rapid growth of epigenetic studies, cases of inheritance deviating from Mendelian patterns have emerged. Growing studies underscore phenotypic variations and increased risk of pathologies that are transgenerationally inherited in a non-Mendelian fashion in the absence of any classically identifiable mutation or predisposing genetic lesion in the genome of individuals who develop the disease. Non-Mendelian inheritance is most often transmitted through the germline in consequence of primary events occurring in somatic cells, implying soma-to-germline transmission of information. While studies of sperm cells suggest that epigenetic variations can potentially underlie phenotypic alterations across generations, no instance of transmission of DNA- or RNA-mediated information from somatic to germ cells has been reported as yet. To address these issues, we have now generated a mouse model xenografted with human melanoma cells stably expressing EGFP-encoding plasmid. We find that EGFP RNA is released from the xenografted human cells into the bloodstream and eventually in spermatozoa of the mice. Tumor-released EGFP RNA is associated with an extracellular fraction processed for exosome purification and expressing exosomal markers, in all steps of the process, from the xenografted cancer cells to the spermatozoa of the recipient animals, strongly suggesting that exosomes are the carriers of a flow of information from somatic cells to gametes. Together, these results indicate that somatic RNA is transferred to sperm cells, which can therefore act as the final recipients of somatic cell-derived information.

  10. Activin type IB receptor signaling in prostate cancer cells promotes lymph node metastasis in a xenograft model

    SciTech Connect

    Nomura, Masatoshi; Tanaka, Kimitaka; Wang, Lixiang; Goto, Yutaka; Mukasa, Chizu; Ashida, Kenji; Takayanagi, Ryoichi

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer ActRIB signaling induces Snail and S100A4 expressions in prostate cancer cells. Black-Right-Pointing-Pointer The prostate cancer cell lines expressing an active form of ActRIB were established. Black-Right-Pointing-Pointer ActRIB signaling promotes EMT and lymph node metastasis in xenograft model. -- Abstract: Activin, a member of the transforming growth factor-{beta} family, has been known to be a growth and differentiating factor. Despite its pluripotent effects, the roles of activin signaling in prostate cancer pathogenesis are still unclear. In this study, we established several cell lines that express a constitutive active form of activin type IB receptor (ActRIBCA) in human prostate cancer cells, ALVA41 (ALVA-ActRIBCA). There was no apparent change in the proliferation of ALVA-ActRIBCA cells in vitro; however, their migratory ability was significantly enhanced. In a xenograft model, histological analysis revealed that the expression of Snail, a cell-adhesion-suppressing transcription factor, was dramatically increased in ALVA-ActRIBCA tumors, indicating epithelial mesenchymal transition (EMT). Finally, mice bearing ALVA-ActRIBCA cells developed multiple lymph node metastases. In this study, we demonstrated that ActRIBCA signaling can promote cell migration in prostate cancer cells via a network of signaling molecules that work together to trigger the process of EMT, and thereby aid in the aggressiveness and progression of prostate cancers.

  11. Myeloid cell leukemia-1 is a key molecular target for mithramycin A-induced apoptosis in androgen-independent prostate cancer cells and a tumor xenograft animal model.

    PubMed

    Choi, Eun-Sun; Jung, Ji-Youn; Lee, Jin-Seok; Park, Jong-Hwan; Cho, Nam-Pyo; Cho, Sung-Dae

    2013-01-01

    Mithramycin A (Mith) is a natural polyketide that has been used in multiple areas of research including apoptosis of various cancer cells. Here, we examined the critical role of Mith in apoptosis and its molecular mechanism in DU145 and PC3 prostate cancer cells and tumor xenografts. Mith decreased cell growth and induced apoptosis in DU145 and PC-3 cells. Myeloid cell leukemia-1 (Mcl-1) was over-expressed in both cell lines compared to RWPE1 cells. Mith inhibited Mcl-1 protein expression in both cells, but only altered Mcl-1 mRNA levels in PC-3 cells. We also found that Mith reduced Mcl-1 protein levels through both proteasome-dependent protein degradation and the inhibition of protein synthesis in DU145 cells. Studies using siRNA confirmed that the knockdown of Mcl-1 induced apoptosis. Mith significantly suppressed TPA-induced neoplastic cell transformation through the down-regulation of the Mcl-1 protein in JB6 cells, and suppressed the transforming activity of both cell types. Mith also inhibited tumor growth and Mcl-1 levels, in addition to inducing apoptosis, in athymic nude mice bearing DU145 cell xenografts without affecting five normal organs. Therefore, Mith inhibits cell growth and induces apoptosis by suppressing Mcl-1 in both prostate cancer cells and xenograft tumors, and thus is a potent anticancer drug candidate for prostate cancer.

  12. Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis

    PubMed Central

    Chi, Zhixia; Du, Shou-Hui; Chen, Can; Tay, Johan C.K.; Toh, Han Chong; Connolly, John E.; Xu, Xue Hu; Wang, Shu

    2017-01-01

    The epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of tumor types, including peritoneal carcinomatosis (PC) from gastrointestinal and gynecological malignancies. To develop a chimeric antigen receptor T (CART) cell therapy approach to treat patients with end-stage PC, we constructed third generation CARs specific to EpCAM using the 4D5MOC-B single chain variable fragment. CART cells were generated with lentiviral transduction and exhibited specific in vitro killing activity against EpCAM-positive human ovarian and colorectal cancer cells. A single intraperitoneal injection of the CART cells eradicated established ovarian xenografts and resulted in significantly prolonged animal survival. Since EpCAM is also expressed on normal epithelium, anti-EpCAM CART cells were generated by mRNA electroporation that display a controlled cytolytic activity with a limited CAR expression duration. Multiple repeated infusions of these RNA CAR-modified T cells delayed disease progression in immunodeficient mice bearing well-established peritoneal ovarian and colorectal xenografts. Thus, our study demonstrates the effectiveness of using anti-EpCAM CAR-expressing T cells for local treatment of PC in mice. The possibility of using this approach for clinical treatment of EpCAM-positive gastrointestinal and gynecological malignancies warrants further validation. PMID:28088790

  13. The dual pathway inhibitor rigosertib is effective in direct-patient tumor xenografts of head and neck squamous cell carcinomas

    PubMed Central

    Anderson, Ryan T.; Keysar, Stephen B.; Bowles, Daniel W.; Glogowska, Magdalena J.; Astling, David P.; Morton, J. Jason; Le, Phuong; Umpierrez, Adrian; Eagles-Soukup, Justin; Gan, Gregory N.; Vogler, Brian W.; Sehrt, Daniel; Takimoto, Sarah M.; Aisner, Dara L.; Wilhelm, Francois; Frederick, Barbara A.; Varella-Garcia, Marileila; Tan, Aik-Choon; Jimeno, Antonio

    2013-01-01

    The dual pathway inhibitor rigosertib inhibits phosphoinositide 3-kinase (PI3K) pathway activation as well as polo-like kinase 1 (PLK1) activity across a broad spectrum of cancer cell lines. The importance of PIK3CA alterations in head and neck squamous cell cancer (HNSCC) has raised interest in exploring agents targeting PI3K, the product of PIK3CA. The genetic and molecular basis of rigosertib treatment response was investigated in a panel of 16 HNSCC cell lines, and direct patient tumor xenografts from 8 HNSCC patients (4 HPV16-positive). HNSCC cell lines and xenografts were characterized by pathway enrichment gene expression analysis, exon sequencing, gene copy number, western blotting, and IHC. Rigosertib had potent antiproliferative effects on 11 of the 16 HPV− HNSCC cell lines. Treatment sensitivity was confirmed in two cell lines using an orthotopic in vivo xenograft model. Growth reduction after rigosertib treatment was observed in 3/8 HNSCC direct patient tumor lines. The responsive tumor lines carried a combination of a PI3KCA activating event (amplification or mutation) and a p53 inactivating event (either HPV16-mediated or mutation-mediated TP53 inactivation). In this study, we evaluated the in vitro and in vivo efficacy of rigosertib in both HPV+ and HPV− HNSCCs focusing on inhibition of the PI3K pathway. Although consistent inhibition of the PI3K pathway was not evident in HNSCC, we identified a combination of PI3K/TP53 events necessary, but not sufficient for rigosertib-sensitivity. PMID:23873848

  14. Enhanced effect of geldanamycin nanocomposite against breast cancer cells growing in vitro and as xenograft with vanquished normal cell toxicity.

    PubMed

    Prabhu, Suma; Ananthanarayanan, Preeta; Aziz, Sajida Kannangar; Rai, Sharada; Mutalik, Srinivas; Sadashiva, Satish Rao Bola

    2017-04-01

    Despite enormous advances in remedies developed for breast cancer, an effective therapeutic strategy by targeting malignant cells with the least normal tissue toxicity is yet to be developed. Hsp90 is considered to be an important therapeutic target to inhibit cell proliferation. Geldanamycin (GDM), a potent inhibitor of Hsp90 was withdrawn from clinical trials due to its undesirable hepatotoxicity. We report a superparamagnetic iron oxide (SPION) based polymeric nanocomposite of GDM augmenting anticancer competence with decreased hepatic toxicity. The particle size of nanocomposite was ascertained to be 76±10nm with acceptable stability. A comparative dose dependent in vitro validation of cytotoxicity showed an enhanced cellular damage and necrosis in breast cancer (MCF-7) cell line at a low dose of 5.49nM (in GDM nanocomposite) in contrast to 20nM of pure GDM, while normal breast epithelial cells (MCF-10A) were least affected. Besides, in vivo study (in breast cancer xenografts) substantiated 2.7 fold delay in tumor progression mediated by redundancy in the downstream functions of p-Akt and MAPK-Erk leading to apoptosis with negligible hepatotoxicity. Pure GDM disrupted the function and morphology of liver with lesser therapeutic efficacy than the GDM nanocomposite. These findings deduce that GDM based polymeric magnetite nanocomposite play a vital role in efficacious therapy while vanquishing normal cells and hepatic toxicity and thereby promising it to be reinstated in clinics.

  15. ABCG2-overexpressing S1-M1-80 cell xenografts in nude mice keep original biochemistry and cell biological properties.

    PubMed

    Wang, Fang; Liang, Yong-Ju; Wu, Xing-Ping; Su, Xiao-Dong; Fu, Li-Wu

    2012-03-01

    S1-M1-80 cells, derived from human colon carcinoma S1 cells, are mitoxantrone-selected ABCG2-overexpressing cells and are widely used in in vitro studies of multidrug resistance(MDR). In this study, S1-M1-80 cell xenografts were established to investigate whether the MDR phenotype and cell biological properties were maintained in vivo. Our results showed that the proliferation, cell cycle, and ABCG2 expression level in S1-M1-80 cells were similar to those in cells isolated from S1-M1-80 cell xenografts (named xS1-M1-80 cells). Consistently, xS1-M1-80 cells exhibited high levels of resistance to ABCG2 substrates such as mitoxantrone and topotecan, but remained sensitive to the non-ABCG2 substrate cisplatin. Furthermore, the specific ABCG2 inhibitor Ko143 potently sensitized xS1-M1-80 cells to mitoxantrone and topotecan. These results suggest that S1-M1-80 cell xenografts in nude mice retain their original cytological characteristics at 9 weeks. Thus, this model could serve as a good system for further investigation of ABCG2-mediated MDR.

  16. mRNA expression profiles of primary high-grade central osteosarcoma are preserved in cell lines and xenografts

    PubMed Central

    2011-01-01

    Background Conventional high-grade osteosarcoma is a primary malignant bone tumor, which is most prevalent in adolescence. Survival rates of osteosarcoma patients have not improved significantly in the last 25 years. Aiming to increase this survival rate, a variety of model systems are used to study osteosarcomagenesis and to test new therapeutic agents. Such model systems are typically generated from an osteosarcoma primary tumor, but undergo many changes due to culturing or interactions with a different host species, which may result in differences in gene expression between primary tumor cells, and tumor cells from the model system. We aimed to investigate whether gene expression profiles of osteosarcoma cell lines and xenografts are still comparable to those of the primary tumor. Methods We performed genome-wide mRNA expression profiling on osteosarcoma biopsies (n = 76), cell lines (n = 13), and xenografts (n = 18). Osteosarcoma can be subdivided into several histological subtypes, of which osteoblastic, chondroblastic, and fibroblastic osteosarcoma are the most frequent ones. Using nearest shrunken centroids classification, we generated an expression signature that can predict the histological subtype of osteosarcoma biopsies. Results The expression signature, which consisted of 24 probes encoding for 22 genes, predicted the histological subtype of osteosarcoma biopsies with a misclassification error of 15%. Histological subtypes of the two osteosarcoma model systems, i.e. osteosarcoma cell lines and xenografts, were predicted with similar misclassification error rates (15% and 11%, respectively). Conclusions Based on the preservation of mRNA expression profiles that are characteristic for the histological subtype we propose that these model systems are representative for the primary tumor from which they are derived. PMID:21933437

  17. Scaffold-integrated microchips for end-to-end in vitro tumor cell attachment and xenograft formation

    PubMed Central

    Lee, Jungwoo; Kohl, Nathaniel; Shanbhang, Sachin; Parekkadan, Biju

    2015-01-01

    Microfluidic technologies have substantially advanced cancer research by enabling the isolation of rare circulating tumor cells (CTCs) for diagnostic and prognostic purposes. The characterization of isolated CTCs has been limited due to the difficulty in recovering and growing isolated cells with high fidelity. Here, we present a strategy that uses a 3D scaffold, integrated into a microfludic device, as a transferable substrate that can be readily isolated after device operation for serial use in vivo as a transplanted tissue bed. Hydrogel scaffolds were incorporated into a PDMS fluidic chamber prior to bonding and were rehydrated in the chamber after fluid contact. The hydrogel matrix completely filled the fluid chamber, significantly increasing the surface area to volume ratio, and could be directly visualized under a microscope. Computational modeling defined different flow and pressure regimes that guided the conditions used to operate the chip. As a proof of concept using a model cell line, we confirmed human prostate tumor cell attachment in the microfluidic scaffold chip, retrieval of the scaffold en masse, and serial implantation of the scaffold to a mouse model with preserved xenograft development. With further improvement in capture efficiency, this approach can offer an end-to-end platform for the continuous study of isolated cancer cells from a biological fluid to a xenograft in mice. PMID:26709385

  18. Scaffold-integrated microchips for end-to-end in vitro tumor cell attachment and xenograft formation.

    PubMed

    Lee, Jungwoo; Kohl, Nathaniel; Shanbhang, Sachin; Parekkadan, Biju

    2015-12-01

    Microfluidic technologies have substantially advanced cancer research by enabling the isolation of rare circulating tumor cells (CTCs) for diagnostic and prognostic purposes. The characterization of isolated CTCs has been limited due to the difficulty in recovering and growing isolated cells with high fidelity. Here, we present a strategy that uses a 3D scaffold, integrated into a microfludic device, as a transferable substrate that can be readily isolated after device operation for serial use in vivo as a transplanted tissue bed. Hydrogel scaffolds were incorporated into a PDMS fluidic chamber prior to bonding and were rehydrated in the chamber after fluid contact. The hydrogel matrix completely filled the fluid chamber, significantly increasing the surface area to volume ratio, and could be directly visualized under a microscope. Computational modeling defined different flow and pressure regimes that guided the conditions used to operate the chip. As a proof of concept using a model cell line, we confirmed human prostate tumor cell attachment in the microfluidic scaffold chip, retrieval of the scaffold en masse, and serial implantation of the scaffold to a mouse model with preserved xenograft development. With further improvement in capture efficiency, this approach can offer an end-to-end platform for the continuous study of isolated cancer cells from a biological fluid to a xenograft in mice.

  19. SVCT-2 determines the sensitivity to ascorbate-induced cell death in cholangiocarcinoma cell lines and patient derived xenografts.

    PubMed

    Wang, Changzheng; Lv, Hongwei; Yang, Wen; Li, Ting; Fang, Tian; Lv, Guishuai; Han, Qin; Dong, Liwei; Jiang, Tianyi; Jiang, Beige; Yang, Guangshun; Wang, Hongyang

    2017-04-04

    Cholangiocarcinoma (CC) is a devastating malignancy with late diagnosis and poor response to conventional chemotherapy. Recent studies have revealed anti-cancer effect of vitamin C (l-ascorbic acid, ascorbate) in several types of cancer. However, the effect of l-ascorbic acid (AA) in CC remains elusive. Herein, we demonstrated that AA induced cytotoxicity in CC cells by generating intracellular reactive oxygen species (ROS), and subsequently DNA damage, ATP depletion, mTOR pathway inhibition. Moreover, AA worked synergistically with chemotherapeutic agent cisplatin to impair CC cells growth both in vitro and in vivo. Intriguingly, sodium-dependent vitamin C transporter 2 (SVCT-2) expression was inversely correlated with IC50 values of AA. Knockdown of SVCT-2 dramatically alleviated DNA damage, ATP depletion, and inhibition of mTOR pathway induced by AA. Furthermore, SVCT-2 knockdown endowed CC cells with the resistance to AA treatment. Finally, the inhibitory effects of AA were further confirmed in patient-derived CC xenograft models. Thus, our results unravel therapeutic potential of AA alone or in combination with cisplatin for CC. SVCT2 expression level may serve as a positive outcome predictor for AA treatment in CC.

  20. Laparoscope resection of retroperitoneal ectopic insulinoma: A rare case

    PubMed Central

    Liu, Jie; Zhang, Cheng-Wu; Hong, De-Fei; Wu, Jia; Yang, Hong-Guo; Chen, Yuan; Zhao, Da-Jian; Zhang, Yu-Hua

    2015-01-01

    Ectopic insulinoma is a very rare and dormant tumor. Here we report the case of a 79-year-old female who presented with repeated episodes of hypoglycemia and was diagnosed with insulinoma based on laboratory and imaging examinations. Computed tomography and positron emission tomography revealed a tumor in the retroperitoneum under and left of the hepatoduodenal ligament, which was resected successfully using a laparoscopic approach. Pathologic results revealed an ectopic insulinoma, which was confirmed immunohistochemically. Ectopic insulinomas are accompanied by hypoglycemia that can be misdiagnosed as drug- or disease-induced. These tumors are difficult to diagnose and locate, particularly in atypical cases or for very small tumors. Synthetic or targeted examinations, including low blood glucose, elevated insulin, proinsulin, and C-peptide levels, 48-h fasting tests, and relevant imaging methods should be considered for suspected cases of insulinoma. Surgery is the treatment of choice for patients with insulinoma, and laparoscopic resection is a feasible and effective method for select ectopic insulinoma cases. PMID:25892896

  1. Quercetin induces cell apoptosis of myeloma and displays a synergistic effect with dexamethasone in vitro and in vivo xenograft models.

    PubMed

    He, Donghua; Guo, Xing; Zhang, Enfan; Zi, Fuming; Chen, Jing; Chen, Qingxiao; Lin, Xuanru; Yang, Li; Li, Yi; Wu, Wenjun; Yang, Yang; He, Jingsong; Cai, Zhen

    2016-07-19

    Quercetin, a kind of dietary flavonoid, has shown its anticancer activity in many kinds of cancers including hematological malignancies (acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and MM) in vitro and in vivo. However, its effects on MM need further investigation. In this study, MM cell lines were treated with quercetin alone or in combination with dexamethasone. In order to observe the effects in vivo, a xenograft model of human myeloma was established. Quercetin inhibited proliferation of MM cells (RPMI8226, ARP-1, and MM.1R) by inducing cell cycle arrest in the G2/M phase and apoptosis. Western blot showed that quercetin downregulated c-myc expression and upregulated p21 expression. Quercetin also activated caspase-3, caspase-9, and poly(ADP-ribose)polymerase 1. Caspase inhibitors partially blocked apoptosis induced by quercetin. Furthermore, quercetin combined with dexamethasone significantly increased MM cell apoptosis. In vivo xenograft models, quercetin obviously inhibited tumor growth. Caspase-3 was activated to a greater extent when quercetin was combined with dexamethasone. In conclusion, quercetin alone or in combination with dexamethasone may be an effective therapy for MM.

  2. Chlorin e6 – polyvinylpyrrolidone mediated photosensitization is effective against human non-small cell lung carcinoma compared to small cell lung carcinoma xenografts

    PubMed Central

    Chin, William WL; Heng, Paul WS; Olivo, Malini

    2007-01-01

    Background Photodynamic therapy (PDT) is an effective local cancer treatment that involves light activation of a photosensitizer, resulting in oxygen-dependent, free radical-mediated cell death. Little is known about the comparative efficacy of PDT in treating non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC), despite ongoing clinical trials treating lung cancers. The present study evaluated the potential use of chlorin e6 – polyvinylpyrrolidone (Ce6-PVP) as a multimodality photosensitizer for fluorescence detection and photodynamic therapy (PDT) on NSCLC and SCLC xenografts. Results Human NSCLC (NCI-H460) and SCLC (NCI-H526) tumor cell lines were used to establish tumor xenografts in the chick chorioallantoic membrane (CAM) model as well as in the Balb/c nude mice. In the CAM model, Ce6-PVP was applied topically (1.0 mg/kg) and fluorescence intensity was charted at various time points. Tumor-bearing mice were given intravenous administration of Ce6-PVP (2.0 mg/kg) and laser irradiation at 665 nm (fluence of 150 J/cm2 and fluence rate of 125 mW/cm2). Tumor response was evaluated at 48 h post PDT. Studies of temporal fluorescence pharmacokinetics in CAM tumor xenografts showed that Ce6-PVP has a selective localization and a good accuracy in demarcating NSCLC compared to SCLC from normal surrounding CAM after 3 h post drug administration. Irradiation at 3 h drug-light interval showed greater tumor necrosis against human NSCLC xenografts in nude mice. SCLC xenografts were observed to express resistance to photosensitization with Ce6-PVP. Conclusion The formulation of Ce6-PVP is distinctly advantageous as a diagnostic and therapeutic agent for fluorescence diagnosis and PDT of NSCLC. PMID:18053148

  3. Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types.

    PubMed

    Iliopoulos, Dimitrios; Hirsch, Heather A; Struhl, Kevin

    2011-05-01

    Metformin, the first-line drug for treating diabetes, selectively kills the chemotherapy resistant subpopulation of cancer stem cells (CSC) in genetically distinct types of breast cancer cell lines. In mouse xenografts, injection of metformin and the chemotherapeutic drug doxorubicin near the tumor is more effective than either drug alone in blocking tumor growth and preventing relapse. Here, we show that metformin is equally effective when given orally together with paclitaxel, carboplatin, and doxorubicin, indicating that metformin works together with a variety of standard chemotherapeutic agents. In addition, metformin has comparable effects on tumor regression and preventing relapse when combined with a four-fold reduced dose of doxorubicin that is not effective as a monotherapy. Finally, the combination of metformin and doxorubicin prevents relapse in xenografts generated with prostate and lung cancer cell lines. These observations provide further evidence for the CSC hypothesis for cancer relapse, an experimental rationale for using metformin as part of combinatorial therapy in a variety of clinical settings, and for reducing the chemotherapy dose in cancer patients.

  4. Pleurocidin-family cationic antimicrobial peptides are cytolytic for breast carcinoma cells and prevent growth of tumor xenografts

    PubMed Central

    2011-01-01

    Introduction Cationic antimicrobial peptides (CAPs) defend against microbial pathogens; however, certain CAPs also exhibit anticancer activity. The purpose of this investigation was to determine the effect of the pleurocidin-family CAPs, NRC-03 and NRC-07, on breast cancer cells. Methods MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) and acid phosphatase cell-viability assays were used to assess NRC-03- and NRC-07-mediated killing of breast carcinoma cells. Erythrocyte lysis was determined with hemolysis assay. NRC-03 and NRC-07 binding to breast cancer cells and normal fibroblasts was assessed with fluorescence microscopy by using biotinylated-NRC-03 and -NRC-07. Lactate dehydrogenase-release assays and scanning electron microscopy were used to evaluate the effect of NRC-03 and NRC-07 on the cell membrane. Flow-cytometric analysis of 3,3'-dihexyloxacarbocyanine iodide- and dihydroethidium-stained breast cancer cells was used to evaluate the effects of NRC-03 and NRC-07 on mitochondrial membrane integrity and reactive oxygen species (ROS) production, respectively. Tumoricidal activity of NRC-03 and NRC-07 was evaluated in NOD SCID mice bearing breast cancer xenografts. Results NRC-03 and NRC-07 killed breast cancer cells, including drug-resistant variants, and human mammary epithelial cells but showed little or no lysis of human dermal fibroblasts, umbilical vein endothelial cells, or erythrocytes. Sublethal doses of NRC-03 and, to a lesser extent, NRC-07 significantly reduced the median effective concentration (EC50) of cisplatin for breast cancer cells. NRC-03 and NRC-07 bound to breast cancer cells but not fibroblasts, suggesting that killing required peptide binding to target cells. NRC-03- and NRC-07-mediated killing of breast cancer cells correlated with expression of several different anionic cell-surface molecules, suggesting that NRC-03 and NRC-07 bind to a variety of negatively-charged cell-surface molecules. NRC-03 and NRC-07 also

  5. Optimizing lutetium 177-anti-carbonic anhydrase IX radioimmunotherapy in an intraperitoneal clear cell renal cell carcinoma xenograft model.

    PubMed

    Muselaers, Constantijn H J; Oosterwijk, Egbert; Bos, Desirée L; Oyen, Wim J G; Mulders, Peter F A; Boerman, Otto C

    2014-01-01

    A new approach in the treatment of clear cell renal carcinoma (ccRCC) is radioimmunotherapy (RIT) using anti-carbonic anhydrase IX (CAIX) antibody G250. To investigate the potential of RIT with lutetium 177 (177Lu)-labeled G250, we conducted a protein dose escalation study and subsequently an RIT study in mice with intraperitoneally growing ccRCC lesions. Mice with intraperitoneal xenografts were injected with 1, 3, 10, 30, or 100 μg of G250 labeled with 10 MBq indium 111 (111In) to determine the optimal protein dose. The optimal protein dose determined with imaging and biodistribution studies was used in a subsequent RIT experiment in three groups of 10 mice with intraperitoneal SK-RC-52 tumors. One group received 13 MBq 177Lu-DOTA-G250, a control group received 13 MBq nonspecific 177Lu-MOPC21, and the second control group was not treated and received 20 MBq 111In-DOTA-G250. The optimal G250 protein dose to target ccRCC in this model was 10 μg G250. Treatment with 13 MBq 177Lu-DOTA-G250 was well tolerated and resulted in significantly prolonged median survival (139 days) compared to controls (49-53 days, p  =  .015), indicating that RIT has potential in this metastatic ccRCC model.

  6. Metoclopramide enhances the effect of ionizing radiation on xenografted squamous cell carcinoma of the head and neck

    SciTech Connect

    Lybak, S.; Kjellen, E.; Wennerberg, J.; Pero, R. )

    1990-12-01

    The commonly used drug metoclopramide, a benzamide derivative, has been shown previously in our laboratory to enhance the effect of cisplatin on xenografted squamous cell carcinomas of the head and neck. In the present study, we show that metoclopramide also enhances the effect of ionizing radiation. Two human squamous cell carcinoma lines of the head and neck xenografted to nude mice have been used. Doses of radiation were chosen (5 and 8 Gy single doses) which caused only a slight retardation of tumor growth when administered alone. Tumor response to ionizing radiation was assessed with and without metoclopramide (2.0 mg kg-1), and administered at the time of radiation and 24 and 48 hr after treatment. The effects of these schedules on the tumors were compared using the reduction of the area under the growth curves and specific growth delay. The dose schedule with metoclopramide alone did not induce any significant reduction in the area under the growth curves. The addition of metoclopramide to the radiated groups caused a significant enhancement of the radiation-induced reduction of the area under the growth curves in both of the tumor lines studied.

  7. Sodium Selenite Radiosensitizes Hormone-Refractory Prostate Cancer Xenograft Tumors but Not Intestinal Crypt Cells In Vivo

    SciTech Connect

    Tian Junqiang; Ning Shouchen; Knox, Susan J.

    2010-09-01

    Purpose: We have previously shown that sodium selenite (SSE) increases radiation-induced cell killing of human prostate carcinoma cells in vitro. In this study we further evaluated the in vivo radiosensitizing effect of SSE in prostate cancer xenograft tumors and normal radiosensitive intestinal crypt cells. Methods and Materials: Immunodeficient (SCID) mice with hormone-independent LAPC-4 (HI-LAPC-4) and PC-3 xenograft tumors (approximately 200 mm{sup 3}) were divided into four groups: control (untreated), radiation therapy (XRT, local irradiation), SSE (2 mg/kg, intraperitoneally, 3 times/week), and XRT plus SSE. The XRT was given at the beginning of the regimen as a single dose of 5 Gy for HI-LAPC-4 tumors and a single dose of 7 Gy followed by a fractional dose of 3 Gy/d for 5 days for PC-3 tumors. The tumor volume was measured 3 times per week. The radiosensitizing effect of SSE on normal intestinal epithelial cells was assessed by use of a crypt cell microcolony assay. Results: In the efficacy study, SSE alone significantly inhibited the tumor growth in HI-LAPC-4 tumors but not PC-3 tumors. Sodium selenite significantly enhanced the XRT-induced tumor growth inhibition in both HI-LAPC-4 and PC-3 tumors. In the toxicity study, SSE did not affect the intestinal crypt cell survival either alone or in combination with XRT. Conclusions: Sodium selenite significantly enhances the effect of radiation on well-established hormone-independent prostate tumors and does not sensitize the intestinal epithelial cells to radiation. These results suggest that SSE may increase the therapeutic index of XRT for the treatment of prostate cancer.

  8. Structure, growth and cell proliferation of human osteosarcoma and malignant fibrous histiocytoma xenografts in serial transplantation in nude mice.

    PubMed

    Broström, L A; Crnalic, S; Löfvenberg, R; Stenling, R; Boquist, L

    1996-11-01

    Tumour specimens from one patient with osteosarcoma and one with malignant fibrous histiocytoma were transplanted in serial passages in nude mice. Structure, growth and cell kinetics of the xenografts were studied in order to assess the validity of the two tumour models. Cell proliferation was analysed using in vivo labelling with the thymidine analogue iododeoxyuridine (IdUrd) and the IdUrd labelling index (LI) was determined by immunohistochemistry. The DNA index (DI) was examined by flow cytometry. The c-myc oncoprotein expression was studied by immunohistochemistry. More intense proliferation was observed in the peripheral parts of the tumours. There was no correlation between tumour growth and cell proliferation in the two tumour groups. Stability of the tumour models was indicated by low intrapassage and interpassage variations of DI, LI, and volume doubling time, and also by retained histopathological characteristics and c-myc staining patterns of donor patients' tumours during serial transplantation.

  9. The Influence of Hypoxia and pH on Bioluminescence Imaging of Luciferase-Transfected Tumor Cells and Xenografts

    PubMed Central

    Khalil, Ashraf A.; Jameson, Mark J.; Broaddus, William C.; Lin, Peck Sun; Dever, Seth M.; Golding, Sarah E.; Rosenberg, Elizabeth; Valerie, Kristoffer; Chung, Theodore D.

    2013-01-01

    Bioluminescence imaging (BLI) is a relatively new noninvasive technology used for quantitative assessment of tumor growth and therapeutic effect in living animal models. BLI involves the generation of light by luciferase-expressing cells following administration of the substrate luciferin in the presence of oxygen and ATP. In the present study, the effects of hypoxia, hypoperfusion, and pH on BLI signal (BLS) intensity were evaluated in vitro using cultured cells and in vivo using a xenograft model in nude mice. The intensity of the BLS was significantly reduced in the presence of acute and chronic hypoxia. Changes in cell density, viability, and pH also affected BLS. Although BLI is a convenient non-invasive tool for tumor assessment, these factors should be considered when interpreting BLS intensity, especially in solid tumors that could be hypoxic due to rapid growth, inadequate blood supply, and/or treatment. PMID:23936647

  10. Therapeutic Antibodies Targeting CSF1 Impede Macrophage Recruitment in a Xenograft Model of Tenosynovial Giant Cell Tumor

    PubMed Central

    Cheng, Hongwei; Clarkson, Paul W.; Gao, Dongxia; Pacheco, Marina; Wang, Yuzhuo; Nielsen, Torsten O.

    2010-01-01

    Tenosynovial giant cell tumor is a neoplastic disease of joints that can cause severe morbidity. Recurrences are common following local therapy, and no effective medical therapy currently exists. Recent work has demonstrated that all cases overexpress macrophage colony-stimulating factor (CSF1), usually as a consequence of an activating gene translocation, resulting in an influx of macrophages that form the bulk of the tumor. New anti-CSF1 drugs have been developed; however there are no preclinical models suitable for evaluation of drug benefits in this disease. In this paper, we describe a novel renal subcapsular xenograft model of tenosynovial giant cell tumor. Using this model, we demonstrate that an anti-CSF1 monoclonal antibody significantly inhibits host macrophage infiltration into this tumor. The results from this model support clinical trials of equivalent humanized agents and anti-CSF1R small molecule drugs in cases of tenosynovial giant cell tumor refractory to conventional local therapy. PMID:20981142

  11. Therapeutic Antibodies Targeting CSF1 Impede Macrophage Recruitment in a Xenograft Model of Tenosynovial Giant Cell Tumor.

    PubMed

    Cheng, Hongwei; Clarkson, Paul W; Gao, Dongxia; Pacheco, Marina; Wang, Yuzhuo; Nielsen, Torsten O

    2010-01-01

    Tenosynovial giant cell tumor is a neoplastic disease of joints that can cause severe morbidity. Recurrences are common following local therapy, and no effective medical therapy currently exists. Recent work has demonstrated that all cases overexpress macrophage colony-stimulating factor (CSF1), usually as a consequence of an activating gene translocation, resulting in an influx of macrophages that form the bulk of the tumor. New anti-CSF1 drugs have been developed; however there are no preclinical models suitable for evaluation of drug benefits in this disease. In this paper, we describe a novel renal subcapsular xenograft model of tenosynovial giant cell tumor. Using this model, we demonstrate that an anti-CSF1 monoclonal antibody significantly inhibits host macrophage infiltration into this tumor. The results from this model support clinical trials of equivalent humanized agents and anti-CSF1R small molecule drugs in cases of tenosynovial giant cell tumor refractory to conventional local therapy.

  12. Episodic confusional state: Due to insulinoma

    PubMed Central

    Jagadheesan, Venkatesan; Suresh, Stelina Sophie Dina

    2008-01-01

    This case report deals with 45-year-old male who came for consultation in the psychiatry department for the persisting symptoms, after consulting various departments with no relief. He had episodes of confusion with disorganized behavior, restlessness, and symptoms like talking irrelevantly once a week lasting up to 10-30 min in the preceding six months. Investigations like computerized tomography scan, electroencephalogram were not contributory. While under observation in our ward for evaluation and diagnosis, one such episode with intense sweating and clouding of consciousness was witnessed and helped in clinching the diagnosis of insulinoma. The case is reported for its rarity and as one of the causes of episodic confusional state. PMID:19742181

  13. Pituitary Adenylate Cyclase-Activating Polypeptide Induces the Voltage-Independent Activation of Inward Membrane Currents and Elevation of Intracellular Calcium in HIT-T15 Insulinoma Cells*

    PubMed Central

    LEECH, COLIN A.; HOLZ, GEORGE G.; HABENER, JOEL F.

    2010-01-01

    The secretion of insulin by pancreatic β-cells is controlled by synergistic interactions of glucose and hormones of the glucagon-related peptide family, of which pituitary adenylate cyclase-activating polypeptide (PACAP) is a member. Here we show by simultaneous recording of intracellular calcium ion ([Ca2+]i) and membrane potential that both PACAP-27 and PACAP-38 depolarize HIT-T15 cells and raise [Ca2+]i. PACAP stimulation can result in membrane depolarization by two distinct mechanisms: 1) PACAP reduces the membrane conductance and increases membrane excitability; and 2) PACAP activates a pronounced inward current that is predominantly a Na+ current, blockable by La3+, and which exhibits a reversal potential of about −28 mV. Activation of this current does not require membrane depolarization, because the response is observed when cells are held under voltage clamp at −70 mV. This current may result from the cAMP-dependent activation of nonspecific cation channels because the current is also observed in response to forskolin or membrane-permeant analogs of cAMP. We also suggest that PACAP raises [Ca2+]i and stimulates insulin secretion by three distinct mechanisms: 1) depolarization activates Ca2+ influx through L-type voltage-dependent calcium channels, 2) mobilization of intracellular Ca2+ stores, and 3) entry of Ca2+ via voltage-independent Ca2+ channels. These effects of PACAP may play an important role in a neuro-entero-endocrine loop regulating insulin secretion from pancreatic β-cells during the transition period from fasting to feeding. PMID:7895663

  14. Relevant infrastructural alterations in a pancreatic neuroendocrine tumor: an insulinoma case.

    PubMed

    Mirancea, Gabriel-Valeriu; Moroşanu, Ana-Maria; Carniciu, Simona; Dima, Simona; Bacalbaşa, Nicolae; Popescu, Irinel; Ionescu-Tîrgovişte, Constantin; Mirancea, Nicolae

    2014-01-01

    In this study, we focus our interest on some peculiar infrastructural abnormalities detected in an insulinoma case. Tumor pancreatic endocrine cells proliferated detrimental to exocrine counterpart, so that extensive areas of prevalent β-tumor cells can be seen. Two phenotypes of β-tumor cells can be identified: (1) β-tumor cells with full euchromatic and nucleolated nuclei and (2) β-tumor cells with heterochromatic and shrink nuclei. Because of stroma alteration, including basement membrane, cell-extracellular matrix junctions are also compromised. The mostly striking and important finding in this report for a case of insulinoma is the high fragility of plasma membrane of both two phenotypes of β-tumor cells. Cell-cell junctions, especially desmosomal junctions are severely altered, almost missing, plasma membranes showed shedding membrane vesicles and extensive dissolutions leading to pseudo-syncytia formation. Extravasated blood cells, including inflammatory cells contribute to the dramatic and extensive destructive areas of epithelial cells as well as stroma counterpart. Moreover, also the inner cell cytomembranes exhibit abnormalities: many β-tumor cells have excessive dilatations of nuclear envelope and endoplasmic reticulum. All above severe infrastructural abnormalities, especially down regulation of cell-cell and cell-extracellular matrix adhesions and plasma membranes fragility might result in aberrant cell behavior and, consequently, much care should be taken for the postoperatory patient evolution.

  15. 90Y-Labeled Anti-ROBO1 Monoclonal Antibody Exhibits Antitumor Activity against Small Cell Lung Cancer Xenografts

    PubMed Central

    Fujiwara, Kentaro; Koyama, Keitaro; Suga, Kosuke; Ikemura, Masako; Saito, Yasutaka; Hino, Akihiro; Iwanari, Hiroko; Kusano-Arai, Osamu; Mitsui, Kenichi; Kasahara, Hiroyuki; Fukayama, Masashi; Kodama, Tatsuhiko; Hamakubo, Takao; Momose, Toshimitsu

    2015-01-01

    Introduction ROBO1 is a membrane protein that contributes to tumor metastasis and angiogenesis. We previously reported that 90Y-labeled anti-ROBO1 monoclonal antibody (90Y-anti-ROBO1 IgG) showed an antitumor effect against ROBO1-positive tumors. In this study, we performed a biodistribution study and radioimmunotherapy (RIT) against ROBO1-positive small cell lung cancer (SCLC) models. Methods For the biodistribution study, 111In-labeled anti-ROBO1 monoclonal antibody (111In-anti-ROBO1 IgG) was injected into ROBO1-positive SCLC xenograft mice via the tail vein. To evaluate antitumor effects, an RIT study was performed, and SCLC xenograft mice were treated with 90Y-anti-ROBO1 IgG. Tumor volume and body weight were periodically measured throughout the experiments. The tumors and organs of mice were then collected, and a pathological analysis was carried out. Results As a result of the biodistribution study, we observed tumor uptake of 111In-anti-ROBO1 IgG. The liver, kidney, spleen, and lung showed comparably high accumulation of 111In-labeled anti-ROBO1. In the RIT study, 90Y-anti-ROBO1 IgG significantly reduced tumor volume compared with baseline. Pathological analyses of tumors revealed coagulation necrosis and fatal degeneration of tumor cells, significant reduction in the number of Ki-67-positive cells, and an increase in the number of apoptotic cells. A transient reduction of hematopoietic cells was observed in the spleen, sternum, and femur. Conclusions These results suggest that RIT with 90Y-anti-ROBO1 IgG is a promising treatment for ROBO1-positive SCLC. PMID:26017283

  16. RECQ1 Helicase Silencing Decreases the Tumour Growth Rate of U87 Glioblastoma Cell Xenografts in Zebrafish Embryos.

    PubMed

    Vittori, Miloš; Breznik, Barbara; Hrovat, Katja; Kenig, Saša; Lah, Tamara T

    2017-09-06

    RECQ1 helicase has multiple roles in DNA replication, including restoration of the replication fork and DNA repair, and plays an important role in tumour progression. Its expression is highly elevated in glioblastoma as compared to healthy brain tissue. We studied the effects of small hairpin RNA (shRNA)-induced silencing of RECQ1 helicase on the increase in cell number and the invasion of U87 glioblastoma cells. RECQ1 silencing reduced the rate of increase in the number of U87 cells by 30%. This corresponded with a 40% reduction of the percentage of cells in the G2 phase of the cell cycle, and an accumulation of cells in the G1 phase. These effects were confirmed in vivo, in the brain of zebrafish ( Daniorerio ) embryos, by implanting DsRed-labelled RECQ1 helicase-silenced and control U87 cells. The growth of resulting tumours was quantified by monitoring the increase in xenograft fluorescence intensity during a three-day period with fluorescence microscopy. The reduced rate of tumour growth, by approximately 30% in RECQ1 helicase-silenced cells, was in line with in vitro measurements of the increase in cell number upon RECQ1 helicase silencing. However, RECQ1 helicase silencing did not affect invasive behaviour of U87 cells in the zebrafish brain. This is the first in vivo confirmation that RECQ1 helicase is a promising molecular target in the treatment of glioblastoma.

  17. The activation of the rat insulin gene II by BETA2 and PDX-1 in rat insulinoma cells is repressed by Pax6.

    PubMed

    Wolf, Gabriele; Hessabi, Behnam; Karkour, Anke; Henrion, Ulrike; Dahlhaus, Meike; Ostmann, Annett; Giese, Bernd; Fraunholz, Martin; Grabarczyk, Piotr; Jack, Robert; Walther, Reinhard

    2010-12-01

    The transcriptional transactivator Pax6 binds the pancreatic islet cell-specific enhancer sequence (PISCES) of the rat insulin I gene. However the human, mouse, and rat insulin gene II promoters do not contain a PISCES element. To analyze the role of Pax6 in those PISCES-less promoters, we investigated its influence on rat insulin gene II expression and included in our studies the main activators: pancreatic and duodenal homeobox protein-1 (PDX-1) and BETA2/E47. Luciferase assays, Northern blots, and RIA were used to study effects of Pax6 overexpression, gel shift and chromatin precipitation assays to study its binding to the DNA, and yeast two-hybrid assays and glutathione S transferase capture assays to investigate its interactions with PDX-1 and BETA2. Finally, glucose-dependent intracellular transport of Pax6 was demonstrated by fluorescence microscopy. Overexpression of Pax6 prevents activation of the rat insulin II gene by BETA2 and PDX-1 and hence suppresses insulin synthesis and secretion. In vitro, Pax6 binds to the A-boxes, thereby blocking binding of PDX-1, and at the same time, its paired domain interacts with BETA2. Fluorescence microscopy demonstrated that the nuclear-cytoplasmic localization of Pax6 and PDX-1 are oppositely regulated by glucose. From the results, it is suggested that at low concentrations of glucose, Pax6 is localized in the nucleus and prevents the activation of the insulin gene by occupying the PDX-1 binding site and by interacting with BETA2.

  18. The in vivo performance of plasmonic nanobubbles as cell theranostic agents in zebrafish hosting prostate cancer xenografts

    PubMed Central

    Wagner, Daniel S.; Delk, Nikki A.; Lukianova-Hleb, Ekaterina Y.; Hafner, Jason H.; Farach-Carson, Mary C.; Lapotko, Dmitri O.

    2010-01-01

    Cell theranostics is a new approach that unites diagnosis, therapy and confirmation (guidance) of the results of therapy in one single process at cell level, thus principally improving both the rapidity and precision of treatment. The ideal theranostic agent will support all three of the above functions in vivo with cellular resolution, allowing individual assessment of disease state and the elimination of diseased cells while leaving healthy cells intact. We have developed and evaluated plasmonic nanobubbles (PNBs) as an in vivo tunable theranostic cellular agent in zebrafish hosting prostate cancer xenografts. PNBs were selectively generated around gold nanoparticles in cancer cells in the zebrafish with short single laser pulses. By varying the energy of the laser pulse, we dynamically tuned the PNB size in a theranostic sequence of two PNBs: an initial small PNB detected a cancer cell through optical scattering, followed by a second bigger PNB, which mechanically ablated this cell without damage to surrounding tissue, while its optical scattering confirmed the destruction of the cell. Thus PNBs supported the diagnosis and guided ablation of individual human cancer cells in a living organism without damage to the host. PMID:20630586

  19. The in vivo performance of plasmonic nanobubbles as cell theranostic agents in zebrafish hosting prostate cancer xenografts.

    PubMed

    Wagner, Daniel S; Delk, Nikki A; Lukianova-Hleb, Ekaterina Y; Hafner, Jason H; Farach-Carson, Mary C; Lapotko, Dmitri O

    2010-10-01

    Cell theranostics is a new approach that unites diagnosis, therapy and confirmation (guidance) of the results of therapy in one single process at cell level, thus principally improving both the rapidity and precision of treatment. The ideal theranostic agent will support all three of the above functions in vivo with cellular resolution, allowing individual assessment of disease state and the elimination of diseased cells while leaving healthy cells intact. We have developed and evaluated plasmonic nanobubbles (PNBs) as an in vivo tunable theranostic cellular agent in zebrafish hosting prostate cancer xenografts. PNBs were selectively generated around gold nanoparticles in cancer cells in the zebrafish with short single laser pulses. By varying the energy of the laser pulse, we dynamically tuned the PNB size in a theranostic sequence of two PNBs: an initial small PNB detected a cancer cell through optical scattering, followed by a second bigger PNB, which mechanically ablated this cell without damage to surrounding tissue, while its optical scattering confirmed the destruction of the cell. Thus PNBs supported the diagnosis and guided ablation of individual human cancer cells in a living organism without damage to the host.

  20. [Compound K suppresses myeloid-derived suppressor cells in a mouse model bearing CT26 colorectal cancer xenograft].

    PubMed

    Wang, Rong; Li, Yalin; Wang, Wuzhou; Zhou, Meijuan; Cao, Zhaohui

    2015-05-01

    To investigate the effect of ginseng-derived compound K (C-K) on apoptosis, immunosuppressive activity, and pro-inflammatory cytokine production of myeloid-derived suppressor cells (MDSCs) from mice bearing colorectal cancer xenograft. Flow-sorted bone marrow MDSCs from Balb/c mice bearing CT26 tumor xenograft were treated with either C-K or PBS for 96 h and examined for apoptosis with Annexin V/7-AAD, Cox-2 and Arg-1 expressions using qRT-PCR, and supernatant IL-1β, IL-6, and IL-17 levels with ELISA. C-K- or PBS-treated MDSCs were subcutaneously implanted along with CT26 tumor cells in WT Balb/c mice, and the tumor size and morphology were evaluated 21 days later. C-K treatment significantly increased the percentages of early and late apoptotic MDSCs in vitro (P<0.01 and P<0.05, respectively), decreased the expressions of immunosuppression-related genes Cox-2 (P<0.05) and Arg-1 (P<0.01), and suppressed the production of IL-1β (P<0.05), IL-6 (P<0.01), and IL-17 (P<0.05) by the MDSCs . Compared with PBS-pre-treated cells, C-K-pretreated MDSCs showed significantly attenuated activity in promoting CT26 tumor growth in mice (P<0.01). C-K can suppress the immunosuppresive effect of MDSCs to inhibit tumor cell proliferation in mice, which suggests a new strategy of tumor therapy by targeting MDSCs.

  1. Acquired resistance to EGFR tyrosine kinase inhibitor in A431 squamous cell carcinoma xenografts is mediated by c-Kit pathway transduction.

    PubMed

    Zhang, Lixia; Yang, Xiaokun; Zhao, Bei; Cai, Zhen

    2015-04-01

    Epidermal growth factor inhibitors (EGFRIs), the first targeted cancer therapy, are currently an essential treatment for many advance-stage epithelial cancers. These agents have the superior ability to target cancers cells and better safety profile compared to conventional chemotherapies. However, all responding patients eventually developed acquired resistance to EGFRIs and the mechanisms of acquired resistance invariably develops. In the current study, we reported the tumor xenografts of the human A431 squamous cell carcinoma, after 25-week consecutive therapy with EGFR inhibitor (gefitinib) that developed resistance as a result of c-Kit overexpression. Moreover, combined therapeutic inhibition of EGFR and c-Kit may abrogate this acquired mechanism of drug resistance due to an enhanced apoptotic effect in gefitinib-resistant xenograft model. Taken together, the results suggest that at least in the A431 xenograft model displaying acquired resistance to gefitinib can emerge in vivo, at least in part, by mechanisms involving the c-Kit overexpression.

  2. Zanthoxylum avicennae extracts induce cell apoptosis through protein phosphatase 2A activation in HA22T human hepatocellular carcinoma cells and block tumor growth in xenografted nude mice.

    PubMed

    Dung, Tran Duc; Chang, Hsien-Cheh; Chen, Chung-Yu; Peng, Wen-Huang; Tsai, Chang-Hai; Tsai, Fuu-Jen; Kuo, Wei-Wen; Chen, Li-Mien; Huang, Chih-Yang

    2011-12-01

    The use of herbs as alternative cancer therapies has attracted a great deal of attention owing to their lower toxicity. Whether Zanthoxylum avicennae (Ying Bu Bo, YBB) induces liver cancer cell apoptosis remains unclear. In this study, we investigated the effect of YBB extracts (YBBEs) on HA22T human hepatocellular carcinoma cells in vitro and in an in vivo mouse xenograft model. HA22T cells were treated with different concentrations of YBBEs and analyzed with Western blot analysis, TUNEL, JC-1 staining and siRNA transfection assays. Additionally, the HA22T-implanted xenograft nude mice model was applied to confirm the cellular effects. YBBEs-induced apoptosis, up-regulated death receptor apoptotic pathway markers as well as mitochondrial proteins, and suppressed the survival proteins in a dose-dependent manner. Pro-survival Bcl-2 family proteins were inhibited and the pro-apoptotic ones were increased. Protein phosphatase 2A (PP2A) siRNA or okadaic acid reversed the YBBEs effects, confirming the role of PP2A in YBBEs-induced HA22T apoptosis. All our experimental evidence indicates that YBBEs significantly promote HA22T apoptosis and reduce tumor sizes in xenograft nude mice via PP2A in a dose-dependent manner.

  3. Rejection of wild-type and genetically engineered major histocompatibility complex-deficient glial cell xenografts in the central nervous system results in bystander demyelination and Wallerian degeneration.

    PubMed

    O'Leary, M T; Bujdoso, R; Blakemore, W F

    1998-07-01

    Mixed glial cell cultures prepared from neonatal wild type and mutant male mice lacking either major histocompatibility complex class I, class II or both class I and II molecules (major histocompatibility complex class I(o/o)II(o/o)), and from syngeneic male rats were transplanted into female rat spinal cord white matter. Graft survival was monitored using DNA probes specific to the Y chromosome. Survival of major histocompatibility complex class-deficient grafts was not prolonged compared to wild-type grafts and in most cases grafts could not be detected at 28 days post-transplantation, at which time syngeneic grafts were still present. However, rejection of xenografts resulted in significant bystander damage to host tissue. In recipients of wild-type and major histocompatibility complex class I(o/o) xenografts the predominant pathology was demyelination. Demyelination was also observed in recipients of major histocompatibility complex class II(o/o) and major histocompatibility complex class I(o/o)II(o/o) xenografts, however in addition there was marked collagen deposition and meningeal cell invasion. Significantly more axons had undergone Wallerian degeneration in recipients of major histocompatibility complex class II(o/o) and major histocompatibility complex class I(o/o)II(o/o) xenografts than recipients of wild-type and major histocompatibility complex class I(o/o) xenografts. These findings were interpreted as evidence of a more destructive immune response associated with rejection of grafts lacking major histocompatibility complex class II molecules. It was proposed that the difference in the severity of bystander damage may be related to the previously demonstrated ability of xenogeneic major histocompatibility complex class II molecules to activate host T cells directly, whereas xenografts lacking major histocompatibility complex class II molecules were capable of activating host T cells only by the indirect pathway.

  4. Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

    PubMed Central

    Rowan, Brian G.; Gimble, Jeffrey M.; Sheng, Mei; Anbalagan, Muralidharan; Jones, Ryan K.; Frazier, Trivia P.; Asher, Majdouline; Lacayo, Eduardo A.; Friedlander, Paul L.; Kutner, Robert; Chiu, Ernest S.

    2014-01-01

    Background Fat grafting is used to restore breast defects after surgical resection of breast tumors. Supplementing fat grafts with adipose tissue-derived stromal/stem cells (ASCs) is proposed to improve the regenerative/restorative ability of the graft and retention. However, long term safety for ASC grafting in proximity of residual breast cancer cells is unknown. The objective of this study was to determine the impact of human ASCs derived from abdominal lipoaspirates of three donors, on a human breast cancer model that exhibits early metastasis. Methodology/Principal Findings Human MDA-MB-231 breast cancer cells represents “triple negative” breast cancer that exhibits early micrometastasis to multiple mouse organs [1]. Human ASCs were derived from abdominal adipose tissue from three healthy female donors. Indirect co-culture of MDA-MB-231 cells with ASCs, as well as direct co-culture demonstrated that ASCs had no effect on MDA-MB-231 growth. Indirect co-culture, and ASC conditioned medium (CM) stimulated migration of MDA-MB-231 cells. ASC/RFP cells from two donors co-injected with MDA-MB-231/GFP cells exhibited a donor effect for stimulation of primary tumor xenografts. Both ASC donors stimulated metastasis. ASC/RFP cells were viable, and integrated with MDA-MB-231/GFP cells in the tumor. Tumors from the co-injection group of one ASC donor exhibited elevated vimentin, matrix metalloproteinase-9 (MMP-9), IL-8, VEGF and microvessel density. The co-injection group exhibited visible metastases to the lung/liver and enlarged spleen not evident in mice injected with MDA-MB-231/GFP alone. Quantitation of the total area of GFP fluorescence and human chromosome 17 DNA in mouse organs, H&E stained paraffin sections and fluorescent microscopy confirmed multi-focal metastases to lung/liver/spleen in the co-injection group without evidence of ASC/RFP cells. Conclusions Human ASCs derived from abdominal lipoaspirates of two donors stimulated metastasis of MDA-MB-231

  5. The novel organic arsenical darinaparsin induces MAPK-mediated and SHP1-dependent cell death in T-cell lymphoma and Hodgkin lymphoma cells and human xenograft models.

    PubMed

    Ravi, Dashnamoorthy; Bhalla, Savita; Gartenhaus, Ronald B; Crombie, Jennifer; Kandela, Irawati; Sharma, Jaya; Mazar, Andrew; Evens, Andrew M

    2014-12-01

    Darinaparsin (Zio-101) is a novel organic arsenical compound with encouraging clinical activity in relapsed/refractory T-cell lymphoma (TCL) and Hodgkin lymphoma (HL); however, little is known about its mechanism of action. TCL cell lines (Jurkat, Hut78, and HH) and HL cell lines (L428, L540, and L1236) were examined for in vitro cell death by MTT assay and Annexin V-based flow cytometry. Jurkat and L540-derived xenografts in SCID mice were examined for in vivo tumor inhibition and survival. Biologic effects of darinaparsin on the MAPK pathway were investigated using pharmacologic inhibitors, RNAi and transient transfection for overexpression for SHP1 and MEK. Darinaparsin treatment resulted in time- and dose-dependent cytotoxicity and apoptosis in all TCL and HL cell lines. In addition, darinaparsin had more rapid, higher, and sustained intracellular arsenic levels compared with arsenic trioxide via mass spectrometry. In vivo experiments with Jurkat (TCL) and L540 (HL)-derived lymphoma xenografts showed significant inhibition of tumor growth and improved survival in darinaparsin-treated SCID mice. Biologically, darinaparsin caused phosphorylation of ERK (and relevant downstream substrates) primarily by decreasing the inhibitory SHP1 phosphatase and coimmunoprecipitation showed significant ERK/SHP1 interaction. Furthermore, ERK shRNA knockdown or constitutive overexpression of SHP1 resulted in increased apoptosis, whereas cotreatment with pharmacologic MEK inhibitors resulted in synergistic cell death. Conversely, SHP1 blockade (via pharmacologic inhibition or RNAi) and MEK constitutive activation decreased darinaparsin-related cell death. Altogether, these data show that darinaparsin is highly active in HL and TCL and its activity is dependent primarily on MAPK mechanisms. ©2014 American Association for Cancer Research.

  6. Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.

    PubMed

    Prével, Camille; Pellerano, Morgan; González-Vera, Juan A; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May C

    2016-11-15

    Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.

  7. A Small Molecule Inhibitor of Human RAD51 Potentiates Breast Cancer Cell Killing by Therapeutic Agents in Mouse Xenografts

    PubMed Central

    Huang, Fei; Mazin, Alexander V.

    2014-01-01

    The homologous recombination pathway is responsible for the repair of DNA double strand breaks. RAD51, a key homologous recombination protein, promotes the search for homology and DNA strand exchange between homologous DNA molecules. RAD51 is overexpressed in a variety of cancer cells. Downregulation of RAD51 by siRNA increases radio- or chemo-sensitivity of cancer cells. We recently developed a specific RAD51 small molecule inhibitor, B02, which inhibits DNA strand exchange activity of RAD51 in vitro. In this study, we used human breast cancer cells MDA-MB-231 to investigate the ability of B02 to inhibit RAD51 and to potentiate an anti-cancer effect of chemotherapeutic agents including doxorubicin, etoposide, topotecan, and cisplatin. We found that the combination of B02 with cisplatin has the strongest killing effect on the cancer cells. We then tested the effect of B02 and cisplatin on the MDA-MB-231 cell proliferation in mouse xenografts. Our results showed that B02 significantly enhances the therapeutic effect of cisplatin on tumor cells in vivo. Our current data demonstrate that use of RAD51-specific small molecule inhibitor represents a feasible strategy of a combination anti-cancer therapy. PMID:24971740

  8. Naltrindole Inhibits Human Multiple Myeloma Cell Proliferation In Vitro and in a Murine Xenograft Model In Vivo

    PubMed Central

    Mundra, Jyoti Joshi; Terskiy, Alexandra

    2012-01-01

    It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrindole, a nonpeptidic δ-opioid receptor-selective antagonist; therefore, we hypothesized that human multiple myeloma (MM) would be a valuable model for studying potential antineoplastic properties of naltrindole. [3H]naltrindole exhibited saturable, low-affinity binding to intact human MM cells; however, the pharmacological profile of the binding site differed considerably from the properties of δ-, κ-, and μ-opioid receptors, and opioid receptor mRNA was not detected in MM cells by reverse transcriptase-polymerase chain reaction. Naltrindole inhibited the proliferation of cultured human U266 MM cells in a time- and dose-dependent manner with an EC50 of 16 μM. The naltrindole-induced inhibition of U266 cell proliferation was not blocked by a 10-fold molar excess of naltrexone, a nonselective opioid antagonist. Additive inhibition of MM cell proliferation was observed when using a combination of naltrindole with the histone deacetylase inhibitor sodium valproate, the proteasome inhibitor bortezomib, the glucocorticoid receptor agonist dexamethasone, and the HMG CoA reductase inhibitor simvastatin. Treatment of U266 cells with naltrindole significantly decreased the level of the active, phosphorylated form of the kinases, extracellular signal-regulated kinase and Akt, which may be related to its antiproliferative activity. The antiproliferative activity of naltrindole toward MM cells was maintained in cocultures of MM and bone marrow-derived stromal cells, mimicking the bone marrow microenvironment. In vivo, naltrindole significantly decreased tumor cell volumes in human MM cell xenografts in severe combined immunodeficient mice. We hypothesize that naltrindole inhibits the proliferation of MM cells through a nonopioid receptor-dependent mechanism. PMID:22537770

  9. Matrix metalloproteinases in human melanoma cell lines and xenografts: increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression

    PubMed Central

    Hofmann, U B; Westphal, J R; Waas, E T; Zendman, A J W; Cornelissen, I M H A; Ruiter, D J; Muijen, G N P van

    1999-01-01

    Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tumour progression and metastasis. In this study, we investigated the in vitro and in vivo expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA and protein in a previously described human melanoma xenograft model. This model consists of eight human melanoma cell lines with different metastatic behaviour after subcutaneous (s.c.) injection into nude mice. MMP-1 mRNA was detectable in all cell lines by reverse transcription polymerase chain reaction (RT-PCR), but the expression was too low to be detected by Northern blot analysis. No MMP-1 protein could be found using Western blotting. MMP-2 mRNA and protein were present in all cell lines, with the highest expression of both latent and active MMP-2 in the highest metastatic cell lines MV3 and BLM. MMP-3 mRNA was expressed in MV3 and BLM, and in the non-metastatic cell line 530, whereas MMP-3 protein was detectable only in MV3 and BLM. None of the melanoma cell lines expressed MMP-9. TIMP-1 and TIMP-2 mRNA and protein, finally, were present in all cell lines. A correlation between TIMP expression level and metastatic capacity of cell lines, however, was lacking. MMP and TIMP mRNA and protein expression levels were also studied in s.c. xenograft lesions derived from a selection of these cell lines. RT-PCR analysis revealed that MMP-1 mRNA was present in MV3 and BLM xenografts, and to a lesser extent in 530. Positive staining for MMP-1 protein was found in xenograft lesions derived from both low and high metastatic cell lines, indicating an in vivo up-regulation of MMP-1. MMP-2 mRNA was detectable only in xenografts derived from the highly metastatic cell lines 1F6m, MV3 and BLM. In agreement with the in vitro results, the highest levels of both latent and activated MMP-2 protein were observed in MV3 and BLM xenografts. With the exception of MMP-9 mRNA expression in 530 xenografts, MMP-3, MMP-9, and TIMP-1 mRNA and

  10. Antitumor effects of deguelin on H460 human lung cancer cells in vitro and in vivo: Roles of apoptotic cell death and H460 tumor xenografts model.

    PubMed

    Hsu, Yu-Chieh; Chiang, Jo-Hua; Yu, Chun-Shu; Hsia, Te-Chun; Wu, Rick Sai-Chuen; Lien, Jin-Cherng; Lai, Kuang-Chi; Yu, Fu-Shun; Chung, Jing-Gung

    2017-01-01

    Deguelin, a naturally occurring rotenoid of the flavonoid family, is known to be an Akt inhibitor, to have chemopreventive activities and anti-tumor effect on several cancers. In this study, investigation to elucidate the effect of deguelin on apoptotic pathways in human lung cancer cells and on the anti-tumor effect in lung cancer xenograft nu/nu mice was performed. In vitro studies, found that deguelin induced cell morphological changes, and decreased the percentage of viability through the induction of apoptosis in H460 lung cancer cells. Deguelin triggered apoptosis in H460 cells was also confirmed by DAPI staining, DNA gel electrophoresis, and Annexin V-FITC staining and these effects are dose-dependent manners. It was also found that deguelin promoted the Ca(2+) production and activation of caspase-3 but decreased the level of ΔΨm in H460 cells. Western blots indicated that the protein levels of cytochrome c, AIF, and pro-apoptotic Bax and Bak protein were increased, but the anti-apoptotic Bcl-2 and Bcl-x were decreased that may have led to apoptosis in H460 cells after exposure to deguelin. It was also confirmed by confocal laser microscope examination that deguelin promoted the release of AIF from mitochondria to cytosol. In vivo studies, found that in immunodeficient nu/nu mice bearing H460 tumor xenografts showed that the deguelin significantly suppressed tumor growth. Deguelin might be a potential therapeutic agent for the treatment of lung cancer in the future. This finding might fully support a critical event for deguelin via induction of apoptotic cell death and H460 tumor xenografts model against human lung cancer. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 84-98, 2017.

  11. Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Head and Neck Cancer Xenografts.

    PubMed

    Rowan, Brian G; Lacayo, Eduardo A; Sheng, Mei; Anbalagan, Muralidharan; Gimble, Jeffrey M; Jones, Ryan K; Joseph, Walter J; Friedlander, Paul L; Chiu, Ernest S

    2016-01-01

    Fat grafting has become popular for repair of postsurgical/postradiation defects after head/neck cancers resection. Fat graft supplementation with adipose tissue-derived stromal/stem cells (ASCs) is proposed to improve graft viability/efficacy, although the impact of ASCs on head/neck cancer cells is unknown. To determine whether ASCs affect growth, migration, and metastasis of human head/neck cancer. Human Cal-27 and SCC-4 head/neck cancer cells were co-cultured human ASCs, or treated with ASC conditioned medium (CM), and cancer cell growth/migration was assessed by MTT, cell count, and scratch/wound healing assays in vitro. Co-injection of 3 × 10(6) Cal-27/green fluorescent protein (GFP) cells and ASCs into the flank of NUDE mice assessed ASC effect on tumor growth/morphology. Quantitation of human chromosome 17 DNA in mouse organs assessed ASC effects on micrometastasis. Primary tumors were evaluated for markers of epithelial-to-mesenchymal transition, matrix metalloproteinases, and angiogenesis by immunohistochemistry. Co-culture of Cal-27 or SCC-4 cells with ASCs from 2 different donors or ASC CM had no effect on cell growth in vitro. However, ASC CM stimulated Cal-27 and SCC-4 migration. Co-injection of ASCs from 2 different donors with Cal-27 cells did not affect tumor volume at 6 weeks, but increased Cal-27 micrometastasis to the brain. Evaluation of tumors sections from 1 ASC donor co-injection revealed that ASCs were viable and well integrated with Cal-27/GFP cells. These tumors exhibited increased MMP2, MMP9, IL-8, and microvessel density. Human ASCs did not alter growth of human head/neck cancer cells or tumor xenografts, but stimulated migration and early micrometastasis to mouse brain. © 2015 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

  12. The isothiocyanate erucin abrogates telomerase in hepatocellular carcinoma cells in vitro and in an orthotopic xenograft tumour model of HCC

    PubMed Central

    Herz, Corinna; Hertrampf, Anke; Zimmermann, Stefan; Stetter, Nadine; Wagner, Meike; Kleinhans, Claudia; Erlacher, Miriam; Schüler, Julia; Platz, Stefanie; Rohn, Sascha; Mersch-Sundermann, Volker; Lamy, Evelyn

    2014-01-01

    In contrast to cancer cells, most normal human cells have no or low telomerase levels which makes it an attractive target for anti-cancer drugs. The small molecule sulforaphane from broccoli is known for its cancer therapeutic potential in vitro and in vivo. In animals and humans it was found to be quickly metabolized into 4-methylthiobutyl isothiocyanate (MTBITC, erucin) which we recently identified as strong selective apoptosis inducer in hepatocellular carcinoma (HCC) cells. Here, we investigated the relevance of telomerase abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective against telomerase, independent from TP53 and MTBITC also blocked telomerase in chemoresistant subpopulations. By using an orthotopic human liver cancer xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d significantly decreased telomerase activity in vivo without affecting enzyme activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was consistent with a dose-dependent switch to anti-survival, cell arrest and apoptosis in our in vitro HCC models. Blocking telomerase by the specific inhibitor TMPyP4 further sensitized cancer cells to MTBITC-mediated cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis induction by MTBITC was prevented by hTERT overexpression. These findings imply that telomerase enzyme activity does not protect against MTBITC-induced DNA damage but impacts signalling processes upstream of apoptosis execution level. PMID:25256442

  13. Photodynamic therapy using methylene blue in lung adenocarcinoma xenograft and hamster cheek pouch induced squamous cell carcinoma.

    PubMed

    Obstoy, Bérengère; Salaun, Mathieu; Bohn, Pierre; Veresezan, Liana; Sesboué, Richard; Thiberville, Luc

    2016-09-01

    Photodynamic therapy (PDT) is used to treat early proximal bronchial cancer during a flexible bronchoscopy. The technique relies on the excitation of a photosensitizer by an appropriate wavelength, which is delivered into the bronchus in close contact with the tumor. To assess methylene blue (MB) as a PDT agent for the treatment of respiratory tract cancer in animal models. MB-induced PDT was performed on 7 subcutaneous NCI-H460 lung adenocarcinoma xenografts in nude mice and 9 induced squamous cell cancer in the hamster cheek pouch model. In mice, PDT was carried out on right-sided tumors after intratumoral injection of methylene blue 1% (w/v) and illumination at 630nm at 200J/cm (Diomed PDT 630), with the left tumor used as control (illumination alone or MB alone). The tumoral volume was assessed before and 15 days after PDT. Fourteen xenografts were treated in mice, including seven treated with MB-PDT, producing a 52% mean tumor volume regression (1568mm(3)vs. 544mm(3)) compared to seven control cases in which tumor volume increased (p=0.007; Mann-Whitney test). Nine cheek pouch induced carcinomas were treated in the hamster group, with a mean volume decrease of 85.8% (from 44.8% to 100%) (initial mean volume=210mm(3)vs. post PDT mean volume=97mm(3)). Histology analysis showed 4/9 complete responses. Intratumoral MB appears efficient as PDT agent for cancer treatment in animal models. Further studies are needed to assess the safety and efficacy of MB-associated PDT for the treatment of lung cancer in humans. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Combining metformin and nelfinavir exhibits synergistic effects against the growth of human cervical cancer cells and xenograft in nude mice

    PubMed Central

    Xia, Chenglai; Chen, Ruihong; Chen, Jinman; Qi, Qianqian; Pan, Yanbin; Du, Lanying; Xiao, Guohong; Jiang, Shibo

    2017-01-01

    Human cervical cancer is the fourth most common carcinoma in women worldwide. However, the emergence of drug resistance calls for continuously developing new anticancer drugs and combination chemotherapy regimens. The present study aimed to investigate the anti-cervical cancer effects of metformin, a first-line therapeutic drug for type 2 diabetes mellitus, and nelfinavir, an HIV protease inhibitor, when used alone or in combination. We found that both metformin and nelfinavir, when used alone, were moderately effective in inhibiting proliferation, inducing apoptosis and suppressing migration and invasion of human cervical cell lines HeLa, SiHa and CaSki. When used in combination, these two drugs acted synergistically to inhibit the growth of human cervical cancer cells in vitro and cervical cancer cell xenograft in vivo in nude mice, and suppress cervical cancer cell migration and invasion. The protein expression of phosphoinositide 3-kinase catalytic subunit PI3K(p110α), which can promote tumor growth, was remarkably downregulated, while the tumor suppressor proteins p53 and p21 were substantially upregulated following the combinational treatment in vitro and in vivo. These results suggest that clinical use of metformin and nelfinavir in combination is expected to have synergistic antitumor efficacy and significant potential for the treatment of human cervical cancer. PMID:28252027

  15. Imatinib mesylate (Glivec) inhibits Schwann cell viability and reduces the size of human plexiform neurofibroma in a xenograft model.

    PubMed

    Demestre, Maria; Herzberg, Jan; Holtkamp, Nikola; Hagel, Christian; Reuss, David; Friedrich, Reinhard E; Kluwe, Lan; Von Deimling, Andreas; Mautner, Victor-F; Kurtz, Andreas

    2010-05-01

    Plexiform neurofibromas (PNF), one of the major features of neurofibromatosis type 1 (NF1), are characterized by complex cellular composition and mostly slow but variable growth patterns. In this study, we examined the effect of imatinib mesylate, a receptor tyrosine kinase inhibitor, on PNF-derived Schwann cells and PNF tumour growth in vitro and in vivo. In vitro, PNF-derived primary Schwann cells express platelet-derived growth factors receptors (PDGFR) alpha and beta, both targets of imatinib, and cell viability was reduced by imatinib mesylate, with 50% inhibition concentration (IC(50)) of 10 microM. For in vivo studies, PNF tumour fragments xenografted onto the sciatic nerve of athymic nude mice were first characterized. The tumours persisted for at least 63 days and maintained typical characteristics of PNFs such as complex cellular composition, low proliferation rate and angiogenesis. A transient enlargement of the graft size was due to inflammation by host cells. Treatment with imatinib mesylate at a daily dose of 75 mg/kg for 4 weeks reduced the graft size by an average of 80% (n = 8), significantly different from the original sizes within the group and from sizes of the grafts in 11 untreated mice in the control group (P < 0.001). We demonstrated that grafting human PNF tumour fragments into nude mice provides an adequate in vivo model for drug testing. Our results provide in vivo and in vitro evidence for efficacy of imatinib mesylate for PNF.

  16. Basal Tumor Cell Isolation and Patient-Derived Xenograft Engraftment Identify High-Risk Clinical Bladder Cancers

    PubMed Central

    Skowron, K. B.; Pitroda, S. P.; Namm, J. P.; Balogun, O.; Beckett, M. A.; Zenner, M. L.; Fayanju, O.; Huang, X.; Fernandez, C.; Zheng, W.; Qiao, G.; Chin, R.; Kron, S. J.; Khodarev, N. N.; Posner, M. C.; Steinberg, G. D.; Weichselbaum, R. R.

    2016-01-01

    Strategies to identify tumors at highest risk for treatment failure are currently under investigation for patients with bladder cancer. We demonstrate that flow cytometric detection of poorly differentiated basal tumor cells (BTCs), as defined by the co-expression of CD90, CD44 and CD49f, directly from patients with early stage tumors (T1-T2 and N0) and patient-derived xenograft (PDX) engraftment in locally advanced tumors (T3-T4 or N+) predict poor prognosis in patients with bladder cancer. Comparative transcriptomic analysis of bladder tumor cells isolated from PDXs indicates unique patterns of gene expression during bladder tumor cell differentiation. We found cell division cycle 25C (CDC25C) overexpression in poorly differentiated BTCs and determined that CDC25C expression predicts adverse survival independent of standard clinical and pathologic features in bladder cancer patients. Taken together, our findings support the utility of BTCs and bladder cancer PDX models in the discovery of novel molecular targets and predictive biomarkers for personalizing oncology care for patients. PMID:27775025

  17. Pig to rat cell transplantation: reduced cellular and antibody responses to xenografts overexpressing PD-L1.

    PubMed

    Plege-Fleck, Annegret; Lieke, Thorsten; Römermann, Dorothee; Düvel, Heike; Hundrieser, Joachim; Buermann, Anna; Kraus, Lilli; Klempnauer, Jürgen; Schwinzer, Reinhard

    2014-01-01

    Programmed death-1 (PD-1) costimulation acts as a negative regulator of T-cell responses to allografts. However, the role of the PD-1 pathway in xenotransplantation is not well defined yet. We have shown previously that human in vitro T-cell responses to porcine transfectants overexpressing PD-Ligand1 (L23-PD-L1 cells) are remarkably weak. In this report, we asked whether the PD-1/PD-L1 pathway has the potential to diminish xenogeneic immune responses also in vivo. L23-PD-L1 or mock transfected control cells (L23-GFP) were transplanted under the kidney capsule of rats. The occurrence of kidney-infiltrating rat leukocytes and the induction of anti-pig antibodies were monitored in grafted animals. Assessment of cellular infiltrates revealed similar numbers of macrophages in kidneys grafted with L23-PD-L1 or L23-GFP control cells. However, the level of MHC class-II molecules was reduced on macrophages responding to L23-PD-L1 grafts, suggesting a lower state of activation. Furthermore, less T cells were found in kidneys receiving L23-PD-L1 cells. In addition, the titers of induced anti-pig antibodies were significantly lower in rats grafted with L23-PD-L1 cells. These data suggest that signals triggered by PD-1-PD-L1 interaction interfere with activation pathways involved in the induction of cellular and antibody-mediated immune responses to xenografts in vivo. Targeting of PD-1 and/or PD-L1 may be a promising approach for immune modulation after xenotransplantation. © 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.

  18. Monoclonal antibodies to an epithelial ovarian adenocarcinoma: distinctive reactivity with xenografts of the original tumor and a cultured cell line.

    PubMed

    Baumal, R; Law, J; Buick, R N; Kahn, H; Yeger, H; Sheldon, K; Colgan, T; Marks, A

    1986-08-01

    Four monoclonal antibodies (mAb) (8C, 10B, M2A, and M2D) were produced against the human epithelial ovarian adenocarcinoma cell line, HEY. The affinity constants of binding of the mAb to cultured HEY cells were 8 X 10(8) M-1 (M2D) and 10(9) M-1 (8C and 10B). mAb 8C reacted with a major glycoprotein of Mr 90,000 on the surface of HEY cells. The four mAb differed from previously reported mAb to epithelial ovarian adenocarcinomas on the basis of their reactivity with cultured ovarian adenocarcinoma cell lines using a cell-binding radioimmunoassay, and their staining of cryostat sections of various human normal and tumor tissues using an immunoperoxidase reaction. All four mAb reacted with s.c. tumors derived by injecting cultured HEY cells into thymectomized CBA/CJ mice. However, only two of the four mAb (8C and 10B) also reacted with s.c. tumors of the original HEY xenograft from which the cultured cell line was derived. In addition, mAb 8C and 10B reacted by immunoperoxidase staining with 2 and 4 different cases, respectively, of 11 epithelial ovarian adenocarcinomas examined. Cultured HEY cells were adapted to grow i.p. in BALB/c-nu/nu mice and the i.p. tumors retained their reactivity with the monoclonal antibodies. These tumor-bearing mice offer a useful model system for studying the potential of mAb, especially 8C and 10B, for the diagnosis and treatment of patients with peritoneal extension of epithelial ovarian adenocarcinomas.

  19. Adenoviral expression of XIAP antisense RNA induces apoptosis in glioma cells and suppresses the growth of xenografts in nude mice.

    PubMed

    Naumann, U; Bähr, O; Wolburg, H; Altenberend, S; Wick, W; Liston, P; Ashkenazi, A; Weller, M

    2007-01-01

    The expression of inhibitor of apoptosis (IAP) family members contributes to the resistance of human cancers to apoptosis induced by radiotherapy and chemotherapy. We report that the infection of malignant glioma cells and several other tumor cell lines with adenoviruses encoding antisense RNA to X-linked IAP (XIAP) depletes endogenous XIAP levels and promotes global caspase activation and apoptosis. In contrast, non-neoplastic SV-FHAS human astrocytes and other non-neoplastic cells express XIAP at very low levels and resist these effects of adenovirus-expressing XIAP antisense RNA (Ad-XIAP-as). Caspase inhibitors such as z-Val-Ala-DL-Asp(OMe)-fluoromethylketone (zVAD-fmk) delay caspase processing and XIAP depletion, suggesting that XIAP depletion results both from antisense-mediated interference with protein synthesis and proteolytic cleavage by activated caspases. However, zVAD-fmk neither prevents nor delays cell death, indicating a caspase-independent pathway to cell death triggered by IAP depletion. Similarly, B-cell lymphoma-X(L) (BCL-X(L)) inhibits caspase activity, but fails to rescue from apoptosis. Loss of p65/nuclear factor-kappaB (NF-kappaB) protein and NF-kappaB activity is an early event triggered by Ad-XIAP-as and probably involved in Ad-XIAP-as-induced apoptosis. Finally, Ad-XIAP-as gene therapy induces cell death in intracranial glioma xenografts, prolongs survival in nude mice and may reduce tumorigenicity in synergy with Apo2L/TNF-related apoptosis-inducing ligand (TRAIL) in vivo. Altogether, these data define a powerful survival function for XIAP and reinforce its possible role as a therapeutic target in human glioma cells.

  20. Concurrent insulinoma with mosaic Turner syndrome: A case report.

    PubMed

    Wang, Shaoyun; Yang, Lijuan; Li, Jie; Mu, Yiming

    2015-03-01

    Turner syndrome is a chromosomal abnormality in which the majority of patients have a 45XO karyotype, while a small number have a 45XO/47XXX karyotype. Congenital adrenal hyperplasia has been previously reported in patients with Turner syndrome. Although insulinomas are the most common type of functioning pancreatic neuroendocrine tumor and have been reported in patients with multiple endocrine neoplasias, the tumors have not been reported in patients with mosaic Turner syndrome. The present study reports the first case of an insulinoma in a patient with 45XO/47XXX mosaic Turner syndrome. The patient suffered from recurrent hypoglycemia, which was relieved following ingestion of glucose or food. A 5-h glucose tolerance test was performed and the levels of glucose, C-Peptide and insulin were detected. In addition, computed tomography (CT) and ultrasound scanning were performed to evaluate the possibility of an insulinoma. Pathological examination and karyotyping were performed on a surgical specimen and a whole blood sample, respectively. The patient was found to suffer from premature ovarian failure, and a physical examination was consistent with a diagnosis of Turner syndrome. An ultrasound scan demonstrated streak ovaries and the patient was found to have a 45XO/47XXX karyotype. Furthermore, a lesion was detected in the pancreas following CT scanning, which was identified as an insulinoma following surgical removal and histological examination. In conclusion, the present study reports the first case of an insulinoma in a patient with mosaic Turner syndrome. Since mosaic Turner syndrome and insulinoma are rare diseases, an association may exist that has not been previously identified.

  1. Effects of green-synthesized silver nanoparticles on lung cancer cells in vitro and grown as xenograft tumors in vivo

    PubMed Central

    He, Yan; Du, Zhiyun; Ma, Shijing; Liu, Yue; Li, Dongli; Huang, Huarong; Jiang, Sen; Cheng, Shupeng; Wu, Wenjing; Zhang, Kun; Zheng, Xi

    2016-01-01

    Silver nanoparticles (AgNPs) have now been recognized as promising therapeutic molecules and are extending their use in cancer diagnosis and therapy. This study demonstrates for the first time the antitumor activity of green-synthesized AgNPs against lung cancer in vitro and in vivo. Cytotoxicity effect was explored on human lung cancer H1299 cells in vitro by MTT and trypan blue assays. Apoptosis was measured by morphological assessment, and nuclear factor-κB (NF-κB) transcriptional activity was determined by a luciferase reporter gene assay. The expressions of phosphorylated stat3, bcl-2, survivin, and caspase-3 were examined by Western blot analysis. AgNPs showed dose-dependent cytotoxicity and stimulation of apoptosis in H1299 cells. The effects on H1299 cells correlated well with the inhibition of NF-κB activity, a decrease in bcl-2, and an increase in caspase-3 and survivin expression. AgNPs significantly suppressed the H1299 tumor growth in a xenograft severe combined immunodeficient (SCID) mouse model. The results demonstrate the anticancer activities of AgNPs, suggesting that they may act as potential beneficial molecules in lung cancer chemoprevention and chemotherapy, especially for early-stage intervention. PMID:27217750

  2. TGF-β signal rewiring sustains epithelial-mesenchymal transition of circulating tumor cells in prostate cancer xenograft hosts

    PubMed Central

    Huang, Guangcun; Osmulski, Pawel A.; Bouamar, Hakim; Mahalingam, Devalingam; Lin, Chun-Lin; Liss, Michael A.; Kumar, Addanki Pratap; Chen, Chun-Liang; Thompson, Ian M.; Sun, Lu-Zhe; Gaczynska, Maria E.; Huang, Tim H.-M.

    2016-01-01

    Activation of TGF-β signaling is known to promote epithelial-mesenchymal transition (EMT) for the development of metastatic castration-resistant prostate cancer (mCRPC). To determine whether targeting TGF-β signaling alone is sufficient to mitigate mCRPC, we used the CRISPR/Cas9 genome-editing approach to generate a dominant-negative mutation of the cognate receptor TGFBRII that attenuated TGF-β signaling in mCRPC cells. As a result, the delicate balance of oncogenic homeostasis is perturbed, profoundly uncoupling proliferative and metastatic potential of TGFBRII-edited tumor xenografts. This signaling disturbance triggered feedback rewiring by enhancing ERK signaling known to promote EMT-driven metastasis. Circulating tumor cells displaying upregulated EMT genes had elevated biophysical deformity and an increase in interactions with chaperone macrophages for facilitating metastatic extravasation. Treatment with an ERK inhibitor resulted in decreased aggressive features of CRPC cells in vitro. Therefore, combined targeting of TGF-β and its backup partner ERK represents an attractive strategy for treating mCRPC patients. PMID:27780930

  3. Effects of green-synthesized silver nanoparticles on lung cancer cells in vitro and grown as xenograft tumors in vivo.

    PubMed

    He, Yan; Du, Zhiyun; Ma, Shijing; Liu, Yue; Li, Dongli; Huang, Huarong; Jiang, Sen; Cheng, Shupeng; Wu, Wenjing; Zhang, Kun; Zheng, Xi

    2016-01-01

    Silver nanoparticles (AgNPs) have now been recognized as promising therapeutic molecules and are extending their use in cancer diagnosis and therapy. This study demonstrates for the first time the antitumor activity of green-synthesized AgNPs against lung cancer in vitro and in vivo. Cytotoxicity effect was explored on human lung cancer H1299 cells in vitro by MTT and trypan blue assays. Apoptosis was measured by morphological assessment, and nuclear factor-κB (NF-κB) transcriptional activity was determined by a luciferase reporter gene assay. The expressions of phosphorylated stat3, bcl-2, survivin, and caspase-3 were examined by Western blot analysis. AgNPs showed dose-dependent cytotoxicity and stimulation of apoptosis in H1299 cells. The effects on H1299 cells correlated well with the inhibition of NF-κB activity, a decrease in bcl-2, and an increase in caspase-3 and survivin expression. AgNPs significantly suppressed the H1299 tumor growth in a xenograft severe combined immunodeficient (SCID) mouse model. The results demonstrate the anticancer activities of AgNPs, suggesting that they may act as potential beneficial molecules in lung cancer chemoprevention and chemotherapy, especially for early-stage intervention.

  4. Setup and characterization of a human head and neck squamous cell carcinoma xenograft model in nude rats.

    PubMed

    Bao, Ande; Phillips, William T; Goins, Beth; McGuff, Howard S; Zheng, Xiangpeng; Woolley, F Ross; Natarajan, Mohan; Santoyo, Cristina; Miller, Frank R; Otto, Randal A

    2006-12-01

    To develop and characterize a new head and neck cancer animal model. A human head and neck squamous cell carcinoma (HNSCC) xenograft model in nude rats was established via subcutaneous inoculation of a human-origin HNSCC cell line, SCC-4. The tumor was evaluated for growth characteristics, pathologic features by hematoxylin-eosin (HE) staining, and immunohistochemistry of epidermal growth factor receptor (EGFR). 2-[18F] fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET) imaging characteristics were studied too. A new HNSCC animal model was successfully established. Tumor sizes reached about 1 cm3 on day 15 after tumor cell inoculation. HE staining pathology has confirmed that this tumor is a typical SCC. EGFR immunohistochemistry demonstrated this tumor model to be strongly EGFR positive. 18F-FDG PET study has shown that 18F-FDG accumulated in tumors. This study has demonstrated that this tumor model is an appropriate HNSCC tumor model for animal studies on HNSCC.

  5. Inhibition of proteasome activity by the dietary flavonoid apigenin is associated with growth inhibition in cultured breast cancer cells and xenografts.

    PubMed

    Chen, Di; Landis-Piwowar, Kristin R; Chen, Marina S; Dou, Q Ping

    2007-01-01

    Proteasome inhibition is an attractive approach to anticancer therapy and may have relevancy in breast cancer treatment. Natural products, such as dietary flavonoids, have been suggested as natural proteasome inhibitors with potential use for cancer prevention and therapeutics. We previously reported that apigenin, a flavonoid widely distributed in many fruits and vegetables, can inhibit proteasome activity and can induce apoptosis in cultured leukemia Jurkat T cells. Whether apigenin has proteasome-inhibitory activity in the highly metastatic human breast MDA-MB-231 cells and xenografts,however, is unknown. MDA-MB-231 breast cancer cell cultures and xenografts were treated with apigenin, followed by measurement of reduced cellular viability/proliferation,proteasome inhibition, and apoptosis induction. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity, by ubiquitinated proteins, and by accumulation of proteasome target proteins in extracts of the treated cells or tumors. Apoptotic cell death was measured by caspase-3/caspase-7 activation, poly(ADP-ribose) polymerase cleavage, and immunohistochemistry for terminal nucleotidyltransferase-mediated nick end labeling positivity. We report for the first time that apigenin inhibits the proteasomal chymotrypsin-like activity and induces apoptosis not only in cultured MDA-MB-231 cells but also in MDA-MB-231 xenografts. Furthermore, while apigenin has antibreast tumor activity, no apparent toxicity to the tested animals was observed. We have shown that apigenin is an effective proteasome inhibitor in cultured breast cancer cells and in breast cancer xenografts. Furthermore, apigenin induces apoptotic cell death in human breast cancer cells and exhibits anticancer activities in tumors. The results suggest its potential benefits in breast cancer prevention and treatment.

  6. p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells.

    PubMed

    Cen, Ling; Carlson, Brett L; Schroeder, Mark A; Ostrem, Jamie L; Kitange, Gaspar J; Mladek, Ann C; Fink, Stephanie R; Decker, Paul A; Wu, Wenting; Kim, Jung-Sik; Waldman, Todd; Jenkins, Robert B; Sarkaria, Jann N

    2012-07-01

    Deregulation of the p16(INK4a)-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16(INK4a)-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16(INK4a)-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16(INK4a) and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18(INK4c) (GBM43). In contrast, 2 GBM lines with p16(INK4a) expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16(INK4a) expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16(INK4a) expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991.

  7. Enhanced cell killing in lewis lung carcinoma and a human pancreatic-carcinoma xenograft by the combination of cytotoxic drugs and misonidazole.

    PubMed

    Stephens, T C; Courtenay, V D; Mills, J; Peacock, J H; Rose, C M; Spooner, D

    1981-04-01

    The "chemosensitizing" properties of the radiosensitizer misonidazole (MISO) were examined in 2 tumour systems, murine Lewis lung carcinoma and human pancreatic adenocarcinoma xenografted into immune-suppressed mice, using a soft-agar colony assay to measure tumour-cell survival. In mice bearing Lewis lung tumour, the administration of MISO simultaneously with melphalan, cyclophosphamide. CCNU, FU or vincristine gave substantial enhancement of cytotoxicity (DEFs from 1.5 to 3.5). However, no enhancement was seen with bleomycin, VP 16-213 or cis-Pt. The same level of enhancement of cyclophosphamide effect (DEF = 2.0) was seen with both cell survival and growth delay end-points effect (DEF = 2.0) was seen with both cell survival and growth delay end-points of tumour response. Enhancement was also seen in the human tumour xenograft with melphalan, cyclophosphamide and MeCCNU, using a cell survival assay, but cis-Pt was again not enhanced.

  8. Hypercalcitoninemia in a patient with a recurrent goitre and insulinoma: a case report.

    PubMed

    Pusztai, P; Sármán, B; Illyés, G; Székely, E; Péter, I; Boer, K; Tihanyi, T; Rácz, K

    2006-05-01

    Serum calcitonin has become a very sensitive and specific marker for medullary thyroid carcinoma that should be determined in patients with nodular thyroid disease. However, a few earlier reports indicated that tumors other than medullary thyroid carcinoma including insulinomas arising from pancreatic islet cells may also produce calcitonin. Of the few cases of calcitonin-producing insulinomas previously reported, most had incomplete data or lack of documentation of the association between raised serum calcitonin concentration and immunohistochemical detection of calcitonin in pancreatic islet cell tumors. In this paper we are reporting a 54-year-old woman with a history of partial thyroidectomy for multinodular goitre at the age of 50 yrs, she was evaluated for a 2-months history of fasting hypoglycemia (plasma glucose 1.9 mmol/L during a supervised fast), raised serum insulin (at the time of hypoglycemia 88.8 microU/ml; normal, 5 - 35 microU/ml) and C-peptide levels (at the time of hypoglycemia 6.1 ng/ml; normal, 1.37 - 3.51 ng/ml), markedly increased serum calcitonin concentration (481 pg/ml; normal, < 9.9 pg/ml), and an enlarged residual thyroid gland. Aspiration biopsy of the thyroid was negative for parafollicular C-cell hyperplasia or medullary thyroid carcinoma. Abdominal ultrasound and CT scan revealed a tumor in the head of the pancreas, which was surgically removed. Histopathological evaluation of the pancreatic tumor showed typical features of a neuroendocrine neoplasm with strong immunostaining for both insulin and calcitonin. After removal of the pancreatic tumor, clinical symptoms resolved and biochemical markers normalized (serum insulin, 14.9 microU/ml; C-peptide, 3.0 ng/ml; calcitonin, 2.9 pg/ml) confirming the causal relationship between insulinoma and markedly increased serum calcitonin levels.

  9. Circulating Tumor Cells as a Biomarker of Response to Treatment in Patient-Derived Xenograft Mouse Models of Pancreatic Adenocarcinoma

    PubMed Central

    Torphy, Robert J.; Tignanelli, Christopher J.; Kamande, Joyce W.; Moffitt, Richard A.; Herrera Loeza, Silvia G.; Soper, Steven A.; Yeh, Jen Jen

    2014-01-01

    Circulating tumor cells (CTCs) are cells shed from solid tumors into circulation and have been shown to be prognostic in the setting of metastatic disease. These cells are obtained through a routine blood draw and may serve as an easily accessible marker for monitoring treatment effectiveness. Because of the rapid progression of pancreatic ductal adenocarcinoma (PDAC), early insight into treatment effectiveness may allow for necessary and timely changes in treatment regimens. The objective of this study was to evaluate CTC burden as a biomarker of response to treatment with a oral phosphatidylinositol-3-kinase inhibitor, BKM120, in patient-derived xenograft (PDX) mouse models of PDAC. PDX mice were randomized to receive vehicle or BKM120 treatment for 28 days and CTCs were enumerated from whole blood before and after treatment using a microfluidic chip that selected for EpCAM (epithelial cell adhesion molecule) positive cells. This microfluidic device allowed for the release of captured CTCs and enumeration of these cells via their electrical impedance signatures. Median CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (26.61 to 2.21 CTCs/250 µL, p = 0.0207) while no significant change was observed in the vehicle group (23.26 to 11.89 CTCs/250 µL, p = 0.8081). This reduction in CTC burden in the treatment group correlated with tumor growth inhibition indicating CTC burden is a promising biomarker of response to treatment in preclinical models. Mutant enriched sequencing of isolated CTCs confirmed that they harbored KRAS G12V mutations, identical to the matched tumors. In the long-term, PDX mice are a useful preclinical model for furthering our understanding of CTCs. Clinically, mutational analysis of CTCs and serial monitoring of CTC burden may be used as a minimally invasive approach to predict and monitor treatment response to guide therapeutic regimens. PMID:24586805

  10. Honokiol inhibits sphere formation and xenograft growth of oral cancer side population cells accompanied with JAK/STAT signaling pathway suppression and apoptosis induction.

    PubMed

    Huang, Jhy-Shrian; Yao, Chih-Jung; Chuang, Shuang-En; Yeh, Chi-Tai; Lee, Liang-Ming; Chen, Ruei-Ming; Chao, Wan-Ju; Whang-Peng, Jacqueline; Lai, Gi-Ming

    2016-03-24

    Eliminating cancer stem cells (CSCs) has been suggested for prevention of tumor recurrence and metastasis. Honokiol, an active compound of Magnolia officinalis, had been proposed to be a potential candidate drug for cancer treatment. We explored its effects on the elimination of oral CSCs both in vitro and in vivo. By using the Hoechst side population (SP) technique, CSCs-like SP cells were isolated from human oral squamous cell carcinoma (OSCC) cell lines, SAS and OECM-1. Effects of honokiol on the apoptosis and signaling pathways of SP-derived spheres were examined by Annexin V/Propidium iodide staining and Western blotting, respectively. The in vivo effectiveness was examined by xenograft mouse model and immunohistochemical tissue staining. The SP cells possessed higher stemness marker expression (ABCG2, Ep-CAM, Oct-4 and Nestin), clonogenicity, sphere formation capacity as well as tumorigenicity when compared to the parental cells. Treatment of these SP-derived spheres with honokiol resulted in apoptosis induction via Bax/Bcl-2 and caspase-3-dependent pathway. This apoptosis induction was associated with marked suppression of JAK2/STAT3, Akt and Erk signaling pathways in honokiol-treated SAS spheres. Consistent with its effect on JAK2/STAT3 suppression, honokiol also markedly inhibited IL-6-mediated migration of SAS cells. Accordingly, honokiol dose-dependently inhibited the growth of SAS SP xenograft and markedly reduced the immunohistochemical staining of PCNA and endothelial marker CD31 in the xenograft tumor. Honokiol suppressed the sphere formation and xenograft growth of oral CSC-like cells in association with apoptosis induction and inhibition of survival/proliferation signaling pathways as well as angiogenesis. These results suggest its potential as an integrative medicine for combating oral cancer through targeting on CSCs.

  11. Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential

    PubMed Central

    Sonke, Eric; Verrydt, Megan; Postenka, Carl O.; Pardhan, Siddika; Willie, Chantalle J.; Mazzola, Clarisse R.; Hammers, Matthew D.; Pluth, Michael D.; Lobb, Ian; Power, Nicholas E.; Chambers, Ann F.; Leong, Hon S.; Sener, Alp

    2016-01-01

    Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel–Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease. PMID:26068241

  12. Chlorella sorokiniana induces mitochondrial-mediated apoptosis in human non-small cell lung cancer cells and inhibits xenograft tumor growth in vivo.

    PubMed

    Lin, Ping-Yi; Tsai, Ching-Tsan; Chuang, Wan-Ling; Chao, Ya-Hsuan; Pan, I-Horng; Chen, Yu-Kuo; Lin, Chi-Chen; Wang, Bing-Yen

    2017-02-01

    Lung cancer is one of the leading causes of cancer related deaths worldwide. Marine microalgae are a source of biologically active compounds and are widely consumed as a nutritional supplement in East Asian countries. It has been reported that Chlorella or Chlorella extracts have various beneficial pharmacological compounds that modulate immune responses; however, no studies have investigated the anti-cancer effects of Chlorella sorokiniana (CS) on non-small cell lung cancer (NSCLC). In this study, we evaluated the anti-cancer effects of CS in two human NSCLC cell lines (A549 and CL1-5 human lung adenocarcinoma cells), and its effects on tumor growth in a subcutaneous xenograft tumor model. We also investigated the possible molecular mechanisms governing the pharmacological function of CS. Our results showed that exposure of the two cell lines to CS resulted in a concentration-dependent reduction in cell viability. In addition, the percentage of apoptotic cells increased in a dose-dependent manner, suggesting that CS might induce apoptosis in human NSCLC cells. Western blot analysis revealed that exposure to CS resulted in increased protein expression of the cleaved/activated forms of caspase-3, caspase-9, and PARP, except caspase-8. ZDEVD (caspase-3 inhibitor) and Z-LEHD (caspase-9 inhibitor) were sufficient at preventing apoptosis in both A549 and CL1-5 cells, proving that CS induced cell death via the mitochondria-mediated apoptotic pathway. Exposure of A549 and CL1-5 cells to CS for 24 h resulted in decreased expression of Bcl-2 protein and increased expression of Bax protein as well as decreased expression of two IAP family proteins, survivin and XIAP. We demonstrated that CS induces mitochondrial-mediated apoptosis in NSCLC cells via downregulation of Bcl-2, XIAP and survivin. In addition, we also found that the tumors growth of subcutaneous xenograft in vivo was markedly inhibited after oral intake of CS.

  13. A triple combination of atorvastatin, celecoxib and tipifarnib strongly inhibits pancreatic cancer cells and xenograft pancreatic tumors.

    PubMed

    Ding, Ning; Cui, Xiao-Xing; Gao, Zhi; Huang, Huarong; Wei, Xingchuan; Du, Zhiyun; Lin, Yong; Shih, Weichung Joe; Rabson, Arnold B; Conney, Allan H; Hu, Chunhong; Zheng, Xi

    2014-06-01

    Because K-Ras mutation and cyclooxygenase-2 (COX-2) overexpression are hallmarks of majority of pancreatic cancer patients, an approach to inhibit the progression and growth of pancreatic cancer using the simultaneous administration of agents that inhibit the function of both targets, should be considered. In the present study, we assessed the effects of atorvastatin (Lipitor), celecoxib (Celebrex) and tipifarnib (Zarnestra) on the growth of human pancreatic cancer. In the in vitro studies, we found that treatment of human pancreatic tumor cells with a combination of atorvastatin, celecoxib and tipifarnib had a stronger inhibitory effect on growth and a stronger stimulatory effect on apoptosis than each drug alone or for any combination of two drugs. We also found that treatment of Panc-1 cells with a combination of all three drugs strongly decreased the levels of phosphorylated Erk1/2 and Akt. In an animal model of xenograft tumors in severe combined immunodeficient (SCID) mice, we found that daily i.p. injections of a combination of atorvastatin, celecoxib and tipifarnib had a stronger inhibitory effect on the growth of the tumors in mice than each drug alone or for any combination of two drugs. The results of our study indicate that a combination of atorvastatin, celecoxib and tipifarnib may be an effective strategy for the treatment of pancreatic cancer.

  14. Patient-Derived Xenograft Models of Non-Small Cell Lung Cancer and Their Potential Utility in Personalized Medicine.

    PubMed

    Morgan, Katherine M; Riedlinger, Gregory M; Rosenfeld, Jeffrey; Ganesan, Shridar; Pine, Sharon R

    2017-01-01

    Traditional preclinical studies of cancer therapeutics have relied on the use of established human cell lines that have been adapted to grow in the laboratory and, therefore, may deviate from the cancer they were meant to represent. With the emphasis of cancer drug development shifting from non-specific cytotoxic agents to rationally designed molecularly targeted therapies or immunotherapy comes the need for better models with predictive value regarding therapeutic activity and response in clinical trials. Recently, the diversity and accessibility of immunodeficient mouse strains has greatly enhanced the production and utility of patient-derived xenograft (PDX) models for many tumor types, including non-small cell lung cancer (NSCLC). Combined with next-generation sequencing, NSCLC PDX mouse models offer an exciting tool for drug development and for studying targeted therapies while utilizing patient samples with the hope of eventually aiding in clinical decision-making. Here, we describe NSCLC PDX mouse models generated by us and others, their ability to reflect the parental tumors' histomorphological characteristics, as well as the effect of clonal selection and evolution on maintaining genomic integrity in low-passage PDXs compared to the donor tissue. We also raise vital questions regarding the practical utility of PDX and humanized PDX models in predicting patient response to therapy and make recommendations for addressing those questions. Once collaborations and standardized xenotransplantation and data management methods are established, NSCLC PDX mouse models have the potential to be universal and invaluable as a preclinical tool that guides clinical trials and standard therapeutic decisions.

  15. Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction in EGFR, pEGFR, and pSrc.

    PubMed

    Jedlinski, Adam; Garvin, Stina; Johansson, Ann-Charlotte; Edqvist, Per-Henrik; Ponten, Fredrik; Roberg, Karin

    2017-10-01

    The aims of this study were to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC) cell lines in an in vivo xenograft model and to identify treatment-induced changes in the epidermal growth factor receptor (EGFR) signaling pathway that could be used as markers for cetuximab treatment response. The in vitro and in vivo cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UT-SCC-45, was assessed using a crystal violet assay and xenografts in nude mice, respectively. The expression of EGFR, phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki-67 was investigated by immunohistochemistry. To verify these results, the in vitro expression of EGFR and pEGFR was analyzed with ELISA in a panel of 10 HNSCC cell lines. A close correlation was found between in vitro and in vivo cetuximab sensitivity data in the two investigated HNSCC cell lines. In treatment sensitive UT-SCC-14 xenografts, there was a decrease in EGFR, pEGFR, and pSrc upon cetuximab treatment. Interestingly, in insensitive UT-SCC-45 xenografts, an increased expression of these three proteins was found. The change in EGFR and pEGFR expression in vivo was confirmed in cetuximab-sensitive and cetuximab-insensitive HNSCC cell lines using ELISA. High sensitivity to cetuximab was strongly associated with a treatment-induced reduction in pEGFR both in vivo and in vitro in a panel of HNSCC cell lines, suggesting that EGFR and pEGFR dynamics could be used as a predictive biomarker for cetuximab treatment response. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Label-free quantitative proteomic analysis of benzo(a)pyrene-transformed 16HBE cells serum-free culture supernatant and xenografted nude mice sera.

    PubMed

    Zhao, Peng; Fu, Juanling; Yao, Biyun; Jia, Yongrui; Zhang, Hongtao; Li, Xuehui; Dong, Lisha; Gao, Ya; Liu, Wenli; Chen, Wen; Zhou, Zongcan

    2016-02-05

    To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC-MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis.

  17. Growth patterns and cell kinetics of human osteosarcoma xenografts in serial passages in nude mice analyzed by in vivo labelling with iododeoxyuridine.

    PubMed

    Broström, L A; Crnalic, S; Löfvenberg, R; Boquist, L; Stenling, R

    1996-01-01

    A human osteoblastic osteosarcoma was transplanted in nude mice and followed in seven serial passages. Tumor cell kinetics was analyzed by in vivo labelling with the thymidine analogue iododeoxyuridine (IdUrd). Immunohistochemistry was used to measure the IdUrd labelling index. Duration of S phase (ts) was estimated by flow cytometry. From these two parameters potential doubling time (tpot) was calculated. Cell kinetic parameters showed low variations between passages and also between xenografts in same passage. Smaller variations of ts compared to labelling index and tpot were found. tpot was generally short with an interpassageal mean of 1.3 days and CV=14.8%. All xenografts showed DNA aneuploidy (mean DNA index --1.6). Homogeneous tumor growth was indicated by low variations of volume doubling time and lag time. There was no correlation between tumor growth and cell proliferation. Histopathological characteristics of the donor patients tumor were retained during serial transplantation.

  18. Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models.

    PubMed

    Wang, Hui Qin; Halilovic, Ensar; Li, Xiaoyan; Liang, Jinsheng; Cao, Yichen; Rakiec, Daniel P; Ruddy, David A; Jeay, Sebastien; Wuerthner, Jens U; Timple, Noelito; Kasibhatla, Shailaja; Li, Nanxin; Williams, Juliet A; Sellers, William R; Huang, Alan; Li, Fang

    2017-04-20

    The efficacy of ALK inhibitors in patients with ALK-mutant neuroblastoma is limited, highlighting the need to improve their effectiveness in these patients. To this end, we sought to develop a combination strategy to enhance the antitumor activity of ALK inhibitor monotherapy in human neuroblastoma cell lines and xenograft models expressing activated ALK. Herein, we report that combined inhibition of ALK and MDM2 induced a complementary set of anti-proliferative and pro-apoptotic proteins. Consequently, this combination treatment synergistically inhibited proliferation of TP53 wild-type neuroblastoma cells harboring ALK amplification or mutations in vitro, and resulted in complete and durable responses in neuroblastoma xenografts derived from these cells. We further demonstrate that concurrent inhibition of MDM2 and ALK was able to overcome ceritinib resistance conferred by MYCN upregulation in vitro and in vivo. Together, combined inhibition of ALK and MDM2 may provide an effective treatment for TP53 wild-type neuroblastoma with ALK aberrations.

  19. Visualizing Human Hematopoietic Stem Cell Trafficking In Vivo Using a Zebrafish Xenograft Model.

    PubMed

    Staal, Frank J T; Spaink, Herman P; Fibbe, Willem E

    2016-02-15

    Zebrafish is gaining increased popularity as a model organism to study stem cell biology. It also is widely used as model system to visualize human leukemic stem cells. However, xenotransplantation of primary human stem/progenitor cells has not been described. Here, we use casper pigmentation mutant fish that are transparent crossed to fli-GFP transgenic fish as recipients of red labeled human CD34(+) cells. We have investigated various conditions and protocols with the aim to monitor and visualize the fate of transplanted human CD34(+) cells. We here report successful use of casper mutant zebrafish embryos for the direct monitoring of human hematopoietic stem cell transplantation, differentiation, and trafficking in vivo.

  20. Anti-JAM-C therapy eliminates tumor engraftment in a xenograft model of mantle cell lymphoma.

    PubMed

    Doñate, Carmen; Vijaya Kumar, Archana; Imhof, Beat A; Matthes, Thomas

    2016-11-01

    Junctional adhesion molecule (JAM)-C is a member of the JAM family, expressed by a variety of different cell types, including human B lymphocytes and some B-cell lymphoma subtypes-in particular, mantle cell lymphoma (MCL). Treatment with anti-JAM-C pAbs reduces homing of human B cells to lymphoid organs in a NOD/SCID mouse model. In the present study, the role of JAM-C in the engraftment of human lymphoma B cells in mice was investigated. Administration of novel anti-JAM-C mAbs reduced tumor growth of JAM-C(+) MCL cells in bone marrow, spleen, liver, and lymph nodes of mice. Treatment with anti-JAM-C antibodies significantly reduced the proliferation of JAM-C-expressing lymphoma B cells. Moreover, the binding of anti-JAM-C antibodies inhibited the phosphorylation of ERK1/2, without affecting other signaling pathways. The results identify for the first time the intracellular MAPK cascade as the JAM-C-driven signaling pathway in JAM-C(+) B cells. Targeting JAM-C could constitute a new therapeutic strategy reducing lymphoma B-cell proliferation and their capacity to reach supportive lymphoid microenvironments.

  1. Reversible epithelial to mesenchymal transition and acquired resistance to sunitinib in patients with renal cell carcinoma: evidence from a xenograft study

    PubMed Central

    Hammers, Hans J; Verheul, Henk M; Salumbides, Brenda; Sharma, Rajni; Rudek, Michelle; Jaspers, Janneke; Shah, Preeti; Ellis, Leigh; Shen, Li; Paesante, Silvia; Dykema, Karl; Furge, Kyle; Teh, Bin T; Netto, George; Pili, Roberto

    2011-01-01

    Tyrosine kinase inhibitors (TKIs) targeting angiogenesis via inhibition of the vascular endothelial growth factor (VEGF) pathway have changed the medical management of metastatic renal cell carcinoma. While the treatment with TKIs has demonstrated clinical benefit these drugs will eventually fail patients. The potential mechanisms of resistance to TKIs are poorly understood. To address this question we obtained an excisional biopsy of a skin metastasis from a patient with clear cell renal carcinoma who initially had a response on sunitinib and eventually progressed on therapy. Tumor pieces were grafted subcutaneously in athymic nude mice. Established xenografts were treated with sunitinib. Tumor size, microvascular density and pericyte coverage were determined. Plasma as well as tissue levels for sunitinib were assessed. A tumor derived cell line was established and assessed in vitro for a potential direct antitumor effects of sunitinib. To our surprise, xenografts from the patient who progressed on sunitinib regained sensitivity to the drug. At a dose of 40 mg/kg sunitinib caused regression of the subcutaneous tumors. Histology showed a marked reduction in microvascular density and pericyte dysfunction. More interestingly, histological examination of the original skin metastasis revealed evidence of epithelial-to-mesenchymal-transition while the xenografts showed reversion to the clear cell phenotype. In vitro studies showed no inhibitory effect on tumor cell growth at pharmacologically relevant concentrations. In conclusion, the histological examination in this xenograft study suggests that reversible epithelial-to mesenchymal-transition may be associated with acquired tumor resistance to TKIs in patients with clear cell renal carcinoma. PMID:20501804

  2. Effect of fixation time on breast biomarker expression: a controlled study using cell line-derived xenografted (CDX) tumours.

    PubMed

    Kao, K R; Hasan, T; Baptista, A; Truong, T; Gai, L; Smith, A C; Li, S; Gonzales, P; Voisey, K; Erivwo, P; Power, J; Denic, N

    2017-03-23

    Altering the length of time specimens are placed in fixative without compromising analytical testing accuracy is a continuous challenge in the anatomical pathology lab. The aim of this study was to determine under controlled conditions the effects of variable fixation time on breast biomarker expression in human breast cancer cell line-derived xenografted (CDX) tumours. CDX tumours using strong oestrogen receptor (ER)-positive, Her2-negative (MCF7) and weak ER-positive, Her2 equivocal (T47D) breast cancer cell lines were fixed for various times ranging from 1 to 336 hours in 10% neutral buffered formalin. CDX tumours were processed according to routine biomarker testing protocols and stained for ER and Her2 immunohistochemistry (IHC) and processed for HER2 fluorescence in situ hybridisation (FISH). The tumours were evaluated using Allred scoring for ER and current ASCO/CAP guidelines for Her2, and by objective cell counting methodology. No differences were found in expression of ER in either MCF7 or T47D CDX tumours under variable fixation. T47D tumours displayed variably equivocal Her2 staining when fixed for 24 hours, but fixation for ≤8 hours resulted in consistently negative staining while tumours fixed for >72 hours demonstrated consistent equivocal staining (p<0.01). Cell counting assays revealed only a significant increase in sensitivity in tumours fixed for >72 hours (p<0.01). As expected, FISH results were unaffected by variable fixation. Neither shortened nor prolonged fixation affects ER expression, consistent with previous findings. In equivocal Her2-expressing tumours, however, increasing fixation increased the sensitivity of Her2 IHC reporting while not affecting FISH. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  3. Scintigraphic depiction of an insulinoma by I-131 metaiodobenzylguanidine

    SciTech Connect

    Geatti, O.; Shapiro, B.; Barillari, B. )

    1989-12-01

    Scintigraphy with I-131 metaiodobenzylguanidine (MIBG) was effective in depicting a pancreatic insulinoma in a patient suffering from intermittent hypoglycemia. This observation widens the range of neuroendocrine tumors that take up to I-131 MIBG and supports the concept that many tumors of the amine precursor uptake and decarboxylation system may be imaged in this way.

  4. Human Milk Protein Production in Xenografts of Genetically Engineered Bovine Mammary Epithelial Stem Cells

    PubMed Central

    Martignani, Eugenio; Eirew, Peter; Accornero, Paolo

    2010-01-01

    Background In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer. Methods and Findings We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo. Conclusions These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial

  5. Gene mutations in primary tumors and corresponding patient-derived xenografts derived from non-small cell lung cancer.

    PubMed

    Hao, Chuncheng; Wang, Li; Peng, Shaohua; Cao, Mengru; Li, Hongyu; Hu, Jing; Huang, Xiao; Liu, Wei; Zhang, Hui; Wu, Shuhong; Pataer, Apar; Heymach, John V; Eterovic, Agda Karina; Zhang, Qingxiu; Shaw, Kenna R; Chen, Ken; Futreal, Andrew; Wang, Michael; Hofstetter, Wayne; Mehran, Reza; Rice, David; Roth, Jack A; Sepesi, Boris; Swisher, Stephen G; Vaporciyan, Ara; Walsh, Garrett L; Johnson, Faye M; Fang, Bingliang

    2015-02-01

    Molecular annotated patient-derived xenograft (PDX) models are useful for the preclinical investigation of anticancer drugs and individualized anticancer therapy. We established 23 PDXs from 88 surgical specimens of lung cancer patients and determined gene mutations in these PDXs and their paired primary tumors by ultradeep exome sequencing on 202 cancer-related genes. The numbers of primary tumors with deleterious mutations in TP53, KRAS, PI3KCA, ALK, STK11, and EGFR were 43.5%, 21.7%, 17.4%, 17.4%, 13.0%, and 8.7%, respectively. Other genes with deleterious mutations in ≥3 (13.0%) primary tumors were MLL3, SETD2, ATM, ARID1A, CRIPAK, HGF, BAI3, EP300, KDR, PDGRRA and RUNX1. Of 315 mutations detected in the primary tumors, 293 (93%) were also detected in their corresponding PDXs, indicating that PDXs have the capacity to recapitulate the mutations in primary tumors. Nevertheless, a substantial number of mutations had higher allele frequencies in the PDXs than in the primary tumors, or were not detectable in the primary tumor, suggesting the possibility of tumor cell enrichment in PDXs or heterogeneity in the primary tumors. The molecularly annotated PDXs generated from this study could be useful for future translational studies. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Nicotine induces resistance to erlotinib via cross-talk between α 1 nAChR and EGFR in the non-small cell lung cancer xenograft model.

    PubMed

    Li, Heyan; Wang, Shuo; Takayama, Koichi; Harada, Taishi; Okamoto, Isamu; Iwama, Eiji; Fujii, Akiko; Ota, Keiichi; Hidaka, Noriko; Kawano, Yuko; Nakanishi, Yoichi

    2015-04-01

    Given our previously published study, α 1 nicotinic acetylcholine receptor (nAChR) plays an essential role in nicotine-induced cell signaling and nicotine-induced resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in non-small cell lung cancer (NSCLC) PC9 cells. The aim of this study was to investigate the potential mechanism between nAChR and EGFR for nicotine-induced resistance to EGFR-TKI erlotinib in the NSCLC xenograft model. We identified the role of nicotine to EGFR/AKT/ERK pathways and to erlotinib-resistance in NSCLC PC9 and HCC827 cells by MTS assay and western blot. Then, we established the PC9 xenograft model with nicotine exposure and treated mice with erlotinib combined with vehicle or nicotine. We confirmed the effects of nicotine on EGFR/AKT/ERK pathways and determined nicotine's potential in preventing from the effect of erlotinib on NSCLC cells. Then, we showed that nicotine exposures can promote tumor growth and induce resistance to erlotinib in the PC9 xenograft model. Our results also indicated that chronic oral administration of nicotine can cause more significant erlotinib-resistance compared with acute i.v. injection of nicotine through activating α 1 nAChR and EGFR pathways. These results suggest that nicotine contributes to the progression and erlotinib-resistance of the NSCLC xenograft model via the cooperation between nAChR and EGFR. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  7. Cisplatin Increases Sensitivity to FGFR Inhibition in Patient-Derived Xenograft Models of Lung Squamous Cell Carcinoma.

    PubMed

    Weeden, Clare E; Holik, Aliaksei Z; Young, Richard J; Ma, Stephen B; Garnier, Jean-Marc; Fox, Stephen B; Antippa, Phillip; Irving, Louis B; Steinfort, Daniel P; Wright, Gavin M; Russell, Prudence A; Ritchie, Matthew E; Burns, Christopher J; Solomon, Benjamin; Asselin-Labat, Marie-Liesse

    2017-08-01

    Lung squamous cell carcinoma (SqCC) is a molecularly complex and genomically unstable disease. No targeted therapy is currently approved for lung SqCC, although potential oncogenic drivers of SqCC have been identified, including amplification of the fibroblast growth factor receptor 1 (FGFR1). Reports from a recently completed clinical trial indicate low response rates in patients treated with FGFR tyrosine kinase inhibitors, suggesting inadequacy of FGFR1 amplification as a biomarker of response, or the need for combination treatment. We aimed to develop accurate models of lung SqCC and determine improved targeted therapies for these tumors. We show that detection of FGFR1 mRNA by RNA in situ hybridization is a better predictor of response to FGFR inhibition than FGFR1 gene amplification using clinically relevant patient-derived xenograft (PDX) models of lung SqCC. FGFR1-overexpressing tumors were observed in all histologic subtypes of non-small cell lung cancers (NSCLC) as assessed on a tissue microarray, indicating a broader range of tumors that may respond to FGFR inhibitors. In FGFR1-overexpressing PDX tumors, we observed increased differentiation and reduced proliferation following FGFR inhibition. Combination therapy with cisplatin was able to increase tumor cell death, and dramatically prolonged animal survival compared to single-agent treatment. Our data suggest that FGFR tyrosine kinase inhibitors can benefit NSCLC patients with FGFR1-overexpressing tumors and provides a rationale for clinical trials combining cisplatin with FGFR inhibitors. Mol Cancer Ther; 16(8); 1610-22. ©2017 AACR. ©2017 American Association for Cancer Research.

  8. Effect of Citrus bergamia juice on human neuroblastoma cells in vitro and in metastatic xenograft models.

    PubMed

    Navarra, M; Ursino, M R; Ferlazzo, N; Russo, M; Schumacher, U; Valentiner, U

    2014-06-01

    Neuroblastoma is the most common extracranial pediatric solid tumor with poor prognosis in children with disseminated stage of disease. A number of studies show that molecules largely distributed in commonly consumed fruits and vegetables may have anti-tumor activity. In this study we evaluate the effect of Citrus bergamia (bergamot) juice (BJ) in vitro and in a spontaneous metastatic neuroblastoma SCID mouse model. Qualitative and quantitative characterizations of BJ flavonoid fractions were performed by RP-HPLC/PDA/MS. We show that BJ significantly affects SK-N-SH and LAN-1 cell proliferation in vitro, but fails to reduce primary tumor weight in vivo. Moreover, BJ reduced cell adhesiveness and invasion of LAN-1 and SK-N-SH cells in vitro and the number of pulmonary metastases under consideration of the number of tumor cells in the blood in mice inoculated with LAN-1 cells in vivo. These effects without any apparent sign of systemic toxicity confirm the potential clinical interest of BJ and lay the basis for further investigation in cancer. Copyright © 2014. Published by Elsevier B.V.

  9. MicroRNA-627 Mediates the Epigenetic Mechanisms of Vitamin D to Suppress Proliferation of Human Colorectal Cancer Cells and Growth of Xenograft Tumors in Mice

    PubMed Central

    Padi, Sathish K.R.; Zhang, Qunshu; Rustum, Youcef M; Morrison, Carl; Guo, Bin

    2013-01-01

    Background & Aims Vitamin D protects against colorectal cancer by unclear mechanisms. We investigated the effects of calcitriol (1α,25-dihydroxyvitamin D3, the active form of vitamin D) on levels of different microRNAs (miRs) in colorectal cancer (CRC) cells from humans and xenograft tumors in mice. Methods Expression of microRNAs in CRC cell lines was examined using the Ambion mirVana miRNA Bioarray. The effects of calcitriol on expression of miR-627 and cell proliferation were determined by real-time PCR and WST-1 assay, respectively; growth of colorectal xenograft tumors was examined in nude mice. Real-time PCR was used to analyze levels of miR-627 in human colon adenocarcinoma samples and non-tumor colon mucosa tissues (controls). Results In HT-29 cells, miR-627 was the only microRNA significantly upregulated by calcitriol. Jumonji domain containing 1A (JMJD1A), which encodes a histone demethylase, was found to be a target of miR-627. By downregulating JMJD1A, miR-627 increased methylation of histone H3K9 and suppressed expression of proliferative factors such as GDF15. Calcitriol induced expression of miR-627, which downregulated JMJD1A and suppressed growth of xenograft tumors from HCT-116 cells in nude mice. Overexpression of miR-627 prevented proliferation of CRC cell lines in culture and growth of xenograft tumors in mice. Conversely, blocking the activity of miR-627 inhibited the tumor suppressive effects of calcitriol in cultured CRC cells and in mice. Levels of miR-627 were decreased in human colon adenocarcinoma samples, compared with controls. Conclusions miR-627 mediates tumor-suppressive epigenetic activities of vitamin D on CRC cells and xenograft tumors in mice. The mRNA that encodes the histone demethylase JMJD1A is a direct target of miR-627. Reagents designed to target JMJD1A or its mRNA, or increase the function of miR-627, might have the same antitumor activities of vitamin D without the hypercalcemic side effects. PMID:23619147

  10. Human intestinal epithelial cells produce proinflammatory cytokines in response to infection in a SCID mouse-human intestinal xenograft model of amebiasis.

    PubMed Central

    Seydel, K B; Li, E; Swanson, P E; Stanley, S L

    1997-01-01

    The protozoan parasite Entamoeba histolytica causes amebic dysentery and amebic liver abscess, diseases associated with significant morbidity and mortality worldwide. E. histolytica infection appears to involve the initial attachment of amebic trophozoites to intestinal epithelial cells, followed by lysis of these cells and subsequent invasion into the submucosa. A recent in vitro study (L. Eckmann, S. L. Reed, J. R. Smith, and M. F. Kagnoff, J. Clin. Invest. 96:1269-1279, 1995) demonstrated that incubation of E. histolytica trophozoites with epithelial cell lines results in epithelial cell production of inflammatory cytokines, including interleukin-1 (IL-1) and IL-8, suggesting that intestinal epithelial cell production of cytokines might play a role in the inflammatory response and tissue damage seen in intestinal amebiasis. To determine whether intestinal epithelial cell production of IL-1 and IL-8 occurs in response to E. histolytica infection in vivo and as an approach to studying the specific interactions between amebic trophozoites and human intestine, we used a SCID mouse-human intestinal xenograft (SCID-HU-INT) model of disease, where human intestinal xenografts were infected with virulent E. histolytica trophozoites. Infection of xenografts with E. histolytica trophozoites resulted in extensive tissue damage, which was associated with the development of an early inflammatory response composed primarily of neutrophils. Using oligonucleotide primers that specifically amplify human IL-1beta and IL-8, we could demonstrate by reverse transcription PCR that mRNA for both IL-1beta and IL-8 is produced by human intestinal xenografts in response to amebic infection. The increase in human intestinal IL-1beta and IL-8 in response to invasive amebiasis was confirmed by enzyme-linked immunosorbent assays specific for human IL-1beta and IL-8. Using immunohistochemistry, we confirmed that human intestinal epithelial cells were the source of IL-8 in infected xenografts

  11. Effect of dietary selenium on T cell immunity and cancer xenograft in nude mice

    USDA-ARS?s Scientific Manuscript database

    Selenium is known to regulate carcinogenesis and immunity at nutritional and supranutritional levels. Because the immune system provides one of the main body defenses against cancer, we asked whether T cell immunity can modulate selenium chemoprevention. Twenty-four homozygous NU/J nude mice were fe...

  12. Photodynamic therapy of human squamous cell carcinoma in vitro and in xenografts in nude mice.

    PubMed

    Megerian, C A; Zaidi, S I; Sprecher, R C; Setrakian, S; Stepnick, D W; Oleinick, N L; Mukhtar, H

    1993-09-01

    Photodynamic therapy (PDT) of cancer is an experimental tumor therapy which is based on the combined use of a systematically administered photosensitizer to a tumor-bearing host and local illumination of the lesion by a high-intensity visible light source, typically a tunable argon dye laser. Human squamous cell carcinoma (HSCC) is the most frequently encountered malignancy of the head and neck. In this study, responses of HSCC cells to PDT were examined in in vitro and in vivo systems. In in vitro studies, the HSCC cells showed a positive photodynamic response with Photofrin-II (Pf-II), chloroaluminum phthalocyanine tetrasulfonate (AlPcTS), and a newly synthesized silicon phthalocyanine (SiPc IV). Single cell suspension of HSCC injected subcutaneously on the back of athymic nude mice resulted in a well-circumscribed tumor mass. The animals required a low tumor dose for the successful establishment of a tumor. The tumor was minimally immunogenic and showed neither macroscopic signs of early metastasis to lung, kidney, liver, or spleen nor evidence of surrounding erythema, fluctuation, or tenderness until the late stages of necrosis. Intraperitoneal administration of AlPcTS or SiPc IV to tumor-bearing mice resulted in rapid uptake of the photosensitizers in liver, skin, and tumor tissue. Twenty-four hours following the intraperitoneal administration of AlPcTS or SiPc IV to tumor-bearing animals, the tumor to normal skin ratio of the photosensitizer was 1.6 or 1.5, respectively. Administration of Pf-II (5 mg/kg) to tumor-bearing animals followed 24 hours later by irradiation of the tumor (135 J/cm2, 630 nm light from an argon pumped-dye laser) resulted in greater than 80% ablation in tumor volume 24 hours post-PDT. These characteristics make this tumor model system suitable for PDT studies of human tumor cells in vitro as well as in vivo.

  13. The MET Inhibitor AZD6094 (Savolitinib, HMPL-504) Induces Regression in Papillary Renal Cell Carcinoma Patient-Derived Xenograft Models.

    PubMed

    Schuller, Alwin G; Barry, Evan R; Jones, Rhys D O; Henry, Ryan E; Frigault, Melanie M; Beran, Garry; Linsenmayer, David; Hattersley, Maureen; Smith, Aaron; Wilson, Joanne; Cairo, Stefano; Déas, Olivier; Nicolle, Delphine; Adam, Ammar; Zinda, Michael; Reimer, Corinne; Fawell, Stephen E; Clark, Edwin A; D'Cruz, Celina M

    2015-06-15

    Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with nonlocalized disease. The HGF receptor MET plays a central role in PRCC and aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 occurring in the majority of cases. The development of effective therapies in PRCC has been hampered in part by a lack of available preclinical models. We determined the pharmacodynamic and antitumor response of the selective MET inhibitor AZD6094 in two PRCC patient-derived xenograft (PDX) models. Two PRCC PDX models were identified and MET mutation status and copy number determined. Pharmacodynamic and antitumor activity of AZD6094 was tested using a dose response up to 25 mg/kg daily, representing clinically achievable exposures, and compared with the activity of the RCC standard-of-care sunitinib (in RCC43b) or the multikinase inhibitor crizotinib (in RCC47). AZD6094 treatment resulted in tumor regressions, whereas sunitinib or crizotinib resulted in unsustained growth inhibition. Pharmacodynamic analysis of tumors revealed that AZD6094 could robustly suppress pMET and the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes, including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose- and time-dependent induction of cleaved PARP, a marker of cell death. Data presented provide the first report testing therapeutics in preclinical in vivo models of PRCC and support the clinical development of AZD6094 in this indication. ©2015 American Association for Cancer Research.

  14. Patient-Derived Xenograft Models of Non-Small Cell Lung Cancer and Their Potential Utility in Personalized Medicine

    PubMed Central

    Morgan, Katherine M.; Riedlinger, Gregory M.; Rosenfeld, Jeffrey; Ganesan, Shridar; Pine, Sharon R.

    2017-01-01

    Traditional preclinical studies of cancer therapeutics have relied on the use of established human cell lines that have been adapted to grow in the laboratory and, therefore, may deviate from the cancer they were meant to represent. With the emphasis of cancer drug development shifting from non-specific cytotoxic agents to rationally designed molecularly targeted therapies or immunotherapy comes the need for better models with predictive value regarding therapeutic activity and response in clinical trials. Recently, the diversity and accessibility of immunodeficient mouse strains has greatly enhanced the production and utility of patient-derived xenograft (PDX) models for many tumor types, including non-small cell lung cancer (NSCLC). Combined with next-generation sequencing, NSCLC PDX mouse models offer an exciting tool for drug development and for studying targeted therapies while utilizing patient samples with the hope of eventually aiding in clinical decision-making. Here, we describe NSCLC PDX mouse models generated by us and others, their ability to reflect the parental tumors’ histomorphological characteristics, as well as the effect of clonal selection and evolution on maintaining genomic integrity in low-passage PDXs compared to the donor tissue. We also raise vital questions regarding the practical utility of PDX and humanized PDX models in predicting patient response to therapy and make recommendations for addressing those questions. Once collaborations and standardized xenotransplantation and data management methods are established, NSCLC PDX mouse models have the potential to be universal and invaluable as a preclinical tool that guides clinical trials and standard therapeutic decisions. PMID:28154808

  15. Ectopic duodenal insulinoma: a very rare and challenging tumor type. Description of a case and review of the literature.

    PubMed

    La Rosa, Stefano; Pariani, Dario; Calandra, Calogera; Marando, Alessandro; Sessa, Fausto; Cortese, Ferdinando; Capella, Carlo

    2013-12-01

    Although most insulinomas are located in the pancreas, very rare ectopic cases have been described in the spleen, perisplenic tissue, duodenohepatic ligament, and adjacent to the ligament of Treitz. Moreover, three cases located in the duodenum have also been reported in the English literature. Ectopic insulinomas represent challenging neoplasms with clinical implications mainly due to the difficulties in their pre-operatory diagnosis and localization. In the present paper, we describe the fourth ectopic duodenal insulinoma so far reported. A 75-year-old woman presented at clinical observation due to neuroglycopenic symptoms that disappeared after glucose intake. Tumor was localized in the second portion of the duodenum in front of the papilla of Vater and was surgically enucleated. Microscopically, it was composed of monomorphic cells with eosinophilic cytoplasm arranged in trabecular and lobular patterns and diffusely positive for insulin, proinsulin, amylin, and PDX1. About 30 % of tumor cells also showed immunoreactivity for somatostatin, while no positivity for glucagon, pancreatic polypeptide, gastrin, serotonin, and somatostatin receptor subtype 2A was found. The Ki67 proliferative index was 1 %. We have also reviewed the literature on this topic to give the reader a comprehensive overview of this very rare tumor type.

  16. Highly efficient IL-21 and feeder cell-driven ex vivo expansion of human NK cells with therapeutic activity in a xenograft mouse model of melanoma.

    PubMed

    Granzin, Markus; Stojanovic, Ana; Miller, Matthias; Childs, Richard; Huppert, Volker; Cerwenka, Adelheid

    2016-01-01

    Natural killer (NK) cells are promising antitumor effector cells, but the generation of sufficient NK cell numbers for adoptive immunotherapy remains challenging. Therefore, we developed a method for highly efficient ex vivo expansion of human NK cells. Ex vivo expansion of NK cells in medium containing IL-2 and irradiated clinical-grade feeder cells (EBV-LCL) induced a 22-fold NK cell expansion after one week that was significantly increased to 53-fold by IL-21. Repeated stimulation with irradiated EBV-LCL and IL-2 and addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with 10(11)-fold NK cell expansion after 6 weeks. Compared to naive NK cells, expanded NK cells upregulated TRAIL, NKG2D, and DNAM-1, had superior cytotoxicity against tumor cell lines in vitro and produced more IFNγ and TNF-α upon PMA/Iono stimulation. Most importantly, adoptive transfer of NK cells expanded using feeder cells, IL-2 and IL-21 led to significant inhibition of tumor growth in a melanoma xenograft mouse model, which was greater than with NK cells activated with IL-2 alone. Intriguingly, adoptively transferred NK cells maintained their enhanced production of IFNγ and TNF-α upon ex vivo restimulation, although they rapidly lost their capacity to degranulate and mediate tumor cytotoxicity after the in vivo transfer. In conclusion, we developed a protocol for ex vivo NK cell expansion that results in outstanding cell yields. The expanded NK cells possess potent antitumor activity in vitro and in vivo and could be utilized at high numbers for adoptive immunotherapy in the clinic.

  17. Highly efficient IL-21 and feeder cell-driven ex vivo expansion of human NK cells with therapeutic activity in a xenograft mouse model of melanoma

    PubMed Central

    Granzin, Markus; Stojanovic, Ana; Miller, Matthias; Childs, Richard; Huppert, Volker; Cerwenka, Adelheid

    2016-01-01

    ABSTRACT Natural killer (NK) cells are promising antitumor effector cells, but the generation of sufficient NK cell numbers for adoptive immunotherapy remains challenging. Therefore, we developed a method for highly efficient ex vivo expansion of human NK cells. Ex vivo expansion of NK cells in medium containing IL-2 and irradiated clinical-grade feeder cells (EBV-LCL) induced a 22-fold NK cell expansion after one week that was significantly increased to 53-fold by IL-21. Repeated stimulation with irradiated EBV-LCL and IL-2 and addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with 1011-fold NK cell expansion after 6 weeks. Compared to naive NK cells, expanded NK cells upregulated TRAIL, NKG2D, and DNAM-1, had superior cytotoxicity against tumor cell lines in vitro and produced more IFNγ and TNF-α upon PMA/Iono stimulation. Most importantly, adoptive transfer of NK cells expanded using feeder cells, IL-2 and IL-21 led to significant inhibition of tumor growth in a melanoma xenograft mouse model, which was greater than with NK cells activated with IL-2 alone. Intriguingly, adoptively transferred NK cells maintained their enhanced production of IFNγ and TNF-α upon ex vivo restimulation, although they rapidly lost their capacity to degranulate and mediate tumor cytotoxicity after the in vivo transfer. In conclusion, we developed a protocol for ex vivo NK cell expansion that results in outstanding cell yields. The expanded NK cells possess potent antitumor activity in vitro and in vivo and could be utilized at high numbers for adoptive immunotherapy in the clinic. PMID:27757317

  18. Comparative Study Between Mesenchymal Stem Cells Derived from Bone Marrow and from Adipose Tissue, Associated with Xenograft, in Appositional Reconstructions: Histomorphometric Study in Rabbit Calvaria.

    PubMed

    Coelho de Faria, Andrea Baptista; Chiantia, Fernando Biolcati; Teixeira, Marcelo Lucchesi; Aloise, Antonio Carlos; Pelegrine, André Antonio

    This study analyzed the use of bone marrow-derived mesenchymal stem cells and adipose tissue-derived stem cells, associated with xenograft, in appositional reconstructions in rabbit calvaria using histomorphometry. Fifteen New Zealand rabbits, weighing 3.5 to 4.0 kg and aged between 10 and 12 months, were randomly divided into three groups. Appositional bone reconstruction situations were created in the calvaria of the animals using titanium cylinders, fitted with titanium occlusive caps. Bone decortication was performed to promote bleeding. Inside the cylinders, only xenograft was positioned in the control group (CG; n = 5); xenograft combined with mesenchymal bone marrow-derived stem cells was positioned in group 1 (G1; n = 5), and a xenograft combined with adult mesenchymal stem cells derived from adipose tissue was positioned in group 2 (G2; n = 5). After 56 days, all rabbits were euthanized and their parietal bones processed for histomorphometric analysis, and the following parameters were evaluated: newly formed bone; residual graft particles; soft tissue; vital bone titanium contact, also called the level of osseointegration; and the level of bone volume contained inside the cylinders, also called the internal bone volume. The histomorphometric study revealed the following for CG, G1, and G2: newly formed bone of 18.96% ± 9.00%, 27.88% ± 9.98%, and 22.32% ± 7.45%; residual graft particles of 28.43% ± 2.44%, 23.31% ± 3.11%, and 27.58% ± 3.98%; soft tissue of 52.61% ± 10.80%, 50.23% ± 8.72%, and 49.90% ± 8.76%; vital bone titanium contact of 4.98% ± 4.30%, 34.91% ± 7.82%, and 20.87% ± 5.43%; and internal bone volume of 88.36% ± 25.97%, 98.73% ± 19.05%, and 98.52% ± 19.87%, respectively. No statistical difference between groups for newly formed bone, residual graft particles, soft tissue, and internal bone volume (P > .05) were verified. Regarding vital bone titanium contact, it was observed that the use of bone marrow mesenchymal stem cells

  19. Immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts.

    PubMed

    Swijnenburg, Rutger-Jan; Schrepfer, Sonja; Govaert, Johannes A; Cao, Feng; Ransohoff, Katie; Sheikh, Ahmad Y; Haddad, Munif; Connolly, Andrew J; Davis, Mark M; Robbins, Robert C; Wu, Joseph C

    2008-09-02

    Given their self-renewing and pluripotent capabilities, human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However, the host immune response against transplanted hESCs is not well characterized. In fact, controversy remains as to whether hESCs have immune-privileged properties. To address this issue, we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death, suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses, resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover, we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally, we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together, these data suggest that hESCs are immunogenic, trigger both cellular and humoral-mediated pathways, and, as a result, are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches.

  20. Vasculature analysis of patient derived tumor xenografts using species-specific PCR assays: evidence of tumor endothelial cells and atypical VEGFA-VEGFR1/2 signalings

    PubMed Central

    2014-01-01

    Background Tumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types. Methods To characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts. Results As expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated. Conclusions Taken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies. PMID:24625025

  1. p53 mutation and cyclin D1 amplification correlate with cisplatin sensitivity in xenografted human squamous cell carcinomas from head and neck.

    PubMed

    Henriksson, Eva; Baldetorp, Bo; Borg, Ake; Kjellen, Elisabeth; Akervall, Jan; Wennerberg, Johan; Wahlberg, Peter

    2006-01-01

    To investigate the response of tumour growth to cisplatin treatment, in relation to p53 mutation and cyclin D1 dysregulation on DNA and protein level, biopsies from seven xenografted human squamous cell carcinomas from the head and neck were analysed with immunohistochemistry for p53 expression and cyclin D1 expression. Polymerase chain reaction-singlestranded conformation polymorphism was used to determine p53 mutations. Fluorescence in situ hybridization was performed to analyse cyclin D1 amplification. The mice were injected i.p. with NaCl (controls) or cisplatin. After injection the tumour volume were measured. The inhibition of tumour growth by cisplatin was defined as the area under the growth curves, and compared with the growth curves of the tumours in the control group. Xenografts with p53 mutation showed significantly higher resistance to cisplatin (p < 0.001) and also tumours with cyclin D1 amplification showed significantly higher resistance (p < 0.001).

  2. mTORC1 Up-Regulates GP73 to Promote Proliferation and Migration of Hepatocellular Carcinoma Cells and Growth of Xenograft Tumors in Mice.

    PubMed

    Chen, Xinxin; Wang, Yanan; Tao, Jun; Shi, Yuzhuo; Gai, Xiaochen; Huang, Fuqiang; Ma, Qian; Zhou, Zhenzhen; Chen, Hongyu; Zhang, Haihong; Liu, Zhibo; Sun, Qian; Peng, Haiyong; Chen, Rongrong; Jing, Yanling; Yang, Huayu; Mao, Yilei; Zhang, Hongbing

    2015-09-01

    Levels of the Golgi protein 73 (GP73) increase during development of hepatocellular carcinoma (HCC); GP73 is a serum marker for HCC. However, little is known about the mechanisms or effects of GP73 during hepatic carcinogenesis. GP73 was overexpressed from a retroviral vector in HepG2 cells, which were analyzed in proliferation and migration assays. Xenograft tumors were grown from these cells in nude mice. The effects of monoclonal antibodies against GP73 were studied in mice and cell lines. GP73(-/-), GP73(+/-), and GP73(+/+) mice were given injections of diethylnitrosamine to induce liver injury. Levels of GP73 were reduced in MHCC97H, HCCLM3, and HepG2.215 cell lines using small hairpin RNAs; xenograft tumors were grown in mice from MHCC97H-small hairpin GP73 or MHCC97H-vector cells. We used microarray analysis to compare expression patterns between GP73-knockdown and control MHCC97H cells. We studied the effects of the mechanistic target of rapamycin (mTOR) inhibitor rapamycin on GP73 expression in different cancer cell lines and on growth of tumors in mice. Levels of GP73 and activated mTOR were quantified in human HCC tissues. Xenograft tumors grown from HepG2 cells that expressed GP73 formed more rapidly and more metastases than control HepG2 cells in mice. A monoclonal antibody against GP73 reduced proliferation of HepG2 cells and growth of xenograft tumors in mice. GP73(-/-) mice had less liver damage after administration of diethylnitrosamine than GP73(+/-) or GP73(+/+) mice. In phosphatase and tensin homolog-null mouse embryonic fibroblasts with constitutively activated mTOR, GP73 was up-regulated compared with control mouse embryonic fibroblasts; this increase was reversed after incubation with rapamycin. Expression of GP73 also was reduced in HCC and other cancer cell lines incubated with rapamycin. mTORC1 appeared to regulate expression of GP73 in cell lines. Activated mTOR correlated with the level of GP73 in human HCC tissues. Injection of

  3. Expressions of cytochrome P450, UDP-glucuronosyltranferase, and transporter genes in monolayer carcinoma cells change in subcutaneous tumors grown as xenografts in immunodeficient nude mice.

    PubMed

    Sugawara, Michiko; Okamoto, Kiyoshi; Kadowaki, Tadashi; Kusano, Kazutomi; Fukamizu, Akiyoshi; Yoshimura, Tsutomu

    2010-03-01

    Human tumors grown as xenografts in immunodeficient nude mice are widely used to investigate the pharmacological activities of anticancer drugs. Drug-metabolizing enzymes and transporters are expressed in tumor cell lines and changes in drug metabolism and pharmacokinetics (DMPK)-related gene expression after inoculation of the tumor cell may affect the pharmacological activity of the drug under consideration. The aims of the current study were to characterize DMPK-related gene expression profiles and responses to typical cytochrome P450 inducers in monolayer carcinoma cells grown in tissue culture versus those inoculated into a xenograft model. We used the human hepatocellular carcinoma cell line PLC/PRF/5 for this study and comprehensively assessed changes in DMPK-related gene expression by reverse transcription-polymerase chain reaction quantitation. CYP3A4 and UDP-glucuronosyltransferase 1A protein amounts were also analyzed by immunoprecipitation followed by immunoblotting. We found that the expression of many DMPK-related genes was elevated in the inoculated tumor compared with the monolayer carcinoma cells, indicating changes in their gene regulation pathways, presumably due to modulation of the nuclear receptor family of transcription factors. In addition, monolayer carcinoma versus inoculated tumor cells showed different responses to rifampicin, but similar responses to dexamethasone or 3-methylcholanthrene. These results suggest that inoculation of tumor cells results in the activation of drug metabolism and transport function, leading to changes in the responses to pregnane X receptor ligands and consequent discrepancies in the pharmacological activities between in vitro monolayer carcinoma cells and in vivo xenograft models.

  4. Bufalin Inhibits HCT116 Colon Cancer Cells and Its Orthotopic Xenograft Tumor in Mice Model through Genes Related to Apoptotic and PTEN/AKT Pathways

    PubMed Central

    Wang, Jie; Chen, Chao; Wang, Shiying; Zhang, Yong; Yin, Peihao; Gao, Zhongxiang; Xu, Jie; Feng, Dianxu; Zuo, Qinsong; Zhao, Ronghua; Chen, Teng

    2015-01-01

    Aims. To investigate the anticolorectal cancer (CRC) effects of Bufalin, a bioactive polyhydroxysteroid from Venenum Bufonis, using HCT116 human CRC cell and an established orthotopic xenograft model in mice, and to explore the mechanisms of action. Material and Methods. Cultured HCT116 cells or BALB/c mice with orthotopic tumor were treated by Bufalin (positive control: 5-FU). Cell proliferation, apoptosis, and cycling were determined by MTT, Annexin V/PI staining, and flow cytometry, respectively. In mice, tumor inhibition rate and animal survival were calculated. The expressions of PTEN/phosphate-PTEN, AKT/phosphate-AKT, Bad, Bcl-xl, Bax, or Caspase-3 in cells and/or tumors were determined by Western blot or immunohistochemical staining. Results. Bufalin significantly inhibited cell proliferation and induced cell apoptosis and cycle arrest in a dose/time-dependent manner. In the animal model, Bufalin treatment resulted in significant inhibition of tumor growth and prolonged survival. In the Bufalin-treated cultured cells and/or xenograft tumors, the expressions of PTEN, Bad, Bax, and Caspase-3 were significantly increased, while p-AKT and Bcl-xL significantly decreased. Conclusions. Our results indicate that Bufalin inhibit cell proliferation and orthotopic tumor growth by inducing cell apoptosis through the intrinsic apoptotic pathway, which is of pivotal significance in the identification of an anticancer drug that may synergize with Bufalin. PMID:26770191

  5. Apigenin induces apoptosis through mitochondrial dysfunction in U-2 OS human osteosarcoma cells and inhibits osteosarcoma xenograft tumor growth in vivo.

    PubMed

    Lin, Chin-Chung; Chuang, Ya-Ju; Yu, Chien-Chih; Yang, Jai-Sing; Lu, Chi-Cheng; Chiang, Jo-Hua; Lin, Jing-Pin; Tang, Nou-Ying; Huang, An-Cheng; Chung, Jing-Gung

    2012-11-14

    The cytostatic drug from natural products has acted as a chemotherapeutic agent used in treatment of a wide variety of cancers. Apigenin, a type of flavonoid, exhibits anticancer actions, but there is no report to show that apigenin induced apoptosis in osteosarcoma cells. The aim of this study was to investigate the effects of apigenin on U-2 OS human osteosarcoma cells and clarify that the apigenin-induced apoptosis-associated signals. The cytotoxic effects of apigenin were examined by culturing U-2 OS cells with or without apigenin. The percentage of viable cells via PI staining, apoptotic cells, productions of ROS and Ca²⁺, and the level of mitochondrial membrane potential (ΔΨm) were assayed by flow cytometry. The levels of apoptosis-related proteins were measured by immunoblotting. Results indicated that apigenin significantly decreased cell viability. Apigenin effectively induced apoptosis through the activations of caspase-3, -8, -9, and BAX and promoted the release of AIF in U-2 OS cells. In nude mice bearing U-2 OS xenograft tumors, apigenin inhibited tumor growth. In conclusion, apigenin has anticancer properties for induction of cell apoptosis in U-2 OS cells and suppresses the xenograft tumor growth. These findings offer novel information that apigenin possibly possesses anticancer activity in human osteosarcoma.

  6. Loquat (Eriobotrya japonica) leaf extract inhibits the growth of MDA-MB-231 tumors in nude mouse xenografts and invasion of MDA-MB-231 cells

    PubMed Central

    You, Mi-Kyoung; Kim, Min-Sook; Jeong, Kyu-Shik; Kim, Eun; Kim, Yong-Jae

    2016-01-01

    BACKGROUND/OBJECTIVES The present study was conducted to examine the inhibitory effect of loquat leaves on MDA-MB-231 cell proliferation and invasion. MATERIALS/METHODS Female athymic nude mice were given a subcutaneous (s.c.) inoculation of MDA-MB-231 cells and randomly grouped to receive a s.c. injection of either 500 mg/kg ethanol, water extract or vehicle five times a week. Tumor growth, mitotic rate and necrosis were examined. MDA-MB-231 cells were cultured with DMSO or with various concentrations of loquat water or ethanol extract. Proliferation, adhesion, migration, invasion and matrix metalloproteinase (MMP) activity were examined. RESULTS Tumor growth of xenograft nude mouse was significantly reduced by loquat extracts. The results of mitotic examination revealed that loquat extracts reduced tumor cell division. Both ethanol and water extracts significantly inhibited MDA-MB-231 cell proliferation. The protein expression of ErbB3 was significantly down-regulated by loquat leaf extracts. Loquat leaf extracts increased apoptosis of MDA-MB-231 cells following 24 hour incubation and the ethanol extract was more potent in inducing apoptosis than the water extract. Furthermore, loquat extracts inhibited adhesion, migration and invasion of MDA-MB-231 cells. MMP activity was significantly inhibited by loquat extracts. CONCLUSION Our results show that extracts of loquat inhibit the growth of tumor in MDA-MB-231 xenograft nude mice and the invasion of human breast cancer cells, indicating the inhibition of tumor cell proliferation and invasion. PMID:27087896

  7. Loquat (Eriobotrya japonica) leaf extract inhibits the growth of MDA-MB-231 tumors in nude mouse xenografts and invasion of MDA-MB-231 cells.

    PubMed

    You, Mi-Kyoung; Kim, Min-Sook; Jeong, Kyu-Shik; Kim, Eun; Kim, Yong-Jae; Kim, Hyeon-A

    2016-04-01

    The present study was conducted to examine the inhibitory effect of loquat leaves on MDA-MB-231 cell proliferation and invasion. Female athymic nude mice were given a subcutaneous (s.c.) inoculation of MDA-MB-231 cells and randomly grouped to receive a s.c. injection of either 500 mg/kg ethanol, water extract or vehicle five times a week. Tumor growth, mitotic rate and necrosis were examined. MDA-MB-231 cells were cultured with DMSO or with various concentrations of loquat water or ethanol extract. Proliferation, adhesion, migration, invasion and matrix metalloproteinase (MMP) activity were examined. Tumor growth of xenograft nude mouse was significantly reduced by loquat extracts. The results of mitotic examination revealed that loquat extracts reduced tumor cell division. Both ethanol and water extracts significantly inhibited MDA-MB-231 cell proliferation. The protein expression of ErbB3 was significantly down-regulated by loquat leaf extracts. Loquat leaf extracts increased apoptosis of MDA-MB-231 cells following 24 hour incubation and the ethanol extract was more potent in inducing apoptosis than the water extract. Furthermore, loquat extracts inhibited adhesion, migration and invasion of MDA-MB-231 cells. MMP activity was significantly inhibited by loquat extracts. Our results show that extracts of loquat inhibit the growth of tumor in MDA-MB-231 xenograft nude mice and the invasion of human breast cancer cells, indicating the inhibition of tumor cell proliferation and invasion.

  8. Acquired resistance to EGFR tyrosine kinase inhibitors alters the metabolism of human head and neck squamous carcinoma cells and xenograft tumours.

    PubMed

    Beloueche-Babari, M; Box, C; Arunan, V; Parkes, H G; Valenti, M; De Haven Brandon, A; Jackson, L E; Eccles, S A; Leach, M O

    2015-03-31

    Acquired resistance to molecularly targeted therapeutics is a key challenge in personalised cancer medicine, highlighting the need for identifying the underlying mechanisms and early biomarkers of relapse, in order to guide subsequent patient management. Here we use human head and neck squamous cell carcinoma (HNSCC) models and nuclear magnetic resonance (NMR) spectroscopy to assess the metabolic changes that follow acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), and which could serve as potential metabolic biomarkers of drug resistance. Comparison of NMR metabolite profiles obtained from control (CAL(S)) and EGFR TKI-resistant (CAL(R)) cells grown as 2D monolayers, 3D spheroids or xenograft tumours in athymic mice revealed a number of differences between the sensitive and drug-resistant models. In particular, we observed elevated levels of glycerophosphocholine (GPC) in CAL(R) relative to CAL(S) monolayers, spheroids and tumours, independent of the growth rate or environment. In addition, there was an increase in alanine, aspartate and creatine+phosphocreatine in resistant spheroids and xenografts, and increased levels of lactate, branched-chain amino acids and a fall in phosphoethanolamine only in xenografts. The xenograft lactate build-up was associated with an increased expression of the glucose transporter GLUT-1, whereas the rise in GPC was attributed to inhibition of GPC phosphodiesterase. Reduced glycerophosphocholine (GPC) and phosphocholine were observed in a second HNSCC model probably indicative of a different drug resistance mechanism. Our studies reveal metabolic signatures associated not only with acquired EGFR TKI resistance but also growth pattern, microenvironment and contributing mechanisms in HNSCC models. These findings warrant further investigation as metabolic biomarkers of disease relapse in the clinic.

  9. Tumor growth inhibition with cetuximab and chemotherapy in non-small cell lung cancer xenografts expressing wild-type and mutated epidermal growth factor receptor.

    PubMed

    Steiner, Philipp; Joynes, Christopher; Bassi, Rajiv; Wang, Su; Tonra, James R; Hadari, Yaron R; Hicklin, Daniel J

    2007-03-01

    Targeting the epidermal growth factor receptor (EGFR) is a validated approach to treat cancer. In non-small cell lung cancer (NSCLC), EGFR contains somatic mutations in 10% of patients, which correlates with increased response rates to small molecule inhibitors of EGFR. We analyzed the effects of the monoclonal IgG1 antibody Erbitux (cetuximab) in NSCLC xenografts with wild-type (wt) or mutated EGFR. NSCLC cell lines were grown s.c. in nude mice. Dose-dependent efficacy was established for cetuximab. To determine whether combination therapy produces tumor regressions, cetuximab was dosed at half-maximal efficacy with chemotherapy used at maximum tolerated dose. Cetuximab showed antitumor activity in wt (A549, NCI-H358, NCI-H292) and mutated [HCC-827 (delE746-A750), NCI-H1975 (L858R, T790M)] EGFR-expressing xenografts. In the H292 model, cetuximab and docetaxel combination therapy was more potent to inhibit tumor growth than cetuximab or docetaxel alone. Cisplatin augmented efficacy of cetuximab to produce 6 of 10 regressions, whereas 1 of 10 regressions was found with cetuximab and no regression was found with cisplatin. Using H1975 xenografts, gemcitabine increased efficacy of cetuximab resulting in 12 of 12 regressions. Docetaxel with cetuximab was more efficacious with seven of nine regressions compared with single treatments. Cetuximab inhibited autophosphorylation of EGFR in both H292 and H1975 tumor lysates. Exploring the underlying mechanism for combination effects in the H1975 xenograft model, docetaxel in combination with cetuximab added to the antiproliferative effects of cetuximab but was the main component in this drug combination to induce apoptosis. Cetuximab showed antitumor activity in NSCLC models expressing wt and mutated EGFR. Combination treatments increased the efficacy of cetuximab, which may be important for the management of patients with chemorefractory NSCLC.

  10. Imaging of small-cell lung cancer xenografts with I-125, In-111, and Re-188 octreotides

    SciTech Connect

    Hosono, M.; Hosono, M.N.; Haberberger, T. ||

    1995-05-01

    Somatostatin receptor imaging has been reported to be valuable for the localization of small-cell lung cancer (SCLC). We estimated the efficiency of I-125-Tyr-3-octreotide(I-125-octreotide), In-111-DTPA-D-Phe-1-octreotide (in-111-octreotide), and Re-188-octreotide in a mouse model of SCLC. Tyr-3-octreotide was labeled with I-125 by the chloramine T method, and In-111-octreotide was supplied by Mallinckrodt Medical (The Netherlands), while Re-188 was obtained from a W-188/Re-188 generator, and octreotide was labeled with Re-188 efficiently by a direct labeling using stannous chloride as a reduction agent. The expression of somatostatin receptor on NCI-H69 cells (a SCLC cell line) had been previously reported and we confirmed it by a cell binding assay. I-125-, In-111-, and Re-188-octreotides were injected i.v. into nude mice bearing NCI-H69 xenografts. Biodistributions were determined at 15 min, 2, 4, 8, and 24 h after injection. Specific binding of radiolabeled octreotides was observed by pretreatment of mice with unlabeled octreotide. Tumor uptake of I-125-, In-111-, and Re-188-octreotides at 2 h was 0.9{plus_minus}0.3, 0.3{plus_minus}0.1, 0.5{plus_minus}0.1% ID/g, respectively. Tumor-to-blood ratios were 0.91, 7.45, 0.41 at 2 h, 1.66, 11.16, 1.23 at 8 h for I-125-, In-111-, and Rej-188-octreotides, respectively. I-125-and Re-188 octreotides showed significant accumulations in the liver and GI tract. By contrast, In-111-octreotide cleared more rapidly from the blood and accumulated in normal tissues less than I-125- and Re-188- octreotides, resulting in high tumor-to-normal tissue ratios. In conclusion, as absolute level of tumor uptake of Re-188-octreotide is higher than that of In-111-octreotide, and Re-188-octreotide can be prepared easily as a kit, Re-188-octreotide is useful for the targeting of SCLC as well as I-125-octreotide, while In-111-octreotide is potent to achieve clear tumor-to-normal tissue contrast.

  11. Novel murine xenograft model for the evaluation of stem cell therapy for profound dysphagia.

    PubMed

    Kuhn, Maggie A; Black, Amanda B; Siddiqui, M Tausif; Nolta, Jan A; Belafsky, Peter C

    2017-10-01

    Dysphagia is common and costly. Treatments are limited and innovative therapies are required. The tongue is essential for safe, effective swallowing and is a natural target for regenerative therapy. Muscle-derived stem cells (MDSCs) hold potential to restore dynamic function, and their application in the damaged tongue is appealing. We examined the safety and efficacy of human MDSC implantation into a novel mouse tongue model. Animal study. Adult immune-deficient mice were randomized to surgical (hemiglossectomy) and nonsurgical groups. Animals underwent lingual injection of human MDSCs or saline (control). Groups were followed for 12 weeks. The primary outcome was MDSC survival measured by an in vivo imaging system (IVIS). Secondary outcomes included animal survival and weight. Comparisons were made using a Mann-Whitney U test with an α of .05. Human MDSCs survived to the endpoint demonstrating 132% ± 465% and 15% ± 11% bioluminescence by IVIS at 12 weeks in hemiglossectomy and nonsurgical groups, respectively. All but one animal (hemiglossectomy with saline injection) survived to the study endpoint. Mean weight increased from baseline in all groups, with the greatest change observed in hemiglossectomy mice with MDSC injection (baseline 24.5 g ± 3.9 g; delta 5.9 g ± 4.6 g), exceeding the weight gain seen in surgical control mice (baseline 24.9 g ± 4.2 g, delta 2.7 g ± 1.4 g) (P = .04). MDSCs exhibited over 100% survival at 3 months when injected into an immune-deficient hemiglossectomy mouse model. Tongue-injured animals injected with MDSCs exhibited superior weight gain after hemiglossectomy than control animals (P < .05). These data support further investigation into the use of autologous MDSCs as a potential treatment for dysphagia secondary to tongue weakness and fibrosis LEVEL OF EVIDENCE: NA Laryngoscope, 127:E359-E363, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

  12. Neuroblastoma patient-derived xenograft cells cultured in stem-cell promoting medium retain tumorigenic and metastatic capacities but differentiate in serum.

    PubMed

    Persson, Camilla U; von Stedingk, Kristoffer; Bexell, Daniel; Merselius, My; Braekeveldt, Noémie; Gisselsson, David; Arsenian-Henriksson, Marie; Påhlman, Sven; Wigerup, Caroline

    2017-08-31

    Cultured cancer cells serve as important models for preclinical testing of anti-cancer compounds. However, the optimal conditions for retaining original tumor features during in vitro culturing of cancer cells have not been investigated in detail. Here we show that serum-free conditions are critical for maintaining an immature phenotype of neuroblastoma cells isolated from orthotopic patient-derived xenografts (PDXs). PDX cells could be grown either as spheres or adherent on laminin in serum-free conditions with retained patient-specific genomic aberrations as well as tumorigenic and metastatic capabilities. However, addition of serum led to morphological changes, neuronal differentiation and reduced cell proliferation. The epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were central for PDX cell proliferation and MYCN expression, and also hindered the serum-induced differentiation. Although serum induced a robust expression of neurotrophin receptors, stimulation with their cognate ligands did not induce further sympathetic differentiation, which likely reflects a block in PDX cell differentiation capacity coupled to their tumor genotype. Finally, PDX cells cultured as spheres or adherent on laminin responded similarly to various cytotoxic drugs, suggesting that both conditions are suitable in vitro screening models for neuroblastoma-targeting compounds.

  13. Bioluminescence imaging of leukemia cell lines in vitro and in mouse xenografts: effects of monoclonal and polyclonal cell populations on intensity and kinetics of photon emission

    PubMed Central

    2013-01-01

    Background We investigated the utility of bioluminescence imaging (BLI) using firefly luciferase in monoclonal and polyclonal populations of leukemia cells in vitro and in vivo. Methods Monoclonal and polyclonal human lymphoid and myeloid leukemia cell lines transduced with firefly luciferase were used for BLI. Results Kinetics and dynamics of bioluminescence signal were cell line dependent. Luciferase expression decreased significantly over time in polyclonal leukemia cells in vitro. Transplantation of polyclonal luciferase-tagged cells in mice resulted in inconsistent signal intensity. After selection of monoclonal cell populations, luciferase activity was stable, equal kinetic and dynamic of bioluminescence intensity and strong correlation between cell number and light emission in vitro were observed. We obtained an equal development of leukemia burden detected by luciferase activity in NOD-scid-gamma mice after transplantation of monoclonal populations. Conclusion The use of monoclonal leukemia cells selected for stable and equal luciferase activity is recommended for experiments in vitro and xenograft mouse models. The findings are highly significant for bioluminescence imaging focused on pre-clinical drug development. PMID:23343252

  14. Anti-tumor activity of Sann-Joong-Kuey-Jian-Tang alone and in combination with 5-fluorouracil in a human colon cancer colo 205 cell xenograft model.

    PubMed

    Cheng, Chun-Yuan; Lin, Yi-Hsiang; Su, Chin-Cheng

    2010-01-01

    Malignant tumors are the leading cause of death in Taiwan; among these, colon cancer ranks third as a cause of cancer-related death. Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicinal prescription, has been used to treat lymph node diseases and infectious lesions, and exhibits cytotoxic activity in many cancer cell lines. Our previous studies demonstrated that SJKJT inhibits the proliferation of human colon cancer colo 205 cells in vitro. The aim of this study was to evaluate the anti-tumor activity of SJKJT alone and in combination with 5-fluorouracil (5-FU) in vivo. SCID mice bearing human colon cancer colo 205 cell xenografts were administered SJKJT alone (30 mg/kg daily, p.o.), SJKJT (30 mg/kg daily, p.o.) in combination with 5-FU (30 mg/kg weekly, i.p.), or vehicle alone. At the end of the 4-week dosing schedule, the tumor and animal body weights were individually measured. The SCID mice were sacrificed with CO2 inhalation, the xenograft tumors were dissected, and the protein expression of microtubule-associated protein light chain 3 (MAP-LC3-II) in colo 205 xenograft tumors was measured by Western blotting. In the control, SJKJT-, and SJKJT plus 5-FU-treated mice, the tumor weights were 6.37±2.57, 0.43±0.35 and 1.63±0.46 g, and the mice body weights were 29±0.55, 29±2.71 and 27±0.77 g, respectively. Treatment with SJKJT resulted in a reduction in tumor weight compared with the control group, indicating that SJKJT inhibits tumor growth in a colo 205 xenograft model. SJKJT also increased LC3-II protein expression as compared to the controls. The present study shows that SJKJT alone or in combination with 5-FU has a positive effect on the treatment of SCID mice bearing human colon cancer colo 205 cell xenografts. This suggests that SJKJT has therapeutic potential in the treatment of human colon cancer.

  15. Evaluation of the NOD/SCID xenograft model for glucocorticoid-regulated gene expression in childhood B-cell precursor acute lymphoblastic leukemia

    PubMed Central

    2011-01-01

    Background Glucocorticoids such as prednisolone and dexamethasone are critical drugs used in multi-agent chemotherapy protocols used to treat acute lymphoblastic leukemia (ALL), and response to glucocorticoids is highly predictive of outcome. The NOD/SCID xenograft mouse model of ALL is a clinically relevant model in which the mice develop a systemic leukemia which retains the fundamental biological characteristics of the original disease. Here we report a study evaluating the NOD/SCID xenograft mouse model to investigate glucocorticoid-induced gene expression. Cells from a glucocorticoid-sensitive xenograft derived from a child with B-cell precursor ALL were inoculated into NOD/SCID mice. When highly engrafted the mice were randomized into groups of 4 to receive dexamethasone 15 mg/kg by intraperitoneal injection or vehicle control. Leukemia cells were harvested from mice spleens at 0, 8, 24 or 48 hours thereafter, and gene expression analyzed on Illumina WG-6_V3 chips, comparing all groups to time 0 hours. Results The 8 hour dexamethasone-treated timepoint had the highest number of significantly differentially expressed genes, with fewer observed at the 24 and 48 hour timepoints, and with minimal changes seen across the time-matched controls. When compared to publicly available datasets of glucocorticoid-induced gene expression from an in vitro cell line study and from an in vivo study of patients with ALL, at the level of pathways, expression changes in the 8 hour xenograft samples showed a similar response to patients treated with glucocorticoids. Replicate analysis revealed that at the 8 hour timepoint, a dataset with high signal and differential expression, using data from 3 replicates instead of 4 resulted in excellent recovery scores of > 0.9. However at other timepoints with less signal very poor recovery scores were obtained with 3 replicates. Conclusions The NOD/SCID xenograft mouse model provides a reproducible experimental system in which to

  16. Transcriptomic alterations in human prostate cancer cell LNCaP tumor xenograft modulated by dietary phenethyl isothiocyanate

    USDA-ARS?s Scientific Manuscript database

    Temporal growth of tumor xenografts in mice on a control diet was compared to mice supplemented daily with 3 µmol/g of the cancer preventive compound phenethyl isothiocyanate. Phenethyl isothiocyanate decreased the rate of tumor growth. The effects of phenethyl isothiocyanate on tumor growth were ex...

  17. A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines

    PubMed Central

    2011-01-01

    Introduction Inhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2. Methods In a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory. Results In vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells. Conclusions G28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development. PMID:22177475

  18. Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

    SciTech Connect

    Kurundkar, Deepali; Srivastava, Ritesh K.; Chaudhary, Sandeep C.; Ballestas, Mary E.; Kopelovich, Levy; Elmets, Craig A.; Athar, Mohammad

    2013-01-15

    Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100 mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. -- Highlights: ► Vorinostat reduces SCC growth in a xenograft murine model. ► Vorinostat dampens proliferation and induces apoptosis in tumor cells. ► Diminution in mTOR, Akt and ERK signaling underlies inhibition in proliferation. ► Vorinostat by inhibiting HDACs inhibits epithelial–mesenchymal transition.

  19. Sporadic Insulinoma Presenting as Early Morning Night Terrors.

    PubMed

    Beisang, Daniel; Forlenza, Gregory P; Luquette, Mark; Sarafoglou, Kyriakie

    2017-06-01

    A 16-year-old boy with a recent diagnosis of night terrors was evaluated for recurrent early morning hypoglycemia after an early morning seizure. Evaluation in clinic with critical laboratories identified hyperinsulinemic hypoglycemia. Additional investigation revealed a sporadic insulinoma as the etiology of his hypoglycemia and all symptoms were resolved after pancreaticoduodenectomy. The importance of obtaining critical laboratory samples is highlighted and appropriate radiologic, medical, and pathologic testing is discussed. We additionally review the medical and surgical management of hyperinsulinemic hypoglycemia. A discussion of multiple endocrine neoplasia type 1 associated insulinomas is included as well. This case highlights the importance of considering hypoglycemia in the evaluation of night terrors and new-onset seizures. Copyright © 2017 by the American Academy of Pediatrics.

  20. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts

    PubMed Central

    Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A.; Verma, Amit; Boultwood, Jacqueline

    2015-01-01

    Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival. PMID:26623729

  1. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

    PubMed

    Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A; Verma, Amit; Boultwood, Jacqueline

    2015-12-29

    Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

  2. Phenethyl isothiocyanate inhibits proliferation and induces apoptosis in pancreatic cancer cells in vitro and in a MIAPaca2 xenograft animal model.

    PubMed

    Stan, Silvia D; Singh, Shivendra V; Whitcomb, David C; Brand, Randall E

    2014-01-01

    Pancreatic cancer is often diagnosed at an advanced stage and it has a poor prognosis that points to an increased need to develop effective chemoprevention strategies for this disease. We examined the ability of phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate found in cruciferous vegetables, to inhibit the growth of pancreatic cancer cells in vitro and in a MIAPaca2 xenograft animal model. Exposure to PEITC inhibited pancreatic cancer cell growth in a dose-dependent manner, with an IC50 of approximately 7 μmol/L. PEITC treatment induced G2/M phase cell cycle arrest, downregulated the antiapoptotic proteins Bcl-2 and Bcl-XL, upregulated the proapoptotic protein Bak, and suppressed Notch 1 and 2 levels. In addition, treatment with PEITC induced cleavage of poly-(ADP-ribose) polymerase and led to increased cytoplasmic histone-associated DNA fragmentation and subdiploid (apoptotic) fraction in pancreatic cancer cells. Oral administration of PEITC suppressed the growth of pancreatic cancer cells in a MIAPaca2 xenograft animal model. Our data show that PEITC exerts its inhibitory effect on pancreatic cancer cells through several mechanisms, including G2/M phase cell cycle arrest and induction of apoptosis, and supports further investigation of PEITC as a chemopreventive agent for pancreatic cancer.

  3. Diagnostic Difficulties in a Pediatric Insulinoma: A Case Report.

    PubMed

    Miron, Ingrith; Diaconescu, Smaranda; Aprodu, Gabriel; Ioniuc, Ileana; Diaconescu, Mihai Radu; Miron, Lucian

    2016-03-01

    Insulinomas are functional neuroendocrine pancreatic tumors rarely encountered in pediatric pathology. Insulinomas are usually solitary and sporadic, but may occur in association with multiple endocrine neoplasia type 1. Whipple's triad-hypoglycemia, simultaneous compatible adrenergic and/or neurological signs, and relief of symptoms upon the administration of glucose-remains the fundamental diagnostic tool. We report a case of insulinoma in an 11-year-old boy with malnutrition and mild psychic retardation. History revealed neuroglycopenic symptoms associated with hypoglycemia that returned to normal values after glucose intravenous infusion; before admission in our unit, the levels of circulating insulin, as well as the abdominal ultrasound and abdominal computed tomography scan, were reported within normal range. During hospitalization in our service, the glycemic curves showed recurring low values associated with low glycated hemoglobin, positive fasting test, and elevated C-peptide. The pancreatic ultrasound was inconclusive, but the magnetic resonance imaging revealed a high signal focal area with a diameter of 1 cm, located in the tail of pancreas. Conventional enucleation of the lesion prompted a spectacular normalization of glucose metabolism and the alleviation of the main clinical symptoms. The child had a favorable evolution in the clinical follow-up, presenting with weight gain and progressive remission to complete disappearance of most symptoms-except for the mental impairments. Although in our case Whipple's triad was apparent from the beginning, the diagnosis was delayed due to the failure of conventional imaging methods in locating the tumor. Weight loss and mental impairment contributed to the diagnosis pitfalls. Pediatricians should be aware of confusing and nonspecific symptoms, especially when children with insulinoma present mental or neurological retardation. Despite the existence of medical regimens, surgery remains the gold standard for the

  4. ONYX-411, a conditionally replicative oncolytic adenovirus, induces cell death in anaplastic thyroid carcinoma cell lines and suppresses the growth of xenograft tumors in nude mice

    PubMed Central

    Reddi, HV; Madde, P; Reichert-Eberhardt, AJ; Galanis, EC; Copland, JA; McIver, B; Grebe, SKG; Eberhardt, NL

    2011-01-01

    Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer variant, accounting for 1–2% of all cases, but 33% of deaths, and exhibiting an average life expectancy of 5 months. ATC is largely unresponsive to radioactive iodine, chemotherapy, external beam radiation or surgery, underscoring the need for new and effective therapies. We evaluated the therapeutic potential of an oncolytic adenovirus, ONYX-411, that replicates selectively in and kills cells with dysfunction of the retinoblastoma (RB) pathway. In the present study, we report that ONYX-411 is able to induce cell death in eight human anaplastic carcinoma cell lines in vitro. The cytopathic effect of the virus is specific to cells with RB dysfunction, which appears to be frequent in ATC. We confirmed the expression of the coxsackie adenovirus receptor, CAR, in all ATC cell lines, demonstrating the potentially universal application of this oncolytic viral therapy to ATC. In addition, the growth of xenograft tumors induced in athymic mice with the ARO and DRO cell lines was significantly reduced by ONYX-411 treatment. These results indicate that ONYX-411 can be a potential therapeutic agent for the treatment of ATC, rendering this class of conditionally replicating adenoviruses an attractive candidate for clinical trials. PMID:18583996

  5. Combined 5-FU and ChoKα Inhibitors as a New Alternative Therapy of Colorectal Cancer: Evidence in Human Tumor-Derived Cell Lines and Mouse Xenografts

    PubMed Central

    de la Cueva, Ana; Ramírez de Molina, Ana; Álvarez-Ayerza, Néstor; Ramos, Ma Angeles; Cebrián, Arancha; del Pulgar, Teresa Gómez; Lacal, Juan Carlos

    2013-01-01

    Background Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy. Methodology/Principal Findings ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action. Conclusion/Significance Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors. PMID:23762272

  6. Anti-tumor activity of high-dose EGFR tyrosine kinase inhibitor and sequential docetaxel in wild type EGFR non-small cell lung cancer cell nude mouse xenografts.

    PubMed

    Tang, Ning; Zhang, Qianqian; Fang, Shu; Han, Xiao; Wang, Zhehai

    2017-02-07

    Treatment of non-small-cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is still a challenge. This study explored antitumor activity of high-dose icotinib (an EGFR tyrosine kinase inhibitor) plus sequential docetaxel against wild-type EGFR NSCLC cells-generated nude mouse xenografts. Nude mice were subcutaneously injected with wild-type EGFR NSCLC A549 cells and divided into different groups for 3-week treatment. Tumor xenograft volumes were monitored and recorded, and at the end of experiments, tumor xenografts were removed for Western blot and immunohistochemical analyses. Compared to control groups (negative control, regular-dose icotinib [IcoR], high-dose icotinib [IcoH], and docetaxel [DTX]) and regular icotinib dose (60 mg/kg) with docetaxel, treatment of mice with a high-dose (1200 mg/kg) of icotinib plus sequential docetaxel for 3 weeks (IcoH-DTX) had an additive effect on suppression of tumor xenograft size and volume (P < 0.05). Icotinib-containing treatments markedly reduced phosphorylation of EGFR, mitogen activated protein kinase (MAPK), and protein kinase B (Akt), but only the high-dose icotinib-containing treatments showed an additive effect on CD34 inhibition (P < 0.05), an indication of reduced microvessel density in tumor xenografts. Moreover, high-dose icotinib plus docetaxel had a similar effect on mouse weight loss (a common way to measure adverse reactions in mice), compared to the other treatment combinations. The study indicate that the high dose of icotinib plus sequential docetaxel (IcoH-DTX) have an additive effect on suppressing the growth of wild-type EGFR NSCLC cell nude mouse xenografts, possibly through microvessel density reduction. Future clinical trials are needed to confirm the findings of this study.

  7. Increased COX-2 expression in epithelial and stromal cells of high mammographic density tissues and in a xenograft model of mammographic density.

    PubMed

    Chew, G L; Huo, C W; Huang, D; Hill, P; Cawson, J; Frazer, H; Hopper, J L; Haviv, I; Henderson, M A; Britt, K; Thompson, E W

    2015-08-01

    Mammographic density (MD) adjusted for age and body mass index is one of the strongest known risk factors for breast cancer. Given the high attributable risk of MD for breast cancer, chemoprevention with a safe and available agent that reduces MD and breast cancer risk would be beneficial. Cox-2 has been implicated in MD-related breast cancer risk, and was increased in stromal cells in high MD tissues in one study. Our study assessed differential Cox-2 expression in epithelial and stromal cells in paired samples of high and low MD human breast tissue, and in a validated xenograft biochamber model of MD. We also examined the effects of endocrine treatment upon Cox-2 expression in high and low MD tissues in the MD xenograft model. Paired high and low MD human breast tissue samples were immunostained for Cox-2, then assessed for differential expression and staining intensity in epithelial and stromal cells. High and low MD human breast tissues were separately maintained in biochambers in mice treated with Tamoxifen, oestrogen or placebo implants, then assessed for percentage Cox-2 staining in epithelial and stromal cells. Percentage Cox-2 staining was greater for both epithelial (p = 0.01) and stromal cells (p < 0.0001) of high compared with low MD breast tissues. In high MD biochamber tissues, percentage Cox-2 staining was greater in stromal cells of oestrogen-treated versus placebo-treated tissues (p = 0.05).

  8. The Volume of Three-Dimensional Cultures of Cancer Cells InVitro Influences Transcriptional Profile Differences and Similarities with Monolayer Cultures and Xenografted Tumors.

    PubMed

    Boghaert, Erwin R; Lu, Xin; Hessler, Paul E; McGonigal, Thomas P; Oleksijew, Anatol; Mitten, Michael J; Foster-Duke, Kelly; Hickson, Jonathan A; Santo, Vitor E; Brito, Catarina; Uziel, Tamar; Vaidya, Kedar S

    2017-09-01

    Improving the congruity of preclinical models with cancer as it is manifested in humans is a potential way to mitigate the high attrition rate of new cancer therapies in the clinic. In this regard, three-dimensional (3D) tumor cultures in vitro have recently regained interest as they have been acclaimed to have higher similarity to tumors in vivo than to cells grown in monolayers (2D). To identify cancer functions that are active in 3D rather than in 2D cultures, we compared the transcriptional profiles (TPs) of two non-small cell lung carcinoma cell lines, NCI-H1650 and EBC-1 grown in both conditions to the TP of xenografted tumors. Because confluence, diameter or volume can hypothetically alter TPs, we made intra- and inter-culture comparisons using samples with defined dimensions. As projected by Ingenuity Pathway Analysis (IPA), a limited number of signal transduction pathways operational in vivo were better represented by 3D than by 2D cultures in vitro. Growth of 2D and 3D cultures as well as xenografts induced major changes in the TPs of these 3 modes of culturing. Alterations of transcriptional network activation that were predicted to evolve similarly during progression of 3D cultures and xenografts involved the following functions: hypoxia, proliferation, cell cycle progression, angiogenesis, cell adhesion, and interleukin activation. Direct comparison of TPs of 3D cultures and xenografts to monolayer cultures yielded up-regulation of networks involved in hypoxia, TGF and Wnt signaling as well as regulation of epithelial mesenchymal transition. Differences in TP of 2D and 3D cancer cell cultures are subject to progression of the cultures. The emulation of the predicted cell functions in vivo is therefore not only determined by the type of culture in vitro but also by the confluence or diameter of the 2D or 3D cultures, respectively. Consequently, the successful implementation of 3D models will require phenotypic characterization to verify the relevance of

  9. Hinokitiol inhibits cell growth through induction of S-phase arrest and apoptosis in human colon cancer cells and suppresses tumor growth in a mouse xenograft experiment.

    PubMed

    Lee, Youn-Sun; Choi, Kyeong-Mi; Kim, Wonkyun; Jeon, Young-Soo; Lee, Yong-Moon; Hong, Jin-Tae; Yun, Yeo-Pyo; Yoo, Hwan-Soo

    2013-12-27

    Hinokitiol (1), a tropolone-related natural compound, induces apoptosis and has anti-inflammatory, antioxidant, and antitumor activities. In this study, the inhibitory effects of 1 were investigated on human colon cancer cell growth and tumor formation of xenograft mice. HCT-116 and SW-620 cells derived from human colon cancers were found to be similarly susceptible to 1, with IC50 values of 4.5 and 4.4 μM, respectively. Compound 1 induced S-phase arrest in the cell cycle progression and decreased the expression levels of cyclin A, cyclin E, and Cdk2. Conversely, 1 increased the expression of p21, a Cdk inhibitor. Compound 1 decreased Bcl-2 expression and increased the expression of Bax, and cleaved caspase-9 and -3. The effect of 1 on tumor formation when administered orally was evaluated in male BALB/c-nude mice implanted intradermally separately with HCT-116 and SW-620 cells. Tumor volumes and tumor weights in the mice treated with 1 (100 mg/kg) were decreased in both cases. These results suggest that the suppression of tumor formation by compound 1 in human colon cancer may occur through cell cycle arrest and apoptosis.

  10. Preoperative tumor studies using MRI or CT in patients with clinically suspected insulinoma.

    PubMed

    Daneshvar, Keivan; Grenacher, Lars; Mehrabi, Arianeb; Kauczor, Hans-Ulrich; Hallscheidt, Peter

    2011-01-01

    Insulinomas are rare tumors that originate from the islet cells of the pancreas. The aims of this study were to localize insulinomas preoperatively using CT and/or MRI in correlation with postoperative pathological results. Between December 2001 and June 2010, 27 consecutive patients with clinically suspected insulinoma were surgically treated in our university hospital. Preoperative CT (14 of 27 patients) and MRI studies (14 of 27 patients, one patient had both MRI and CT), operation reports, intraoperative ultrasonography reports, and pathological diagnoses were analyzed retrospectively. For each lesion, images were analyzed based on the presence of enhancement or the characteristics of signal intensities. Pathologic correlation was available for all the lesions. The female: male ratio was 2.9, with a mean age of 47.5 years (range 12-82) . Preoperative tumor localization was achieved by means of MRI and CT. A focal pancreatic lesion, which was hypointense on T(1)-weighted sequences, was detected on all the MR images (14 of 27 patients; 100%). These lesions were isointense (4 cases) to slightly hyperintense (10 of 14 cases) on T(2)-weighted sequences. In T(1)-weighted fat-suppressed contrast-enhanced sequences, there were two types of enhancement: homogeneously hyperintense lesions (in 10 of 14 cases) or peripherally hyper-, centrally isointense (in 4 of 14 cases). On all the CT images (14 of 27 patients), there was no detectable lesion on precontrast series; on arterial series in 13 of 14 patients (arterial series has not been done in one patient), lesions enhanced hypervascular in contrast to the rest of the pancreas with a mean enhancement of 147 HU (range 113-248) and 95 HU (range 65-141), respectively. On venous series in 13 of 14 patients (venous series has not been done in one patient), there was an enhanced lesion in contrast to the rest of the pancreas with a mean enhancement of 110 HU (range 91-151) and 86 HU (range 65-137), respectively. Intraoperative

  11. Comparison of basic fibroblast growth factor levels in clone A human colon cancer cells in vitro with levels in xenografted tumours.

    PubMed Central

    McCarty, L. P.; Karr, S. M.; Harris, B. Z.; Michelson, S. G.; Leith, J. T.

    1995-01-01

    We measured levels of basic fibroblast growth factor (FGF-2) in human colon cancer cells (clone A) in vitro and in xenografted solid tumours using a commercial enzyme-linked immunoassay. In Vitro, levels in unfed plateau phase or exponentially growing cells were low, averaging respectively about 2 and 8 pg 10(-6) cells. However, when solid tumours (average volumes 787 mm3) were cut into halves and either enzymatically disaggregated to obtain a cellular fraction or extracted in toto, levels were much higher. In the cellular fraction, values averaged 110 pg 10(-6) cells, while in whole tumour extracts, average values were 24 pg mg-1 tumour tissue. These results indicate that growth factor levels in solid neoplasms may differ markedly from those predicted from in vitro measurements. We hypothesise that the apparent increase in FGF-2 levels in vivo results primarily from the presence of a significant fraction of host cells (in particular, macrophages, which may contain high levels of FGF-2) within xenografted clone A neoplasms. PMID:7599036

  12. Genetically engineered pre-microRNA-34a prodrug suppresses orthotopic osteosarcoma xenograft tumor growth via the induction of apoptosis and cell cycle arrest

    PubMed Central

    Zhao, Yong; Tu, Mei-Juan; Wang, Wei-Peng; Qiu, Jing-Xin; Yu, Ai-Xi; Yu, Ai-Ming

    2016-01-01

    Osteosarcoma (OS) is the most common primary malignant bone tumor in children, and microRNA-34a (miR-34a) replacement therapy represents a new treatment strategy. This study was to define the effectiveness and safety profiles of a novel bioengineered miR-34a prodrug in orthotopic OS xenograft tumor mouse model. Highly purified pre-miR-34a prodrug significantly inhibited the proliferation of human 143B and MG-63 cells in a dose dependent manner and to much greater degrees than controls, which was attributed to induction of apoptosis and G2 cell cycle arrest. Inhibition of OS cell growth and invasion were associated with release of high levels of mature miR-34a from pre-miR-34a prodrug and consequently reduction of protein levels of many miR-34a target genes including SIRT1, BCL2, c-MET, and CDK6. Furthermore, intravenous administration of in vivo-jetPEI formulated miR-34a prodrug significantly reduced OS tumor growth in orthotopic xenograft mouse models. In addition, mouse blood chemistry profiles indicated that therapeutic doses of bioengineered miR-34a prodrug were well tolerated in these animals. The results demonstrated that bioengineered miR-34a prodrug was effective to control OS tumor growth which involved the induction of apoptosis and cell cycle arrest, supporting the development of bioengineered RNAs as a novel class of large molecule therapeutic agents. PMID:27216562

  13. DMAPT inhibits NF-κB activity and increases sensitivity of prostate cancer cells to X-rays in vitro and in tumor xenografts in vivo.

    PubMed

    Mendonca, Marc S; Turchan, William T; Alpuche, Melanie E; Watson, Christopher N; Estabrook, Neil C; Chin-Sinex, Helen; Shapiro, Jeremy B; Imasuen-Williams, Imade E; Rangel, Gabriel; Gilley, David P; Huda, Nazmul; Crooks, Peter A; Shapiro, Ronald H

    2017-11-01

    Constitutive activation of the pro-survival transcription factor NF-κB has been associated with resistance to both chemotherapy and radiation therapy in many human cancers, including prostate cancer. Our lab and others have demonstrated that the natural product parthenolide can inhibit NF-κB activity and sensitize PC-3 prostate cancers cells to X-rays in vitro; however, parthenolide has poor bioavailability in vivo and therefore has little clinical utility in this regard. We show here that treatment of PC-3 and DU145 human prostate cancer cells with dimethylaminoparthenolide (DMAPT), a parthenolide derivative with increased bioavailability, inhibits constitutive and radiation-induced NF-κB binding activity and slows prostate cancer cell growth. We also show that DMAPT increases single and fractionated X-ray-induced killing of prostate cancer cells through inhibition of DNA double strand break repair and also that DMAPT-induced radiosensitization is, at least partially, dependent upon the alteration of intracellular thiol reduction-oxidation chemistry. Finally, we demonstrate that the treatment of PC-3 prostate tumor xenografts with oral DMAPT in addition to radiation therapy significantly decreases tumor growth and results in significantly smaller tumor volumes compared to xenografts treated with either DMAPT or radiation therapy alone, suggesting that DMAPT might have a potential clinical role as a radiosensitizing agent in the treatment of prostate cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1.

    PubMed

    Michelhaugh, Sharon K; Guastella, Anthony R; Varadarajan, Kaushik; Klinger, Neil V; Parajuli, Prahlad; Ahmad, Aamir; Sethi, Seema; Aboukameel, Amro; Kiousis, Sam; Zitron, Ian M; Ebrahim, Salah A; Polin, Lisa A; Sarkar, Fazlul H; Bollig-Fischer, Aliccia; Mittal, Sandeep

    2015-07-15

    There is a paucity of effective therapies for recurrent/aggressive meningiomas. Establishment of improved in vitro and in vivo meningioma models will facilitate development and testing of novel therapeutic approaches. A primary meningioma cell line was generated from a patient with an olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and comparative genomic hybridization. Xenograft models using immunocompromised SCID mice were also developed. Histopathology of the patient tumor was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and occasional psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile consistent with low-grade, good prognosis meningioma. Low passage KCI-MENG1 cells were composed of two cell types with spindle and round morphologies, showed linear growth curve, had very low telomerase activity, and were composed of two distinct unrelated clones on cytogenetic analysis. In contrast, high passage cells were homogeneously round, rapidly growing, had high telomerase activity, and were composed of a single clone with a near triploid karyotype containing 64-66 chromosomes with numerous aberrations. Following subcutaneous and orthotopic transplantation of low passage cells into SCID mice, firm tumors positive for vimentin and progesterone receptor (PR) formed, while subcutaneous implant of high passage cells yielded vimentin-positive, PR-negative tumors, concordant with a high-grade meningioma. Although derived from a benign meningioma specimen, the newly-established spontaneously immortal KCI-MENG1 meningioma cell line can be utilized to generate xenograft tumor models with either low- or high-grade features, dependent on the cell passage number (likely due to the relative abundance of the round, near-triploid cells). These human

  15. Stromal platelet-derived growth factor receptor α (PDGFRα) provides a therapeutic target independent of tumor cell PDGFRα expression in lung cancer xenografts

    PubMed Central

    Gerber, David E.; Gupta, Puja; Dellinger, Michael T.; Toombs, Jason E.; Peyton, Michael; Duignan, Inga; Malaby, Jennifer; Bailey, Timothy; Burns, Colleen; Brekken, Rolf A.; Loizos, Nick

    2012-01-01

    In lung cancer, platelet-derived growth factor receptor α (PDGFRα) is expressed frequently by tumor-associated stromal cells and by cancer cells in a subset of tumors. We sought to determine the effect of targeting stromal PDGFRα in preclinical lung tumor xenograft models (human tumor, mouse stroma). Effects of anti-human (IMC-3G3) and anti-mouse (1E10) PDGFRα mAbs on proliferation and PDGFRα signaling were evaluated in lung cancer cell lines and mouse fibroblasts. Therapy studies were performed using established PDGFRα-positive H1703 cells and PDGFRα-negative Calu-6, H1993, and A549 subcutaneous tumors in immunocompromised mice treated with vehicle, anti-PDGFRα mAbs, chemotherapy, or combination therapy. Tumors were analyzed for growth and levels of growth factors. IMC-3G3 inhibited PDGFRα activation and the growth of H1703 cells in vitro and tumor growth in vivo, but had no effect on PDGFRα-negative cell lines or mouse fibroblasts. 1E10 inhibited growth and PDGFRα activation of mouse fibroblasts, but had no effect on human cancer cell lines in vitro. In vivo, 1E10-targeted inhibition of murine PDGFRα reduced tumor growth as single-agent therapy in Calu-6 cells and enhanced the effect of chemotherapy in xenografts derived from A549 cells. We also identified that low expression cancer cell expression of VEGF-A and elevated expression of PDGF-AA were associated with response to stromal PDGFRα targeting. We conclude that stromal PDGFRα inhibition represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression. PMID:22933705

  16. Antitumor effects with apoptotic death in human promyelocytic leukemia HL-60 cells and suppression of leukemia xenograft tumor growth by irinotecan HCl.

    PubMed

    Chen, Yung-Liang; Chueh, Fu-Shin; Yang, Jai-Sing; Hsueh, Shu-Ching; Lu, Chi-Cheng; Chiang, Jo-Hua; Lee, Ching-Sung; Lu, Hsu-Feng; Chung, Jing-Gung

    2015-07-01

    Irinotecan HCl (CPT-11) is an anticancer prodrug, but there is no available information addressing CPT-11-inhibited leukemia cells in in vitro and in vivo studies. Therefore, we investigated the cytotoxic effects of CPT-11 in promyelocytic leukemia HL-60 cells and in vivo and tumor growth in a leukemia xenograft model. Effects of CPT-11 on HL-60 cells were determined using flow cytometry, immunofluorescence staining, comet assay, real-time PCR, and Western blotting. CPT-11 demonstrated a dose- and time-dependent inhibition of cell growth, induction of apoptosis, and cell-cycle arrest at G0/G1 phase in HL-60 cells. CPT-11 promoted the release of AIF from mitochondria and its translocation to the nucleus. Bid, Bax, Apaf-1, caspase-9, AIF, Endo G, caspase-12, ATF-6b, Grp78, CDK2, Chk2, and cyclin D were all significantly upregulated and Bcl-2 was down-regulated by CPT-11 in HL-60 cells. Induction of cell-cycle arrest by CPT-11 was associated with changes in expression of key cell-cycle regulators such as CDK2, Chk2, and cyclin D in HL-60 cells. To test whether CPT-11 could augment antitumor activity in vivo, athymic BALB/c(nu/nu) nude mice were inoculated with HL-60 cells, followed by treatment with either CPT-11. The treatments significantly inhibited tumor growth and reduced tumor weight and volume in the HL-60 xenograft mice. The present study demonstrates the schedule-dependent antileukemia effect of CPT-11 using both in vitro and in vivo models. CPT-11 could potentially be a promising agent for the treatment of promyelocytic leukemia and requires further investigation.

  17. Quercetin induces mitochondrial-derived apoptosis via reactive oxygen species-mediated ERK activation in HL-60 leukemia cells and xenograft.

    PubMed

    Lee, Wei-Jiunn; Hsiao, Michael; Chang, Junn-Liang; Yang, Shun-Fa; Tseng, Tsui-Hwa; Cheng, Chao-Wen; Chow, Jyh-Ming; Lin, Ke-Hsun; Lin, Yung-Wei; Liu, Chung-Chi; Lee, Liang-Ming; Chien, Ming-Hsien

    2015-07-01

    Quercetin is a plant-derived bioflavonoid that was recently shown to have multiple anticancer activities in various solid tumors. Here, novel molecular mechanisms through which quercetin exerts its anticancer effects in acute myeloid leukemia (AML) cells were investigated. Results from Western blot and flow cytometric assays revealed that quercetin significantly induced caspase-8, caspase-9, and caspase-3 activation, poly ADP-ribose polymerase (PARP) cleavage, and mitochondrial membrane depolarization in HL-60 AML cells. The induction of PARP cleavage by quercetin was also observed in other AML cell lines: THP-1, MV4-11, and U937. Moreover, treatment of HL-60 cells with quercetin induced sustained activation of extracellular signal-regulated kinase (ERK), and inhibition of ERK by an ERK inhibitor significantly abolished quercetin-induced cell apoptosis. MitoSOX red and 2',7'-dichlorofluorescin fluorescence, respectively, showed that mitochondrial superoxide and intracellular peroxide levels were higher in quercetin-treated HL-60 cells compared with the control group. Moreover, both N-acetylcysteine and the superoxide dismutase mimetic, MnTBAP, reversed quercetin-induced intracellular reactive oxygen species production, ERK activation, and subsequent cell death. The in vivo xenograft mice experiments revealed that quercetin significantly reduced tumor growth through inducing intratumoral oxidative stress while activating the ERK pathway and subsequent cell apoptosis in mice with HL-60 tumor xenografts. In conclusions, our results indicated that quercetin induced cell death of HL-60 cells in vitro and in vivo through induction of intracellular oxidative stress following activation of an ERK-mediated apoptosis pathway.

  18. Embolization of an Insulinoma of the Pancreas with Trisacryl Gelatin Microspheres as Definitive Treatment

    SciTech Connect

    Rott, Gernot Biggemann, Martin; Pfohl, Martin

    2008-05-15

    Insulinomas are rare, mostly benign neuroendocrine tumors, originating in 99% of cases from the pancreas, that synthesize and secrete insulin, causing symptomatic hypoglycemia. Today the treatment of choice is surgical removal. We present the case of an 84-year-old woman with a symptomatic insulinoma who refused surgery and was treated with arterial embolization using trisacryl gelatin microspheres as definitive treatment.

  19. Diallyl trisulfide inhibits migration, invasion and angiogenesis of human colon cancer HT-29 cells and umbilical vein endothelial cells, and suppresses murine xenograft tumour growth.

    PubMed

    Lai, Kuang-Chi; Hsu, Shu-Chun; Yang, Jai-Sing; Yu, Chien-Chih; Lein, Jin-Cherng; Chung, Jing-Gung

    2015-02-01

    Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis-dependent diseases including cancer. We examined the cytotoxic, anti-metastatic, anti-cancer and anti-angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases-2, -7 and -9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary-like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex-vivo angiogenesis. We investigated the anti-tumour effects of DATS against human colon cancer xenografts in BALB/c(nu/nu) mice and its anti-angiogenic activity in vivo. In this in-vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  20. Diallyl trisulfide inhibits migration, invasion and angiogenesis of human colon cancer HT-29 cells and umbilical vein endothelial cells, and suppresses murine xenograft tumour growth

    PubMed Central

    Lai, Kuang-Chi; Hsu, Shu-Chun; Yang, Jai-Sing; Yu, Chien-Chih; Lein, Jin-Cherng; Chung, Jing-Gung

    2015-01-01

    Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis-dependent diseases including cancer. We examined the cytotoxic, anti-metastatic, anti-cancer and anti-angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases-2, -7 and -9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary-like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex-vivo angiogenesis. We investigated the anti-tumour effects of DATS against human colon cancer xenografts in BALB/cnu/nu mice and its anti-angiogenic activity in vivo. In this in-vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS. PMID:25403643

  1. [Osteostimulating effect of bone xenograft on bone tissue regeneration].

    PubMed

    Balin, V N; Balin, D V; Iordanishvili, A K; Musikin, M I

    2015-01-01

    The aim of experimental case-control study performed in 28 dogs divided in 2 groups was to assess local tissue reactions on bone xenograft transplantation; dynamics of bone remodeling and formation at the site of bone defect wall contacting with bone xenograft; dynamics and mechanisms of xenograft remodeling. Transplantation of xenograft in conventional bone defects did not cause inflammatory of destructive reactions because of high biocompatibility of the material. At transplantation site active fibrous bone trabeculae formation filling the spaces between xenograft participles was observed. On the 90th day newly formed bone showed lammelar structure. Simultaneously from the 42d day the invasion of cell elements from recipient bed into the material was seen leading to xenograft resorption. The observed dynamics may be assessed as gradual substitution of xenograft with newly formed host bone structures.

  2. Probe-Based Confocal Laser Endomicroscopy for Imaging TRAIL-Expressing Mesenchymal Stem Cells to Monitor Colon Xenograft Tumors In Vivo

    PubMed Central

    Zhang, Zhen; Li, Ming; Chen, Feixue; Li, Lixiang; Liu, Jun; Li, Zhen; Ji, Rui; Zuo, Xiuli; Li, Yanqing

    2016-01-01

    Introduction Mesenchymal stem cells (MSCs) can serve as vehicles for therapeutic genes. However, little is known about MSC behavior in vivo. Here, we demonstrated that probe-based confocal laser endomicroscopy (pCLE) can be used to track MSCs in vivo and individually monitor tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) gene expression within carcinomas. Methods Isolated BALB/c nu/nu mice MSCs (MSCs) were characterized and engineered to co-express the TRAIL and enhanced green fluorescent protein (EGFP) genes. The number of MSCs co-expressing EGFP and TRAIL (TRAIL-MSCs) at tumor sites was quantified with pCLE in vivo, while their presence was confirmed using immunofluorescence (IF) and quantitative polymerase chain reaction (qPCR). The therapeutic effects of TRAIL-MSCs were evaluated by measuring the volumes and weights of subcutaneous HT29-derived xenograft tumors. Results Intravital imaging of the subcutaneous xenograft tumors revealed that BALB/c mice treated with TRAIL-MSCs exhibited specific cellular signals, whereas no specific signals were observed in the control mice. The findings from the pCLE images were consistent with the IF and qPCR results. Conclusion The pCLE results indicated that endomicroscopy could effectively quantify injected MSCs that homed to subcutaneous xenograft tumor sites in vivo and correlated well with the therapeutic effects of the TRAIL gene. By applying pCLE for the in vivo monitoring of cellular trafficking, stem cell-based anticancer gene therapeutic approaches might be feasible and attractive options for individualized clinical treatments. PMID:27617958

  3. Polyphenol-rich Avicennia marina leaf extracts induce apoptosis in human breast and liver cancer cells and in a nude mouse xenograft model.

    PubMed

    Huang, Cheng; Lu, Chung-Kuang; Tu, Ming-Chin; Chang, Jia-Hua; Chen, Yen-Ju; Tu, Yu-Hsuan; Huang, Hsiu-Chen

    2016-06-14

    Avicennia marina is the most abundant and common mangrove species and has been used as a traditional medicine for skin diseases, rheumatism, ulcers, and smallpox. However, its anticancer activities and polyphenol contents remain poorly characterized. Thus, here we investigated anticancer activities of secondary A. marina metabolites that were purified by sequential soxhlet extraction in water, ethanol, methanol, and ethyl acetate (EtOAc). Experiments were performed in three human breast cancer cell lines (AU565, MDA-MB-231, and BT483), two human liver cancer cell lines (HepG2 and Huh7), and one normal cell line (NIH3T3). The chemotherapeutic potential of A. marina extracts was evaluated in a xenograft mouse model. The present data show that EtOAc extracts of A. marina leaves have the highest phenolic and flavonoid contents and anticancer activities and, following column chromatography, the EtOAc fractions F2-5, F3-2-9, and F3-2-10 showed higher cytotoxic effects than the other fractions. 1H-NMR and 13C-NMR profiles indicated that the F3-2-10 fraction contained avicennones D and E. EtOAc extracts of A. marina leaves also suppressed xenograft MDA-MB-231 tumor growth in nude mice, suggesting that EtOAc extracts of A. marina leaves may provide a useful treatment for breast cancer.

  4. Metformin impairs Rho GTPase signaling to induce apoptosis in neuroblastoma cells and inhibits growth of tumors in the xenograft mouse model of neuroblastoma

    PubMed Central

    Kumar, Ambrish; Al-Sammarraie, Nadia; DiPette, Donald J.; Singh, Ugra S.

    2014-01-01

    Metformin has been shown to inhibit tumor growth in xenograft rodent models of adult cancers, and various human clinical trials are in progress. However, the precise molecular mechanisms of metformin action are largely unknown. In the present study we examined the anti-tumor activity of metformin against neuroblastoma, and determined the underlying signaling mechanisms. Using human neuroblastoma xenograft mice, we demonstrated that oral administration of metformin (100 and 250 mg/kg body weight) significantly inhibited the growth of tumors. The interference of metformin in spheroid formation further confirmed the anti-tumor activity of metformin. In tumors, the activation of Rac1 (GTP-Rac1) and Cdc42 (GTP-Cdc42) was increased while RhoA activation (GTP-RhoA) was decreased by metformin. It also induced phosphorylation of JNK and inhibited the phosphorylation of ERK1/2 without affecting p38 MAP Kinase. Infection of cells by adenoviruses expressing dominant negative Rac1 (Rac1-N17), Cdc42 (Cdc42-N17) or constitutively active RhoA (RhoA-V14), or incubation of cells with pharmacological inhibitors of Rac1 (NSC23766) or Cdc42 (ML141) significantly protected neuroblastoma cells from metformin-induced apoptosis. Additionally, inhibition of JNK activity along with Rac1 or Cdc42 attenuated cytotoxic effects of metformin. These studies demonstrated that metformin impairs Rho GTPases signaling to induce apoptosis via JNK pathway. PMID:25365944

  5. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    SciTech Connect

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  6. Polyphenol-rich Avicennia marina leaf extracts induce apoptosis in human breast and liver cancer cells and in a nude mouse xenograft model

    PubMed Central

    Tu, Ming-Chin; Chang, Jia-Hua; Chen, Yen-Ju; Tu, Yu-Hsuan; Huang, Hsiu-Chen

    2016-01-01

    Avicennia marina is the most abundant and common mangrove species and has been used as a traditional medicine for skin diseases, rheumatism, ulcers, and smallpox. However, its anticancer activities and polyphenol contents remain poorly characterized. Thus, here we investigated anticancer activities of secondary A. marina metabolites that were purified by sequential soxhlet extraction in water, ethanol, methanol, and ethyl acetate (EtOAc). Experiments were performed in three human breast cancer cell lines (AU565, MDA-MB-231, and BT483), two human liver cancer cell lines (HepG2 and Huh7), and one normal cell line (NIH3T3). The chemotherapeutic potential of A. marina extracts was evaluated in a xenograft mouse model. The present data show that EtOAc extracts of A. marina leaves have the highest phenolic and flavonoid contents and anticancer activities and, following column chromatography, the EtOAc fractions F2-5, F3-2-9, and F3-2-10 showed higher cytotoxic effects than the other fractions. 1H-NMR and 13C-NMR profiles indicated that the F3-2-10 fraction contained avicennones D and E. EtOAc extracts of A. marina leaves also suppressed xenograft MDA-MB-231 tumor growth in nude mice, suggesting that EtOAc extracts of A. marina leaves may provide a useful treatment for breast cancer. PMID:27078842

  7. Novel phyto-derivative BRM270 inhibits hepatocellular carcinoma cells proliferation by inducing G2/M phase cell cycle arrest and apoptosis in xenograft mice model.

    PubMed

    Kumar Mongre, Raj; Sharma, Neelesh; Singh Sodhi, Simrinder; Ghosh, Mrinmoy; Kumar Singh, Amit; Kim, Nameun; Park, Yang Ho; Shin, Young Gyu; Kim, Sung Jin; Jiao Jiao, Zhang; Huynh, Do Luong; Jeong, Dong Kee

    2017-03-01

    Hepatocellular carcinoma (HCC) is a major threat to human health worldwide and development of novel antineoplastic drug is demanding task. BRM270 is a proprietary combination of traditional medicinal herbs, has been shown to be effective against a wide range of stem-like cancer initiating cells (SLCICs). However, the underlying mechanism and antitumor efficacy of BRM270 in human hepatocellular carcinoma (HCC) cells have not been well elucidated till date. Here we studied the tumoricidal effect of BRM270 on human-CD133(+) expressing stem-like HepG-2 and SNU-398 cells. Gene expression profiling by qPCR and specific cellular protein expressions was measured using immunocytochemistry/western blot analysis. In vivo efficacy of BRM270 has been elucidated in the SLCICs induced xenograft model. In addition, 2DG-(2-Deoxy-d-Glucose) optical-probe guided tumor monitoring was performed to delineate the size and extent of metastasized tumor. Significant (P<0.05) induction of Annexin-V positive cell population and dose-dependent upregulation of caspase-3 confirmed apoptotic cell death by pre/late apoptosis. In addition, bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA fragmentation in Hoechst33342 staining. Levels of c-Myc, Bcl-2 and c-Jun as invasive potential apoptotic marker were detected using qPCR/Western blot. Moreover, BRM270 significantly (P<0.05) increased survival rate that observed by Kaplan-Meier log rank test. In conclusion, these results indicate that BRM270 can effectively inhibit proliferation and induce apoptosis in hepatoma cells by down-regulating CyclinD1/Bcl2 mediated c-Jun apoptotic pathway.

  8. A novel orally available inhibitor of focal adhesion signaling increases survival in a xenograft model of diffuse large B-cell lymphoma with central nervous system involvement.

    PubMed

    Bosch, Rosa; Moreno, María José; Dieguez-Gonzalez, Rebeca; Céspedes, María Virtudes; Gallardo, Alberto; Trias, Manuel; Grañena, Albert; Sierra, Jorge; Casanova, Isolda; Mangues, Ramon

    2013-08-01

    Central nervous system dissemination is a relatively uncommon but almost always fatal complication in diffuse large B-cell lymphoma patients. Optimal therapy for central nervous involvement in this malignancy has not been established. In this paper, we aimed to evaluate the therapeutic effect of E7123, a celecoxib derivative that inhibits focal adhesion signaling, in a novel xenograft model of diffuse large B-cell lymphoma with central nervous system involvement. Cells obtained after disaggregation of HT subcutaneous tumors (HT-SC cells) were intravenously injected in NOD/SCID mice. These mice received oral vehicle or 75 mg/kg of E7123 daily until they were euthanized for weight loss or signs of sickness. The antitumor effect of E7123 was validated in an independent experiment using a bioluminescent mouse model. Intravenously injected HT-SC cells showed higher take rate and higher central nervous system tropism (associated with increased expression of β1-integrin and p130Cas proteins) than HT cells. The oral administration of E7123 significantly increased survival time in 2 independent experiments using mice injected with unmodified or bioluminescent HT-SC cells. We have developed a new xenograft model of diffuse large B-cell lymphoma with central nervous system involvement that can be used in the pre-clinical evaluation of new drugs for this malignancy. E7123 is a new, well-tolerated and orally available therapeutic agent that merits further investigation since it may improve current management of diffuse large B-cell lymphoma patients with central nervous system involvement.

  9. Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model.

    PubMed

    Lin, Jen-Jyh; Hsu, Hui-Ying; Yang, Jai-Sing; Lu, Kung-Wen; Wu, Rick Sai-Chuen; Wu, King-Chuen; Lai, Tung-Yuan; Chen, Po-Yuan; Ma, Chia-Yu; Wood, W Gibson; Chung, Jing-Gung

    2011-09-15

    We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca(2+) levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca(2+) release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo. Copyright © 2011 Elsevier GmbH. All rights reserved.

  10. Multifunctional effects of honokiol as an anti-inflammatory and anti-cancer drug in human oral squamous cancer cells and xenograft.

    PubMed

    Cho, Jin Hyoung; Jeon, Young-Joo; Park, Seon-Min; Shin, Jae-Cheon; Lee, Tae-Hoon; Jung, Seunggon; Park, Hongju; Ryu, Joohyun; Chen, Hanyong; Dong, Zigang; Shim, Jung-Hyun; Chae, Jung-Il

    2015-01-01

    The aim of this study was to investigate anti-inflammatory and anti-cancer effects of honokiol (HK) in two oral squamous cancer cell carcinoma (OSCC) cell lines, HN22 and HSC4, through the regulation of inducible nitric oxide synthase (iNOS) and endoplasmic reticulum resident protein 44 (ERp44). Griess assay, zymography, and quantitative PCR were performed to study iNOS expression and subsequent nitric oxide (NO) production in OSCC cell lines. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis was used to elucidate the proteins associated with ER stress and cellular cytotoxic response induced by HK. Pull-down assay and molecular modeling were performed to better understand how HK interacts with ERp44. In vitro and in vivo experiments in which ERp44 expression was knocked down were performed to better understand the effects of ERp44 on a cellular level and anti-cancer effects of HK. Expression levels of iNOS and subsequent NO secretion were reduced in OSCC cell lines treated with HK. ERp44 was significantly decreased in OSCC cell lines by HK treatment. HK directly bound to ERp44, and ERp44 knock-down significantly inhibited oral cancer cell proliferation and colony formation. Moreover, HK treatment effectively inhibited tumor growth and ERp44 levels in BALB/c nude mice bearing HN22 cell xenografts. Our findings suggest that HK inhibited inflammation and induced apoptosis by suppressing both iNOS/NO and ERp44 expression in HN22 and HSC4 cells and xenograft tumors, and thus could be a potent anti-inflammatory and anti-cancer drug candidate for human oral cancer treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Ingestion of an isothiocyanate metabolite from cruciferous vegetables inhibits growth of human prostate cancer cell xenografts by apoptosis and cell cycle arrest.

    PubMed

    Chiao, Jen Wei; Wu, Hongyan; Ramaswamy, Gita; Conaway, C Clifford; Chung, Fung-Lung; Wang, Longgui; Liu, Delong

    2004-08-01

    Epidemiological surveys indicate that intake of cruciferous vegetables is inversely related to prostate cancer incidence, although the responsible dietary factors have not been identified. Our studies demonstrated that exposure of human prostate cancer cells in culture to the N-acetylcysteine (NAC) conjugate of phenethyl isothiocyanate (PEITC-NAC), the major metabolite of PEITC that is abundant in watercress, inhibited proliferation and tumorigenesis. The PEITC-NAC is known to mediate cytoprotection at initiation of carcinogenesis. The relevance of PEITC-NAC in diets on the growth of prostate tumor cells has been evaluated in immunodeficient mice with xenografted tumors of human prostate cancer PC-3 cells. The daily PEITC-NAC (8 micromol/g) supplemented diet group showed a significant reduction in tumor size in 100% of the mice during the 9-week treatment period. Tumor weight at autopsy was reduced by 50% compared with mice on the diet without PEITC-NAC (P = 0.05). Mitosis and in vivo 5-bromo-2'-deoxyuridine labeled proliferating cells were reduced in these tumors. The PEITC-NAC diet up-regulated the inhibitors of cyclin-dependent kinases p21WAF-1/Cip-1 and p27Kip1, and reduced the expression of cyclins D and E, indicating they were potential molecular targets. As a result, phosphorylated Rb was significantly decreased and the G1- to S-phase transition retarded. The treated tumors also showed a significant increase in apoptosis as determined by in situ end-labeling, and by poly ADP-ribose polymerase cleavage. This study demonstrates the first in vivo evidence of dietary PEITC-NAC inhibiting tumorigenesis of prostate cancer cells. PEITC-NAC may prevent initiation of carcinogenesis and modulate the post-initiation phase by targeting cell cycle regulators and apoptosis induction.

  12. Protracted dosing of the lipophilic camptothecin analogue AR-67 in non-small cell lung cancer xenografts and humans.

    PubMed

    Tsakalozou, Eleftheria; Adane, Eyob D; Liang, Yali; Arnold, Susanne M; Leggas, Markos

    2014-07-01

    Although preclinical studies on camptothecin antitumor effect have demonstrated the superiority of low-dose protracted dosing, these findings were not replicated in the clinic. 7-t-butyldimethylsilyl-10-hydroxycamptothecin (AR-67) is a camptothecin analogue currently under investigation in early phase clinical trials. To maximize the therapeutic potential of AR-67, we sought to identify factors that affect response to treatment. After determining the maximum tolerated dose using neutropenia as a toxicity endpoint, xenografts received AR-67 under varying dosing schedules and were monitored for survival. On the last treatment day, tumor tissue was collected and topoisomerase 1 (Top1), γH2AX, caspase 3 and PARP protein content was evaluated. AR-67 plasma and tumor pharmacokinetics were also studied in mice and cancer patients who were administered AR-67 as a 1-h intravenous infusion on days 1, 4, 8, 12 and 15 every 21 days. Low-dose protracted dosing schedules increased animal survival compared to less frequent, but higher-dose courses and the expression of Top1 and γH2AX were schedule dependent. Fatigue and neutropenia were the dose-limiting toxicities identified in patients receiving AR-67. Finally, elimination of AR-67 from the tumor site was slower in both xenografts and tumor of a patient enrolled in the pilot clinical trial. We demonstrated that low-dose protracted dosing schedules of AR-67 are therapeutically effective and Top1 reflects the biological activity of AR-67 in xenografts. Moreover, the tumor pharmacokinetics as well as the efficacy and safety of AR-67 given intermittently to cancer patients warrant further investigation.

  13. Treatment of small-cell lung cancer xenografts with iodine-313-anti-neural cell adhesion molecule monoclonal antibody and evaluation of absorbed dose in tissue

    SciTech Connect

    Hosono, Makoto; Endo, Keigo; Hosono, Masako N.

    1994-02-01

    Human small-cell lung cancer (SCLC) is considered a feasible target for immunotherapy using a radiolabeled monoclonal antibody (Mab). A murine Mab, NE150 (IgG1), reacts with the neural cell adhesion molecule, which is identical to cluster 1 antigen of SCLC. To estimate their therapeutic effects, NE150 and an isotype-matched control Mab were labeled with {sup 131}I and administered intravenously as a single dose into athymic mice inoculated with a NCI-H69 SCLC xenograft. The absorbed dose in organs was also examined based upon a long-term biodistribution study of {sup 131}I-NE150. Tumors initial volume 563.4 {plus_minus} 223.5 mm{sup 3} treated with 11.1 MBq (300 {mu}Ci) of {sup 131}I-NE150 diminished and became invisible at days 30-33, demonstrating a 60-day mean growth delay to reach a tripled initial volume compared with sham-treated tumors. Cumulative absorbed doses were estimated to be 2310, 410, 500, 330, and 790 cGy for the tumor, liver, kidney, spleen and lung, respectively. Iodine-131-NE150 had potent therapeutic effects against SCLC transplants in athymic mice, however, careful assessment of the side effects, improvement of radioiodination and chimerization of the Mab might be necessary to achieve efficient targeting in clinical therapeutic applications. 25 refs., 2 figs., 3 tabs.

  14. Human amniotic fluid-derived stem cells expressing cytosine deaminase and thymidine kinase inhibits the growth of breast cancer cells in cellular and xenograft mouse models.

    PubMed

    Kang, N-H; Hwang, K-A; Yi, B-R; Lee, H J; Jeung, E-B; Kim, S U; Choi, K-C

    2012-06-01

    As human amniotic fluid-derived stem cells (hAFSCs) are capable of multiple lineage differentiation, extensive self-renewal and tumor targeting, they may be valuable for clinical anticancer therapies. In this study, we used hAFSCs as vehicles for targeted delivery of therapeutic suicide genes to breast cancer cells. hAFSCs were engineered to produce AF2.CD-TK cells in order to express two suicide genes encoding bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) that convert non-toxic prodrugs, 5-fluorocytosine (5-FC) and mono-phosphorylate ganciclovir (GCV-MP), into cytotoxic metabolites, 5-fluorouracil (5-FU) and triphosphate ganciclovir (GCV-TP), respectively. In cell viability test in vitro, AF2.CD-TK cells inhibited the growth of MDA-MB-231 human breast cancer cells in the presence of the 5-FC or GCV prodrugs, or a combination of these two reagents. When the mixture of 5-FC and GCV was treated together, an additive cytotoxic effect was observed in the cell viability. In animal experiments using female BALB/c nude mouse xenografts, which developed by injecting MDA-MB-231 cells, treatment with AF2.CD-TK cells in the presence of 5-FC and GCV significantly reduced tumor volume and weight to the same extent seen in the mice treated with 5-FU. Histopathological and fluorescent staining assays further showed that AF2.CD-TK cells were located exactly at the site of tumor formation. Furthermore, breast tissues treated with AF2.CD-TK cells and two prodrugs maintained their normal structures (for example, the epidermis and reticular layers) while breast tissue structures in 5-FU-treated mice were almost destroyed by the potent cytotoxicity of the drug. Taken together, these results indicate that AF2.CD-TK cells can serve as excellent vehicles in a novel therapeutic cell-based gene-directed prodrug system to selectively target breast malignancies.

  15. Detection of cadherin-17 in human colon cancer LIM1215 cell secretome and tumour xenograft-derived interstitial fluid and plasma.

    PubMed

    Bernhard, Oliver K; Greening, David W; Barnes, Thomas W; Ji, Hong; Simpson, Richard J

    2013-11-01

    Colorectal cancer (CRC), one of the most prevalent cancers in the western world, is treatable if detected early. However, 70% of CRC is detected at an advanced stage. This is largely due to the inadequacy of current faecal occult blood screening testing and costs involved in conducting population-based colonoscopy, the 'gold standard' for CRC detection. Another biomarker for CRC, carcinoembryonic antigen, while useful for monitoring CRC recurrence, is ineffective, lacking the specificity required early detection of CRC. For these reasons there is a need for more effective blood-based markers for early CRC detection. In this study we targeted glycoproteins secreted from the human colon carcinoma cell line LIM1215 as a source of potential CRC biomarkers. Secreted candidate glycoproteins were confirmed by MS and validated by Western blot analysis of tissue/tumour interstitial fluid (Tif) from LIM1215 xenograft tumours grown in immunocompromised mice. Overall, 39 glycoproteins were identified in LIM1215 culture media (CCM) and 5 glycoproteins in LIM1215 tumour xenograft Tif; of these, cadherin-17 (CDH17), galectin-3 binding protein (LGALS3BP), and tyrosine-protein kinase-like 7 (PTK7) were identified in both CM and glycosylation motifs. Swiss-Prot was used to annotate Tif. Many of the glycoproteins identified in this study (e.g., AREG, DSG2, EFNA1, EFNA3, EFNA4, EPHB4, ST14, and TIMP1) have been reported to be implicated in CRC biology. Interestingly, the cadherin-17 ectodomain, but not full length cadherin-17, was identified in CM, Tif and plasma derived from mice bearing the LIM1215 xenograft tumour. To our knowledge, this is the first report of the cadherin-17 ectodomain in plasma. In this study, we report for the first time that the presence of full-length cadherin-17 in exosomes released into the CM. This article is part of a Special Issue entitled: An Updated Secretome.

  16. A Novel Compound ARN-3236 Inhibits Salt-Inducible Kinase 2 and Sensitizes Ovarian Cancer Cell Lines and Xenografts to Paclitaxel.

    PubMed

    Zhou, Jinhua; Alfraidi, Albandri; Zhang, Shu; Santiago-O'Farrill, Janice M; Yerramreddy Reddy, Venkata Krishna; Alsaadi, Abdulkhaliq; Ahmed, Ahmed A; Yang, Hailing; Liu, Jinsong; Mao, Weiqun; Wang, Yan; Takemori, Hiroshi; Vankayalapati, Hariprasad; Lu, Zhen; Bast, Robert C

    2017-04-15

    Purpose: Salt-inducible kinase 2 (SIK2) is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here, we examine the effects of a novel small-molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer.Experimental Design: SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear-centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death, and reduce AKT/survivin signaling.Results: SIK2 is overexpressed in approximately 30% of high-grade serous ovarian cancers. ARN-3236 inhibited the growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 μmol/L, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson r = -0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip (P = 0.028) and OVCAR8 xenografts. In at least three cell lines, a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest, and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression.Conclusions: ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity. Clin Cancer Res; 23(8); 1945-54. ©2016 AACR. ©2016 American Association for Cancer Research.

  17. Involvement of doublesex and mab-3-related transcription factors in human female germ cell development demonstrated by xenograft and interference RNA strategies.

    PubMed

    Poulain, Marine; Frydman, Nelly; Tourpin, Sophie; Muczynski, Vincent; Mucsynski, Vincent; Souquet, Benoit; Benachi, Alexandra; Habert, René; Rouiller-Fabre, Virginie; Livera, Gabriel

    2014-10-01

    We identified three doublesex and mab-3-related transcription factors (DMRT) that were sexually differentially expressed in human fetal gonads and present in the ovaries at the time of meiotic initiation. These were also identified in murine embryonic female germ cells. Among these, we focused on DMRTA2 (DMRT5), whose function is unknown in the developing gonads, and clarified its role in human female fetal germ cells, using an original xenograft model. Early human fetal ovaries (8-11 weeks post-fertilization) were grafted into nude mice. Grafted ovaries developed normally, with no apparent overt changes, when compared with ungrafted ovaries at equivalent developmental stages. Appropriate germ cell density, mitotic/meiotic transition, markers of meiotic progression and follicle formation were evident. Four weeks after grafting, mice were treated with siRNA, specifically targeting human DMRTA2 mRNA. DMRTA2 inhibition triggered an increase in undifferentiated FUT4-positive germ cells and a decrease in the percentage of meiotic γH2AX-positive germ cells, when compared with mice that were injected with control siRNA. Interestingly, the expression of markers associated with pre-meiotic germ cell differentiation was also impaired, as was the expression of DMRTB1 (DMRT6) and DMRTC2 (DMRT7). This study reveals, for the first time, the requirement of DMRTA2 for normal human female embryonic germ cell development. DMRTA2 appears to be necessary for proper differentiation of oogonia, prior to entry into meiosis, in the human species. Additionally, we developed a new model of organ xenografting, coupled with RNA interference, which provides a useful tool for genetic investigations of human germline development. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. [Anesthesia in insulinoma surgery: report of 18 cases].

    PubMed

    Cortés, C; Mora, A; Torres, J; Olivé, M; Cabarrocas, E

    1991-01-01

    We reviewed 18 patients operated on insulinoma during the last 16 years (1974-1990). The previous symptoms, the clinical and biochemical diagnostic procedures, the localization before and during operation, and the medical and surgical treatment are reported. Constant and continuous glucose solution perfusion at the preoperative stage prevented the occurrence of hypoglicemia without interference with the rebound hyperglicemia that follows tumor exeresis. Neuroleptanalgesia with nitrogen protoxide and competitive muscular relaxant agents was satisfactory in all instances. Prevention and treatment of postoperative complications are also discussed in this study.

  19. PKM2 Thr454 phosphorylation increases its nuclear translocation and promotes xenograft tumor growth in A549 human lung cancer cells

    SciTech Connect

    Yu, Zhenhai; Huang, Liangqian; Qiao, Pengyun; Jiang, Aifang; Wang, Li; Yang, Tingting; Tang, Shengjian; Zhang, Wei; Ren, Chune

    2016-05-13

    Pyruvate kinase M2 (PKM2) is a key enzyme of glycolysis which is highly expressed in many tumor cells, and plays an important role in the Warburg effect. In previous study, we found PIM2 phosphorylates PKM2 at Thr454 residue (Yu, etl 2013). However, the functions of PKM2 Thr454 modification in cancer cells still remain unclear. Here we find PKM2 translocates into the nucleus after Thr454 phosphorylation. Replacement of wild type PKM2 with a mutant (T454A) enhances mitochondrial respiration, decreases pentose phosphate pathway, and enhances chemosensitivity in A549 cells. In addition, the mutant (T454A) PKM2 reduces xenograft tumor growth in nude mice. These findings demonstrate that PKM2 T454 phosphorylation is a potential therapeutic target in lung cancer.

  20. Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality.

    PubMed

    Jiang, Yanyan; Verbiest, Tom; Devery, Aoife M; Bokobza, Sivan M; Weber, Anika M; Leszczynska, Katarzyna B; Hammond, Ester M; Ryan, Anderson J

    2016-06-01

    Poly(ADP-ribose) polymerase (PARP) inhibitors potentiate radiation therapy in preclinical models of human non-small cell lung cancer (NSCLC) and other types of cancer. However, the mechanisms underlying radiosensitization in vivo are incompletely understood. Herein, we investigated the impact of hypoxia on radiosensitization by the PARP inhibitor olaparib in human NSCLC xenograft models. NSCLC Calu-6 and Calu-3 cells were irradiated in the presence of olaparib or vehicle under normoxic (21% O2) or hypoxic (1% O2) conditions. In vitro radiosensitivity was assessed by clonogenic survival assay and γH2AX foci assay. Established Calu-6 and Calu-3 subcutaneous xenografts were treated with olaparib (50 mg/kg, daily for 3 days), radiation (10 Gy), or both. Tumors (n=3/group) were collected 24 or 72 hours after the first treatment. Immunohistochemistry was performed to assess hypoxia (carbonic anhydrase IX [CA9]), vessels (CD31), DNA double strand breaks (DSB) (γH2AX), and apoptosis (cleaved caspase 3 [CC3]). The remaining xenografts (n=6/group) were monitored for tumor growth. In vitro, olaparib showed a greater radiation-sensitizing effect in Calu-3 and Calu-6 cells in hypoxic conditions (1% O2). In vivo, Calu-3 tumors were well-oxygenated, whereas Calu-6 tumors had extensive regions of hypoxia associated with down-regulation of the homologous recombination protein RAD51. Olaparib treatment increased unrepaired DNA DSB (P<.001) and apoptosis (P<.001) in hypoxic cells of Calu-6 tumors following radiation, whereas it had no significant effect on radiation-induced DNA damage response in nonhypoxic cells of Calu-6 tumors or in the tumor cells of well-oxygenated Calu-3 tumors. Consequently, olaparib significantly increased radiation-induced growth inhibition in Calu-6 tumors (P<.001) but not in Calu-3 tumors. Our data suggest that hypoxia potentiates the radiation-sensitizing effects of olaparib by contextual synthetic killing, and that tumor hypoxia may be a

  1. Selective antitumor effect of neural stem cells expressing cytosine deaminase and interferon-beta against ductal breast cancer cells in cellular and xenograft models.

    PubMed

    Yi, Bo-Rim; Hwang, Kyung-A; Aboody, Karen S; Jeung, Eui-Bae; Kim, Seung U; Choi, Kyung-Chul

    2014-01-01

    Due to their inherent tumor-tropic properties, genetically engineered stem cells may be advantageous for gene therapy treatment of various human cancers, including brain, liver, ovarian, and prostate malignancies. In this study, we employed human neural stem cells (HB1.F3; hNSCs) transduced with genes expressing Escherichia coli cytosine deaminase (HB1.F3.CD) and human interferon-beta (HB1.F3.CD.IFN-β) as a treatment strategy for ductal breast cancer. CD can convert the prodrug 5-fluorocytosine (5-FC) to its active chemotherapeutic form, 5-fluorouracil (5-FU), which induces a tumor-killing effect through DNA synthesis inhibition. IFN-β also strongly inhibits tumor growth by the apoptotic process. RT-PCR confirmed that HB1.F3.CD cells expressed CD and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β. A modified transwell migration assay showed that HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward MCF-7 and MDA-MB-231 human breast cancer cells. In hNSC-breast cancer co-cultures the viability of breast cancer cells which were significantly reduced by HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. The tumor inhibitory effect was greater with the HB1.F3.CD.IFN-β cells, indicating an additional effect of IFN-β to 5-FU. In addition, the tumor-tropic properties of these hNSCs were found to be attributed to chemoattractant molecules secreted by breast cancer cells, including stem cell factor (SCF), c-kit, vascular endothelial growth factor (VEGF), and VEGF receptor 2. An in vivo assay performed using MDA-MB-231/luc breast cancer mammary fat pad xenografts in immunodeficient mice resulted in 50% reduced tumor growth and increased long-term survival in HB1.F3.CD and HB1.F3.CD.IFN-β plus 5-FC treated mice relative to controls. Our results suggest that hNSCs genetically modified to express CD and/or IFN-β genes can be used as a novel targeted cancer gene therapy.

  2. Pediatric medulloblastoma xenografts including molecular subgroup 3 and CD133+ and CD15+ cells are sensitive to killing by oncolytic herpes simplex viruses

    PubMed Central

    Friedman, Gregory K.; Moore, Blake P.; Nan, Li; Kelly, Virginia M.; Etminan, Tina; Langford, Catherine P.; Xu, Hui; Han, Xiaosi; Markert, James M.; Beierle, Elizabeth A.; Gillespie, G. Yancey

    2016-01-01

    Background Childhood medulloblastoma is associated with significant morbidity and mortality that is compounded by neurotoxicity for the developing brain caused by current therapies, including surgery, craniospinal radiation, and chemotherapy. Innate therapeutic resistance of some aggressive pediatric medulloblastoma has been attributed to a subpopulation of cells, termed cancer-initiating cells or cancer stemlike cells (CSCs), marked by the surface protein CD133 or CD15. Brain tumors characteristically contain areas of pathophysiologic hypoxia, which has been shown to drive the CSC phenotype leading to heightened invasiveness, angiogenesis, and metastasis. Novel therapies that target medulloblastoma CSCs are needed to improve outcomes and decrease toxicity. We hypothesized that oncolytic engineered herpes simplex virus (oHSV) therapy could effectively infect and kill pediatric medulloblastoma cells, including CSCs marked by CD133 or CD15. Methods Using 4 human pediatric medulloblastoma xenografts, including 3 molecular subgroup 3 tumors, which portend worse patient outcomes, we determined the expression of CD133, CD15, and the primary HSV-1 entry molecule nectin-1 (CD111) by fluorescence activated cell sorting (FACS) analysis. Infectability and cytotoxicity of clinically relevant oHSVs (G207 and M002) were determined in vitro and in vivo by FACS, immunofluorescent staining, cytotoxicity assays, and murine survival studies. Results We demonstrate that hypoxia increased the CD133+ cell fraction, while having the opposite effect on CD15 expression. We established that all 4 xenografts, including the CSCs, expressed CD111 and were highly sensitive to killing by G207 or M002. Conclusions Pediatric medulloblastoma, including Group 3 tumors, may be an excellent target for oHSV virotherapy, and a clinical trial in medulloblastoma is warranted. PMID:26188016

  3. Determination of Rottlerin, a Natural Protein Kinases C Inhibitor, in Pancreatic Cancer Cells and Mouse Xenografts by RP-HPLC Method

    PubMed Central

    Lu, Qing-Yi; Zhang, Lifeng; Lugea, Aurelia; Moro, Aune; Edderkaoui, Mouad; Eibl, Guido; Pandol, Stephen J.; Go, Vay-Liang W

    2014-01-01

    Rottlerin is a natural polyphenolic ketone isolated from the pericarps of Mallotus phillippinensis. In previous studies we showed that parenteral administration of rottlerin reduced tumor growth in murine xenograft models of pancreatic cancer. The aim of this study was to develop a simple and validated method for the quantitative determination of rottlerin in plasma and tumor tissues of mice fed a rottlerin diet. A xenograft model of pancreatic cancer was prepared by injection of 2×106 HPAF-II cells subcutaneously into nude mice. One week before tumor implantation, mice were randomly allocated to standard diet (AIN76A) and standard diet supplement with 0.012% rottlerin (n=6 per group). Mice were sacrificed after 6 weeks on diets. Rottlerin was extracted from the plasma and tissues using protein precipitation-extraction and analyzed by reverse-phase HPLC-DAD method. The same HPLC method was also applied to determine rottlerin levels in conditioned culture media and in cell lysates from HPAF-II cells exposed to 25 µM concentration of rottlerin. A substantial amount of rottlerin was detected in tumor (2.11 ± 0.25 nmol/g tissue) and plasma (2.88 ± 0.41 µM) in mice fed rottlerin diet. In addition, significant levels of rottlerin (57.4 ± 5.4 nmol/mg protein) were detected in cell lysates from rottlerin-treated HPAF-II cells. These data indicate that rottlerin is efficiently absorbed in cells and tissues both in vivo and in vitro and suggest a strong potential for rottlerin as a preventive or adjuvant supplement for pancreatic cancer. PMID:24482742

  4. The imaging of insulinomas using a radionuclide-labelled molecule of the GLP-1 analogue liraglutide: a new application of liraglutide.

    PubMed

    Lv, Jing; Pan, Yu; Li, Xiao; Cheng, Dengfeng; Liu, Shuai; Shi, Hongcheng; Zhang, Yifan

    2014-01-01

    This study explores a new, non-invasive imaging method for the specific diagnosis of insulinoma by providing an initial investigation of the use of 125I-labelled molecules of the glucagon-like peptide-1 (GLP-1) analogue liraglutide for in vivo and in vitro small-animal SPECT/CT (single-photon emission computed tomography/computed tomography) imaging of insulinomas. Liraglutide was labelled with 125I by the Iodogen method. The labelled 125I-liraglutide compound and insulinoma cells from the INS-1 cell line were then used for in vitro saturation and competitive binding experiments. In addition, in a nude mouse model, the use of 125I-liraglutide for the in vivo small-animal SPECT/CT imaging of insulinomas and the resulting distribution of radioactivity across various organs were examined. The labelling of liraglutide with 125I was successful, yielding a labelling rate of approximately 95% and a radiochemical purity of greater than 95%. For the binding between 125I-liraglutide and the GLP-1 receptor on the surface of INS-1 cells, the equilibrium dissociation constant (Kd) was 128.8 ± 30.4 nmol/L(N = 3), and the half-inhibition concentration (IC50) was 542.4 ± 187.5 nmol/L(N = 3). Small-animal SPECT/CT imaging with 125I-liraglutide indicated that the tumour imaging was clearest at 90 min after the 125I-liraglutide treatment. An examination of the in vivo distribution of radioactivity revealed that at 90 min after the 125I-liraglutide treatment, the target/non-target (T/NT) ratio for tumour and muscle tissue was 4.83 ± 1.30(N = 3). Our study suggested that 125I-liraglutide was predominantly metabolised and cleared by the liver and kidneys. The radionuclide 125I-liraglutide can be utilised for the specific imaging of insulinomas, representing a new non-invasive approach for the in vivo diagnosis of insulinomas.

  5. Specific knockdown of uPA/uPAR attenuates invasion in glioblastoma cells and xenografts by inhibition of cleavage and trafficking of Notch -1 receptor

    PubMed Central

    2011-01-01

    Background uPA/uPAR is a multifunctional system that is over expressed in many cancers and plays a critical role in glioblastoma (GBM) invasion. Previous studies from our lab have also shown that uPA/uPAR down regulation inhibits cancer cell invasion in SNB 19 GBM cells. Methods As Notch 1 is known to be over expressed and promotes invasion in glioblastoma, we therefore tested our hypothesis of whether down regulation of uPA/uPAR, singly or in tandem, attenuates GBM invasion via Notch 1 receptor. Targeted down regulation of uPA/uPAR, either singly or simultaneously, inhibited the anchorage independent growth of U251MG and GBM xenograft cell lines 4910 and 5310 as assessed by soft agar colony formation assay. Expression of all four Notch receptors was confirmed in GBM tissue array analysis by immunohistochemistry. Results Down regulation of uPA/uPAR, either singly or simultaneously, in U251 MG and tumor xenografts inhibited the cleavage of the Notch receptor between the Gly 1743 and Val 1744 positions, thereby suggesting inhibition of activated cytosolic fragment-related Notch gene transcription. Morphological analysis confirmed inhibition of NICD when U251 MG cells were treated with puPA, puPAR or pU2. uPA/uPAR down regulation inhibited Notch 1 mRNA in all three examined cell lines. uPA/uPAR shRNA down regulated nuclear activation of NF-κB subunits and phosphorylation of AKT/mTOR pathway in U251 MG and GBM xenografts. puPA down regulated NICD and HES induced phosphorylation of AKT/ERK and NF-κB. Down regulation of Notch 1 using siRNA inhibited uPA activity as shown by fibrinogen zymography. It also decreased uPA expression levels as shown by western blotting. Exogenous addition of uPA activated Notch 1 in uPAR antisense U251 MG cells and also in uPAR antisense cells transfected with siRNA against Delta and Jagged. The Notch 1 receptor co-localized with LAMP-1, a marker for lysosomes in uPA, uPAR and U2, down regulated U251 MG cells which probably indicates

  6. Patient-derived xenografts from non-small cell lung cancer brain metastases are valuable translational platforms for the development of personalized targeted therapy.

    PubMed

    Lee, Hye Won; Lee, Jung-Il; Lee, Se Jeong; Cho, Hyun Jung; Song, Hye Jin; Jeong, Da Eun; Seo, Yun Jee; Shin, Sang; Joung, Je-Gun; Kwon, Yong-Jun; Choi, Yoon-La; Park, Woong-Yang; Lee, Hyun Moo; Seol, Ho Jun; Shim, Young Mog; Joo, Kyeung Min; Nam, Do-Hyun

    2015-03-01

    The increasing prevalence of distant metastases from non-small cell lung cancer (NSCLC) indicates an urgent need for novel therapeutic modalities. Brain metastasis is particularly common in NSCLC, with severe adverse effects on clinical prognosis. Although the molecular heterogeneity of NSCLC and availability of various targeted agents suggest personalized therapeutic approaches for such brain metastases, further development of appropriate preclinical models is needed to validate the strategies. We established patient-derived xenografts (PDX) using NSCLC brain metastasis surgical samples and elucidated their possible preclinical and clinical implications for personalized treatment. NSCLC brain metastases (n = 34) showed a significantly higher successful PDX establishment rate than primary specimens (n = 64; 74% vs. 23%). PDXs derived from NSCLC brain metastases recapitulated the pathologic, genetic, and functional properties of corresponding parental tumors. Furthermore, tumor spheres established in vitro from the xenografts under serum-free conditions maintained their in vivo brain metastatic potential. Differential phenotypic and molecular responses to 20 targeted agents could subsequently be screened in vitro using these NSCLC PDXs derived from brain metastases. Although PDX establishment from primary NSCLCs was significantly influenced by histologic subtype, clinical aggressiveness, and genetic alteration status, the brain metastases exhibited consistently adequate in vivo tumor take rate and in vitro tumor sphere formation capacity, regardless of clinical and molecular conditions. Therefore, PDXs from NSCLC brain metastases may better represent the heterogeneous advanced NSCLC population and could be utilized as preclinical models to meet unmet clinical needs such as drug screening for personalized treatments. ©2014 American Association for Cancer Research.

  7. SLC25A22 Promotes Proliferation and Survival of Colorectal Cancer Cells With KRAS Mutations and Xenograft Tumor Progression in Mice via Intracellular Synthesis of Aspartate.

    PubMed

    Wong, Chi Chun; Qian, Yun; Li, Xiaona; Xu, Jiaying; Kang, Wei; Tong, Joanna H; To, Ka-Fai; Jin, Ye; Li, Weilin; Chen, Huarong; Go, Minnie Y Y; Wu, Jian-Lin; Cheng, Ka Wing; Ng, Simon S M; Sung, Joseph J Y; Cai, Zongwei; Yu, Jun

    2016-11-01

    Many colorectal cancer (CRC) cells contain mutations in KRAS. Analyses of CRC cells with mutations in APC or CTNNB1 and KRAS identified SLC25A22, which encodes mitochondrial glutamate transporter, as a synthetic lethal gene. We investigated the functions of SLC25A22 in CRC cells with mutations in KRAS. We measured levels of SLC25A22 messenger RNA and protein in paired tumor and nontumor colon tissues collected from 130 patients in Hong Kong and 17 patients in China and compared protein levels with patient survival times. Expression of SLC25A22 was knocked down in KRAS mutant CRC cell lines (DLD1, HCT116, LOVO, SW480, SW620, and SW1116) and CRC cell lines without mutations in KRAS (CACO-2, COLO205, HT29, and SW48); cells were analyzed for colony formation, proliferation, glutaminolysis and aspartate synthesis, and apoptosis in Matrigel and polymerase chain reaction array analyses. DLD1 and HCT116 cells with SLC25A22 knockdown were grown as xenograft tumors in nude mice; tumor growth and metastasis were measured. SLC25A22 was expressed ectopically in HCT116 cells, which were analyzed in vitro and grown as xenograft tumors in nude mice. Levels of SLC25A22 messenger RNA and protein were increased in colorectal tumor tissues compared with matched nontumor colon tissues; increased protein levels were associated with shorter survival times of patients (P = .01). Knockdown of SLC25A22 in KRAS mutant CRC cells reduced their proliferation, migration, and invasion in vitro, and tumor formation and metastasis in mice, compared with cells without SLC25A22 knockdown. Knockdown of SLC25A22 reduced aspartate biosynthesis, leading to apoptosis, decreased cell proliferation in KRAS mutant CRC cells. Incubation of KRAS mutant CRC cells with knockdown of SLC25A22 with aspartate increased proliferation and reduced apoptosis, which required GOT1, indicating that oxaloacetate is required for cell survival. Decreased levels of oxaloacetate in cells with knockdown of SLC25A22 reduced

  8. Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.

    PubMed

    Fischer, Walter; Gustafsson, Lotta; Mossberg, Ann-Kristin; Gronli, Janne; Mork, Sverre; Bjerkvig, Rolf; Svanborg, Catharina

    2004-03-15

    Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human alpha-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from alpha-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and alpha-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all alpha-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm(3)). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.

  9. Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model.

    PubMed

    Maisel, Daniela; Birzele, Fabian; Voss, Edgar; Nopora, Adam; Bader, Sabine; Friess, Thomas; Goller, Bernhard; Laifenfeld, Daphna; Weigand, Stefan; Runza, Valeria

    2016-01-01

    CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.

  10. Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model

    PubMed Central

    Maisel, Daniela; Birzele, Fabian; Voss, Edgar; Nopora, Adam; Bader, Sabine; Friess, Thomas; Goller, Bernhard; Laifenfeld, Daphna; Weigand, Stefan; Runza, Valeria

    2016-01-01

    CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages. PMID:27463372

  11. Usefulness of artificial endocrine pancreas during resection of insulinoma.

    PubMed

    Hirose, Kayo; Kawahito, Shinji; Mita, Naoji; Takaishi, Kazumi; Kawahara, Tomiya; Soga, Tomohiro; Katayama, Toshiko; Imura, Satoru; Morine, Yuji; Ikemoto, Tetsuya; Shimada, Mitsuo; Matsuhisa, Munehide; Kitahata, Hiroshi

    2014-01-01

    A 71-year-old woman had an episode of syncope due to hypoglycemia of 27 mg/dl. She was diagnosed with insulinoma and scheduled for laparoscopic enucleation along with the use of an artificial endocrine pancreas (STG-22, Nikkiso Co., Ltd., Tokyo, Japan). Anesthesia was maintained with sevoflurane and remifentanil. Her blood glucose level was controlled using the artificial endocrine pancreas, which enabled continuous blood glucose monitoring and computer-operated glucose and insulin infusion to maintain the blood glucose level at a steady state. The target concentration of blood glucose was set at 80-120 mg/dl during surgery. Until removal of the tumor, the blood glucose level was kept at around 80-100 mg/dl. After removal of the tumor, the blood glucose level gradually increased, but it was kept in the normal range by the artificial endocrine pancreas. The artificial endocrine pancreas was useful to monitor and maintain blood glucose levels during and after the removal of insulinoma, without any hyper- or hypoglycemia.

  12. Early events in xenograft development from the human embryonic stem cell line HS181--resemblance with an initial multiple epiblast formation.

    PubMed

    Gertow, Karin; Cedervall, Jessica; Jamil, Seema; Ali, Rouknuddin; Imreh, Marta P; Gulyas, Miklos; Sandstedt, Bengt; Ahrlund-Richter, Lars

    2011-01-01

    Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart.

  13. Silencing Op18/stathmin by RNA Interference Promotes the Sensitivity of Nasopharyngeal Carcinoma Cells to Taxol and High-Grade Differentiation of Xenografted Tumours in Nude Mice.

    PubMed

    Lin, Xuechi; Yu, Ting; Zhang, Lingxi; Chen, Sangyan; Chen, Xian; Liao, Ying; Long, Dan; Shen, Fang

    2016-12-01

    Nasopharyngeal carcinoma (NPC) is a refractory tumour, and chemotherapy is one of the primary treatment modalities. Oncoprotein 18 (Op18)/stathmin is a conserved small cytosolic phosphoprotein and highly expressed in tumours, which plays a vital role in maintaining the malignant phenotype of tumours. Taxol is a clinically widely used chemotherapeutic agent for a broad range of taxol-resistant tumours. This study showed that Op18/stathmin silencing by RNA interference (RNAi) combined taxol cooperatively improved cellular apoptosis in CNE1 cells mainly via initiating endogenous death receptor pathway, impaired the capabilities of cellular proliferation and cellular migration and down-regulated the half maximal inhibitory concentration (IC50 ) of taxol, meanwhile decreased the expression of the upstream extracellular regulated kinase 1 (ERK1) in vitro. Evidence also showed that taxol cytotoxicity was markedly augmented for Op18/stathmin RNAi in other NPC cells. In vivo animal experiments have demonstrated that early combination of Op18/stathmin silencing and taxol evidently inhibited tumourigenicity of CNE1 cells and growth of xenografted tumours in nude mice. Remarkably, silencing Op18/stathmin by RNAi still promoted transformation of late-stage CNE1 cells in NPC-xenografted tumours from moderately to highly differentiated and inhibited the pleiotropic cytokine interleukin-10 (IL-10) autocrine by transplanted tumours. These findings suggest that silencing Op18/stathmin by RNAi promotes chemosensitization of NPC to taxol and reverses malignant phenotypes of NPC, which provides a new clue for treating drug-resistant tumours. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  14. In Silico cancer cell versus stroma cellularity index computed from species-specific human and mouse transcriptome of xenograft models: towards accurate stroma targeting therapy assessment

    PubMed Central

    2014-01-01

    Background The current state of the art for measuring stromal response to targeted therapy requires burdensome and rate limiting quantitative histology. Transcriptome measures are increasingly affordable and provide an opportunity for developing a stromal versus cancer ratio in xenograft models. In these models, human cancer cells are transplanted into mouse host tissues (stroma) and together coevolve into a tumour microenvironment. However, profiling the mouse or human component separately remains problematic. Indeed, laser capture microdissection is labour intensive. Moreover, gene expression using commercial microarrays introduces significant and underreported cross-species hybridization errors that are commonly overlooked by biologists. Method We developed a customized dual-species array, H&M array, and performed cross-species and species-specific hybridization measurements. We validated a new methodology for establishing the stroma vs cancer ratio using transcriptomic data. Results In the biological validation of the H&M array, cross-species hybridization of human and mouse probes was significantly reduced (4.5 and 9.4 fold reduction, respectively; p < 2x10-16 for both, Mann-Whitney test). We confirmed the capability of the H&M array to determine the stromal to cancer cells ratio based on the estimation of cellularity index of mouse/human mRNA content in vitro. This new metrics enable to investigate more efficiently the stroma-cancer cell interactions (e.g. cellularity) bypassing labour intensive requirement and biases of laser capture microdissection. Conclusion These results provide the initial evidence of improved and cost-efficient analytics for the investigation of cancer cell microenvironment, using species-specificity arrays specifically designed for xenografts models. PMID:25079962

  15. Effects of PHA-665752 and cetuximab combination treatment on in vitro and murine xenograft growth of human colorectal cancer cells with KRAS or BRAF mutations.

    PubMed

    Jia, Yi-Tao; Yang, Dong-Hai; Zhao, Zhaolong; Bi, Xiao-Hui; Han, Wei-Hua; Feng, Bo; Zhi, Jie; Gu, Bin; Duan, Zhihui; Wu, Jian-Hua; Ju, Ying-Chao; Wang, Ming-Xia; Li, Zhong-Xin

    2017-03-30

    It remains unknown whether blockade of c-Met signaling and epidermal growth factor receptor signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. In this study, we investigated the effects of the c-Met inhibitor PHA-665752 alone and in combination with cetuximab on the growth of human CRC cells in vitro and in mouse xenografts. Human CRC cell lines (Caco2, HCT-116, and HT-29) and mice bearing HCT-116 xenografts were treated with cetuximab in the absence or presence of PHA-665752. Cell viability and apoptosis were examined using the MTT and TUNEL assays, respectively. Vimentin was measured by immunohistochemistry as a marker for epithelial-to-mesenchymal transition. Western blotting was used to determine signaling protein expression levels. The MTT assay showed that the growth of Caco2, HCT-116, and HT-29 cells was inhibited by PHA-665752 in a dose-dependent manner, but only Caco2 cell growth was suppressed by cetuximab. Combination treatment with PHA-665752 and cetuximab inhibited the proliferation of Caco2 cells and RAS mutant CRC cell lines. However, relative to the PHA-665752-alone treatment group, HT-29 cells with a BRAF mutation showed no noticeable effect. The mean tumor volume in mice treated with cetuximab in combination with PHA-665752 was significantly smaller than that in the mice treated with only cetuximab (P = 0.033) or PHA-665752 (P < 0.01). Similarly, the expression of vimentin in the mice treated with PHA-665752 in combination with cetuximab was significantly lower than that in the mice treated with cetuximab or PHA-665752 alone (P < 0.05 in each case). TUNEL assays revealed that treatment with PHA-665752 in combination with cetuximab markedly increased CRC cell apoptosis. Western blotting analysis of signaling protein expression showed that PHA-665752 inhibited Met phosphorylation (P < 0.05). In addition, treatment with cetuximab alone or in combination with PHA-665752 effectively inhibited EGFR

  16. Intra-tumour molecular heterogeneity of clear cell renal cell carcinoma reveals the diversity of the response to targeted therapies using patient-derived xenograft models

    PubMed Central

    Hong, Baoan; Yang, Yong; Guo, Sheng; Duoerkun, Shayiremu; Deng, Xiaohu; Chen, Dawei; Yu, Shijun; Qian, Wubin; Li, Qixiang; Li, Qing; Gong, Kan; Zhang, Ning

    2017-01-01

    Inter- and intra-tumour molecular heterogeneity is increasingly recognized in clear cell renal cell carcinoma (ccRCC). It may partially explain the diversity of responses to targeted therapies and the various clinical outcomes. In this study, a 56-year-old male ccRCC patient with multiple metastases received radical nephrectomy and resection of the metastatic tumour in chest wall. The surgical specimens were implanted into nude mice to establish patient-derived xenograft (PDX) models with KI2367 model derived from the primary tumour and KI2368 model from the metastastic tumour. The two modles were treated with Sorafenib, Sunitinib, Axitinib, combined Sorafenib/Sunitinib, or alternating therapy of Sorafenib and Sunitinib. Significant anti-tumour activity was found in KI2367 treated with Sorafenib/Sunitinib monotherapy, combined Sorafenib/Sunitinib, and alternating therapy of Sorafenib/Sunitinib (P<0.05) but not in that treated with Axitinib monotherapy. In contrast, KI2368 was significantly responsive to Sunitinib monotherapy, combined Sorafenib/Sunitinib therapy and alternating therapy of Sorafenib/Sunitinib but not responsive to Sorafenib and Axitinib monotherapy (P<0.05). RNAseq of the two models demonstrated that the expression levels of 1,725 genes including the drug targeted genes of PDGFA, PDGFB and PDGFRA were >5-fold higher in KI2367 than in KI2368 and the expression levels of 994 genes were > 5-fold higher in KI2368 than in KI2367. These results suggest the presence of intra-tumour molecular heterogeneity in this patient. This heterogeneity may influence the response to targeted therapies. Multiple biopsy, liquid biopsy and genomic analysis of intra- tumour molecular heterogeneity may help guide a more precise and effective plan in selecting targeted therapies for ccRCC patients. PMID:28548943

  17. Intra-tumour molecular heterogeneity of clear cell renal cell carcinoma reveals the diversity of the response to targeted therapies using patient-derived xenograft models.

    PubMed

    Hong, Baoan; Yang, Yong; Guo, Sheng; Duoerkun, Shayiremu; Deng, Xiaohu; Chen, Dawei; Yu, Shijun; Qian, Wubin; Li, Qixiang; Li, Qing; Gong, Kan; Zhang, Ning

    2017-07-25

    Inter- and intra-tumour molecular heterogeneity is increasingly recognized in clear cell renal cell carcinoma (ccRCC). It may partially explain the diversity of responses to targeted therapies and the various clinical outcomes. In this study, a 56-year-old male ccRCC patient with multiple metastases received radical nephrectomy and resection of the metastatic tumour in chest wall. The surgical specimens were implanted into nude mice to establish patient-derived xenograft (PDX) models with KI2367 model derived from the primary tumour and KI2368 model from the metastastic tumour. The two modles were treated with Sorafenib, Sunitinib, Axitinib, combined Sorafenib/Sunitinib, or alternating therapy of Sorafenib and Sunitinib. Significant anti-tumour activity was found in KI2367 treated with Sorafenib/Sunitinib monotherapy, combined Sorafenib/Sunitinib, and alternating therapy of Sorafenib/Sunitinib (P<0.05) but not in that treated with Axitinib monotherapy. In contrast, KI2368 was significantly responsive to Sunitinib monotherapy, combined Sorafenib/Sunitinib therapy and alternating therapy of Sorafenib/Sunitinib but not responsive to Sorafenib and Axitinib monotherapy (P<0.05). RNAseq of the two models demonstrated that the expression levels of 1,725 genes including the drug targeted genes of PDGFA, PDGFB and PDGFRA were >5-fold higher in KI2367 than in KI2368 and the expression levels of 994 genes were > 5-fold higher in KI2368 than in KI2367. These results suggest the presence of intra-tumour molecular heterogeneity in this patient. This heterogeneity may influence the response to targeted therapies. Multiple biopsy, liquid biopsy and genomic analysis of intra- tumour molecular heterogeneity may help guide a more precise and effective plan in selecting targeted therapies for ccRCC patients.

  18. Preliminary evaluation of [18F]AlF-NOTA-MAL-Cys39-exendin-4 in insulinoma with PET.

    PubMed

    Xu, Qing; Zhu, Chen; Xu, Yuping; Pan, Donghui; Liu, Ping; Yang, Runlin; Wang, Lizhen; Chen, Fei; Sun, Xinchen; Luo, Shineng; Yang, Min

    2015-01-01

    High expression of glucagon-like peptide-1 receptor (GLP-1R) in insulinoma supplies a potential drug target for tumor imaging. Exendin-4 can specifically bind to GLP-1R as an agonist and its analogs are extensively used in receptor imaging studies. A new GLP-1R imaging agent, [(18)F]AlF-NOTA-MAL-Cys(39)-exendin-4, was designed and prepared for insulinoma imaging. Cys(39)-exendin-4 was conjugated with NOTA-MAL, then the compound was radiolabeled with [(18)F]AlF complex to obtained [(18)F]AlF-NOTA-MAL-Cys(39)-exendin-4. The tumor-targeting characters of the tracer were evaluated in INS-1 cells and BALB/c nude mice models. [(18)F]AlF-NOTA-MAL-Cys(39)-exendin-4 can be efficiently produced with a yield of 17.5 ± 3.2% (non-decay corrected) and radiochemical purity of >95%. The IC50 value of displacement [(18)F]AlF-NOTA-MAL-Cys(39)-exendin-4 with Cys(39)-exendin-4 was 13.52 ± 1.36 nM. PET images showed excellent tumor visualization with high uptake (9.15 ± 1.6%ID/g at 30 min and 7.74 ± 0.87%ID/g at 60 min). The tumor to muscle, pancreas and liver ratios were 63.25, 3.85 and 7.29 at 60 min after injection. GLP-1R binding specificity was demonstrated by co-injection with an excess of unlabeled Cys(39)-exendin-4 and the tumor uptake was found to be reduced significantly. [(18)F]AlF-NOTA-MAL-Cys(39)-exendin-4 shows favorable characteristics for insulinoma imaging and may be translated to clinical studies.

  19. Genes regulated by estrogen in breast tumor cells in vitro are similarly regulated in vivo in tumor xenografts and human breast tumors

    PubMed Central

    Creighton, Chad J; Cordero, Kevin E; Larios, Jose M; Miller, Rebecca S; Johnson, Michael D; Chinnaiyan, Arul M; Lippman, Marc E; Rae, James M

    2006-01-01

    Background Estrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors. Results We generated DNA microarray-based gene expression profiles from three estrogen receptor α (ERα)-positive breast cancer cell lines stimulated by 17β-estradiol (E2) in vitro over a time course, as well as from MCF-7 cells grown as xenografts in ovariectomized athymic nude mice with E2 supplementation and after its withdrawal. When the patterns of genes regulated by E2 in vitro were compared to those obtained from xenografts, we found a remarkable overlap (over 40%) of genes regulated by E2 in both contexts. These patterns were compared to those obtained from published clinical data sets. We show that, as a group, E2-regulated genes from our preclinical models were co-expressed with ERα in a panel of ERα+ breast tumor mRNA profiles, when corrections were made for patient age, as well as with progesterone receptor. Furthermore, the E2-regulated genes were significantly enriched for transcriptional targets of the myc oncogene and were found to be coordinately expressed with Myc in human tumors. Conclusion Our results provide significant validation of a widely used in vitro model of estrogen signaling as being pathologically relevant to breast cancers in vivo. PMID:16606439

  20. Human Umbilical Cord Stem Cell Xenografts Improve Cognitive Decline and Reduce the Amyloid Burden in a Mouse Model of Alzheimer’s Disease

    PubMed Central

    Boutajangout, Allal; Noorwali, Abdulwahab; Atta, Hazem; Wisniewski, Thomas

    2017-01-01

    Introduction Alzheimer’s disease (AD) is the most common cause of dementia. The search for new treatments is made more urgent given its increasing prevalence resulting from the aging of the global population. Over the past 20 years, stem cell technologies have become an increasingly attractive option to both study and potentially treat neurodegenerative diseases. Several investigators reported a beneficial effect of different types of stem or progenitor cells on the pathology and cognitive function in AD models. Mouse models are one of the most important research tools for finding new treatment for AD. We aimed to explore the possible therapeutic potential of human umbilical cord mesenchymal stem cell xenografts in a transgenic (Tg) mouse model of AD. Methods APP/PS1 Tg AD model mice received human umbilical cord stem cells, directly injected into the carotid artery. To test the efficacy of the umbilical cord stem cells in this AD model, behavioral tasks (sensorimotor and cognitive tests) and immunohistochemical quantitation of the pathology was performed. Results Treatment of the APP/PS1 AD model mice, with human umbilical cord stem cells, produced a reduction of the amyloid beta burden in the cortex and the hippocampus which correlated with a reduction of the cognitive loss. Conclusion Human umbilical cord mesenchymal stem cells appear to reduce AD pathology in a transgenic mouse model as documented by a reduction of the amyloid plaque burden compared to controls. This amelioration of pathology correlates with improvements on cognitive and sensorimotor tasks. PMID:27719629

  1. The Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor Cediranib (Recentin; AZD2171) Inhibits Endothelial Cell Function and Growth of Human Renal Tumor Xenografts

    SciTech Connect

    Siemann, Dietmar W. Brazelle, W.D.; Juergensmeier, Juliane M.

    2009-03-01

    Purpose: The goal of this study was to examine the therapeutic potential of the vascular endothelial growth factor (VEGF) signaling inhibitor cediranib in a human model of renal cell carcinoma (Caki-1). Methods and Materials: The effects of cediranib treatment on in vitro endothelial cell function (proliferation, migration, and tube formation), as well as in vivo angiogenesis and tumor growth, were determined. Results: In vitro, cediranib significantly impaired the proliferation and migration of endothelial cells and their ability to form tubes, but had no effect on the proliferation of Caki-1 tumor cells. In vivo, cediranib significantly reduced Caki-1 tumor cell-induced angiogenesis, reduced tumor perfusion, and inhibited the growth of Caki-1 tumor xenografts. Conclusions: The present results are consistent with the notion that inhibition of VEGF signaling leads to an indirect (i.e., antiangiogenic) antitumor effect, rather than a direct effect on tumor cells. These results further suggest that inhibition of VEGF signaling with cediranib may impair the growth of renal cell carcinoma.

  2. Human Umbilical Cord Stem Cell Xenografts Improve Cognitive Decline and Reduce the Amyloid Burden in a Mouse Model of Alzheimer's Disease.

    PubMed

    Boutajangout, Allal; Noorwali, Abdulwahab; Atta, Hazem; Wisniewski, Thomas

    2017-01-01

    Alzheimer's disease (AD) is the most common cause of dementia. The search for new treatments is made more urgent given its increasing prevalence resulting from the aging of the global population. Over the past 20 years, stem cell technologies have become an increasingly attractive option to both study and potentially treat neurodegenerative diseases. Several investigators reported a beneficial effect of different types of stem or progenitor cells on the pathology and cognitive function in AD models. Mouse models are one of the most important research tools for finding new treatment for AD. We aimed to explore the possible therapeutic potential of human umbilical cord mesenchymal stem cell xenografts in a transgenic (Tg) mouse model of AD. APP/PS1 Tg AD model mice received human umbilical cord stem cells, directly injected into the carotid artery. To test the efficacy of the umbilical cord stem cells in this AD model, behavioral tasks (sensorimotor and cognitive tests) and immunohistochemical quantitation of the pathology was performed. Treatment of the APP/PS1 AD model mice, with human umbilical cord stem cells, produced a reduction of the amyloid beta burden in the cortex and the hippocampus which correlated with a reduction of the cognitive loss. Human umbilical cord mesenchymal stem cells appear to reduce AD pathology in a transgenic mouse model as documented by a reduction of the amyloid plaque burden compared to controls. This amelioration of pathology correlates with improvements on cognitive and sensorimotor tasks.

  3. The effect of single agent oral fusaric acid (FA) on the growth of subcutaneously xenografted SCC-1 cells in a nude mouse model.

    PubMed

    Ruda, James M; Beus, Kirt S; Hollenbeak, Christopher S; Wilson, Ronald P; Stack, Brendan C

    2006-09-01

    To determine whether oral administration of fusaric acid (FA) inhibits tumor growth in an animal model of head and neck cancer (HNSCC). In vivo murine model, two arm controlled study. Thirty-eight (38) 5-week-old athymic nude mice were randomly assigned to a fusaric acid treatment group (1 mg/mL) (n = 19) or a sterile saline group (n = 19). A left, lateral flank subcutaneous injection of 2.0 x 10(6) UM-SCC-1 cells were administered to all mice on day 1. Both groups were gavaged daily with either 0.25 mLs of oral FA or sterile saline throughout the experiment (32 days). Latency to a measurable tumor (> or =65 mm3), and tumor volumes were recorded after tumor xenografting. Tumor weights were recorded at the conclusion of the experiment. Tumor volume growth curves were modeled as polynomial functions of time with treatment interaction effects. Survivorship functions for time to measurable tumor were estimated using the Kaplan-Meier product limit estimator. Survival analysis showed mice treated with FA developed measurable tumors after a significantly longer interval post-xenografting than control mice (p = 0.00451). By Day 9, all mice in the control group had developed measurable tumors in comparison to only 78% of mice in the FA group. Likewise, estimated growth curves for both groups suggested that mice receiving FA demonstrated significantly slower tumor growth rates throughout the entire study period (p < 0.0001). At the conclusion of the experiment, tumor weights from both the control and FA groups were also significantly different (p = 0.0142). Single agent oral fusaric acid (1 mg/mL) is an inhibitor of UM-SCC-1 in a murine model. As an orally active agent, it may have a potential role in the treatment of human squamous cell carcinoma of the head and neck.

  4. Biosynthesized Platinum Nanoparticles Inhibit the Proliferation of Human Lung-Cancer Cells in vitro and Delay the Growth of a Human Lung-Tumor Xenograft in vivo

    PubMed Central

    Yogesh, Bendale; Vineeta, Bendale; Rammesh, Natu; Saili, Paul

    2016-01-01

    Objectives: Lung cancer remains a deadly disease with unsatisfactory overall survival. Cisplatin, a standard platinum (Pt)-based chemotherapeutic agent, has the potential to inhibit the growth of lung cancer. Its use, however, is occasionally limited by severe organ toxicity. However, until now, no systematic study has been conducted to verify its efficacy with proper experimental support in vivo. Therefore, we examined whether biosynthesized Pt nanoparticles (NPs) inhibited human lung cancer in vitro and in vivo to validate their use in alternative and complementary medicine. Methods: We evaluated the in vitro and the in vivo anticancer efficiencies of biosynthesized Pt NPs in a subcutaneous xenograft model with A549 cells. Severe combined immune deficient mice (SCID) were divided into four groups: group 1 being the vehicle control group and groups 2, 3 and 4 being the experimental groups. Once the tumor volume had reached 70 ─ 75 mm3, the progression profile of the tumor growth kinetics and the body weights of the mice were measured every week for 6 weeks after oral administration of Pt NPs. Doses of Pt NPs of 500, 1,000 and 2,000 mg/kg of body weight were administered to the experimental groups and a dose of honey was administered to the vehicle control group. The efficacy was quantified by using the delay in tumor growth following the administration of Pt NPs of A549 human-lung-cancer xenografts growing in SCID mice. Results: The in vitro cytotoxicity evaluation indicated that Pt NPs, in a dose-dependent manner, inhibited the growth of A549 cells, and the in vivo evaluation showed that Pt NPs at the mid and high doses effectively inhibited and delayed the growth of lung cancer in SCID mice. Conclusion: These findings confirm the antitumor properties of biosynthesized Pt NPs and suggest that they may be a cost-effective alternative for the treatment of patients with lung cancer. PMID:27386144

  5. Anti-Tumor Effect of Adipose Tissue Derived-Mesenchymal Stem Cells Expressing Interferon-β and Treatment with Cisplatin in a Xenograft Mouse Model for Canine Melanoma

    PubMed Central

    Ahn, Jin ok; Lee, Hee woo; Seo, Kyoung won; Kang, Sung keun; Ra, Jeong chan; Youn, Hwa young

    2013-01-01

    Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are attractive cell-therapy vehicles for the delivery of anti-tumor molecules into the tumor microenvironment. The innate tropism of AT-MSCs for tumors has important implications for effective cellular delivery of anti-tumor molecules, including cytokines, interferon, and pro-drugs. The present study was designed to determine the possibility that the combination of stem cell-based gene therapy with low-dose cisplatin would improve therapeutic efficacy against canine melanoma. The IFN-β transduced canine AT-MSCs (cAT-MSC-IFN-β) inhibited the growth of LMeC canine melanoma cells in direct and indirect in vitro co-culture systems. In animal experiments using BALB/c nude mouse xenografts, which developed by injecting LMeC cells, the combination treatment of cAT-MSC-IFN-β and low-dose cisplatin significantly reduced tumor volume compared with the other treatment groups. Fluorescent microscopic analysis with a TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) assay of tumor section provided evidence for homing of cAT-MSC-IFN-β to the tumor site and revealed that the combination treatment of cAT-MSC-IFN-β with low-dose cisplatin induced high levels of cell apoptosis. These findings may prove useful in further explorations of the application of these combined approaches to the treatment of malignant melanoma and other tumors. PMID:24040358

  6. Anti-tumor effect of adipose tissue derived-mesenchymal stem cells expressing interferon-β and treatment with cisplatin in a xenograft mouse model for canine melanoma.

    PubMed

    Ahn, Jin ok; Lee, Hee woo; Seo, Kyoung won; Kang, Sung keun; Ra, Jeong chan; Youn, Hwa young

    2013-01-01

    Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are attractive cell-therapy vehicles for the delivery of anti-tumor molecules into the tumor microenvironment. The innate tropism of AT-MSCs for tumors has important implications for effective cellular delivery of anti-tumor molecules, including cytokines, interferon, and pro-drugs. The present study was designed to determine the possibility that the combination of stem cell-based gene therapy with low-dose cisplatin would improve therapeutic efficacy against canine melanoma. The IFN-β transduced canine AT-MSCs (cAT-MSC-IFN-β) inhibited the growth of LMeC canine melanoma cells in direct and indirect in vitro co-culture systems. In animal experiments using BALB/c nude mouse xenografts, which developed by injecting LMeC cells, the combination treatment of cAT-MSC-IFN-β and low-dose cisplatin significantly reduced tumor volume compared with the other treatment groups. Fluorescent microscopic analysis with a TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) assay of tumor section provided evidence for homing of cAT-MSC-IFN-β to the tumor site and revealed that the combination treatment of cAT-MSC-IFN-β with low-dose cisplatin induced high levels of cell apoptosis. These findings may prove useful in further explorations of the application of these combined approaches to the treatment of malignant melanoma and other tumors.

  7. Frankincense essential oil prepared from hydrodistillation of Boswellia sacra gum resins induces human pancreatic cancer cell death in cultures and in a xenograft murine model

    PubMed Central

    2012-01-01

    Background Regardless of the availability of therapeutic options, the overall 5-year survival for patients diagnosed with pancreatic cancer remains less than 5%. Gum resins from Boswellia species, also known as frankincense, have been used as a major ingredient in Ayurvedic and Chinese medicine to treat a variety of health-related conditions. Both frankincense chemical extracts and essential oil prepared from Boswellia species gum resins exhibit anti-neoplastic activity, and have been investigated as potential anti-cancer agents. The goals of this study are to identify optimal condition for preparing frankincense essential oil that possesses potent anti-tumor activity, and to evaluate the activity in both cultured human pancreatic cancer cells and a xenograft mouse cancer model. Methods Boswellia sacra gum resins were hydrodistilled at 78°C; and essential oil distillate fractions were collected at different durations (Fraction I at 0–2 h, Fraction II at 8–10 h, and Fraction III at 11–12 h). Hydrodistillation of the second half of gum resins was performed at 100°C; and distillate was collected at 11–12 h (Fraction IV). Chemical compositions were identified by gas chromatography–mass spectrometry (GC-MS); and total boswellic acids contents were quantified by high-performance liquid chromatography (HPLC). Frankincense essential oil-modulated pancreatic tumor cell viability and cytotoxicity were determined by colorimetric assays. Levels of apoptotic markers, signaling molecules, and cell cycle regulators expression were characterized by Western blot analysis. A heterotopic (subcutaneous) human pancreatic cancer xenograft nude mouse model was used to evaluate anti-tumor capability of Fraction IV frankincense essential oil in vivo. Frankincense essential oil-induced tumor cytostatic and cytotoxic activities in animals were assessed by immunohistochemistry. Results Longer duration and higher temperature hydrodistillation produced more abundant high molecular

  8. Frankincense essential oil prepared from hydrodistillation of Boswellia sacra gum resins induces human pancreatic cancer cell death in cultures and in a xenograft murine model.

    PubMed

    Ni, Xiao; Suhail, Mahmoud M; Yang, Qing; Cao, Amy; Fung, Kar-Ming; Postier, Russell G; Woolley, Cole; Young, Gary; Zhang, Jingzhe; Lin, Hsueh-Kung

    2012-12-13

    Regardless of the availability of therapeutic options, the overall 5-year survival for patients diagnosed with pancreatic cancer remains less than 5%. Gum resins from Boswellia species, also known as frankincense, have been used as a major ingredient in Ayurvedic and Chinese medicine to treat a variety of health-related conditions. Both frankincense chemical extracts and essential oil prepared from Boswellia species gum resins exhibit anti-neoplastic activity, and have been investigated as potential anti-cancer agents. The goals of this study are to identify optimal condition for preparing frankincense essential oil that possesses potent anti-tumor activity, and to evaluate the activity in both cultured human pancreatic cancer cells and a xenograft mouse cancer model. Boswellia sacra gum resins were hydrodistilled at 78°C; and essential oil distillate fractions were collected at different durations (Fraction I at 0-2 h, Fraction II at 8-10 h, and Fraction III at 11-12 h). Hydrodistillation of the second half of gum resins was performed at 100°C; and distillate was collected at 11-12 h (Fraction IV). Chemical compositions were identified by gas chromatography-mass spectrometry (GC-MS); and total boswellic acids contents were quantified by high-performance liquid chromatography (HPLC). Frankincense essential oil-modulated pancreatic tumor cell viability and cytotoxicity were determined by colorimetric assays. Levels of apoptotic markers, signaling molecules, and cell cycle regulators expression were characterized by Western blot analysis. A heterotopic (subcutaneous) human pancreatic cancer xenograft nude mouse model was used to evaluate anti-tumor capability of Fraction IV frankincense essential oil in vivo. Frankincense essential oil-induced tumor cytostatic and cytotoxic activities in animals were assessed by immunohistochemistry. Longer duration and higher temperature hydrodistillation produced more abundant high molecular weight compounds, including boswellic

  9. Additional effects of engineered stem cells expressing a therapeutic gene and interferon-β in a xenograft mouse model of endometrial cancer.

    PubMed

    Yi, Bo-Rim; Kim, Seung U; Choi, Kyung-Chul

    2015-07-01

    Endometrial cancer is the most common gynecologic malignancy in women worldwide. In the present study, we evaluated the effects of neural stem cell-directed enzyme/prodrug therapy (NDEPT) designed to more selectively target endometrial cancer. For this, we employed two different types of neural stem cells (NSCs), HB1.F3.CD and HB1.F3.CD.IFN-β cells. Cytosine deaminase (CD) can convert the non-toxic prodrug, 5-fluorocytosine (5-FC), into a toxic agent, 5-fluorouracil (5-FU), which inhibits DNA synthesis. IFN-β is a powerful cytotoxic cytokine that is released by activated immune cells or lymphocytes. In an animal model xenografted with endometrial Ishikawa cancer cells, the stem cells stained with CM-DiI were injected into nearby tumor masses and 5-FC was delivered by intraperitoneal injection. Co-expression of CD and IFN-β significantly inhibited the growth of cancer (~50-60%) in the presence of 5-FC. Among migration-induced factors, VEGF gene was highly expressed in endometrial cancer cells. Histological analysis showed that the aggressive nature of cancer was inhibited by 5-FC in the mice treated with the therapeutic stem cells. Furthermore, PCNA expression was more decreased in HB1.F3.CD.IFN-β treated mice rather than HB1.F3.CD treated mice. To confirm the in vitro combined effects of 5-FU and IFN-β, 5-FU was treated in Ishikawa cells. 5-FU increased the IFN-β/receptor 2 (IFNAR2) and BXA levels, indicating that 5-FU increased sensitivity of endometrial cancer cells to IFN-β, leading to apoptosis of cancer cells. Taken together, these results provide evidence for the efficacy of therapeutic stem cell-based immune therapy involving the targeted expression of CD and IFN-β genes at endometrial cancer sites.

  10. Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model

    PubMed Central

    Gu, Yuan; Körbel, Christina; Scheuer, Claudia; Nenicu, Anca; Menger, Michael D.; Laschke, Matthias W.

    2016-01-01

    Tubeimoside-1 (TBMS1) is a potent anti-tumor phytochemical. Its functional and molecular mode of action, however, remains elusive so far. Since angiogenesis is essential for tumor progression and metastasis, we herein investigated the anti-angiogenic effects of the compound. In a non-small cell lung cancer (NSCLC) xenograft model we found that treatment of CD1 nu/nu mice with TBMS1 (5mg/kg) significantly suppressed the growth and vascularization of NCI-H460 flank tumors. Moreover, TBMS1 dose-dependently reduced vascular sprouting in a rat aortic ring assay. In vitro, TBMS1 induced endothelial cell apoptosis without decreasing the viability of NSCLC tumor cells and inhibited the migration of endothelial cells by disturbing their actin filament organization. TBMS1 further stimulated the proteasomal degradation of vascular endothelial growth factor receptor-2 (VEGFR2) and Tie2 in endothelial cells, which down-regulated AKT/mTOR signaling. These findings indicate that TBMS1 represents a novel phytochemical for anti-angiogenic treatment of cancer and other angiogenesis-related diseases. PMID:26701724

  11. Anticancer activity of pyrithione zinc in oral cancer cells identified in small molecule screens and xenograft model: Implications for oral cancer therapy.

    PubMed

    Srivastava, Gunjan; Matta, Ajay; Fu, Guodong; Somasundaram, Raj Thani; Datti, Alessandro; Walfish, Paul G; Ralhan, Ranju

    2015-10-01

    Oral squamous cell carcinoma (OSCC) patients diagnosed in late stages have limited chemotherapeutic options, underscoring the great need for development of new anticancer agents for more effective disease management. We aimed to identify novel anticancer agents for OSCC using quantitative high throughput assays for screening six chemical libraries consisting of 5170 small molecule inhibitors. In depth characterization resulted in identification of pyrithione zinc (PYZ) as the most effective cytotoxic agent inhibiting cell proliferation and inducing apoptosis in OSCC cells in vitro. Further, treatment with PYZ reduced colony forming, migration and invasion potential of oral cancer cells in a dose-dependent manner. PYZ treatment also led to altered expression of several key components of the major signaling pathways including PI3K/AKT/mTOR and WNT/β-catenin in OSCC cells. In addition, treatment with PYZ also reduced expression of 14-3-3ζ, 14-3-3σ, cyclin D1, c-Myc and pyruvate kinase M2 (PKM2), proteins identified in our earlier studies to be involved in development and progression of OSCCs. Importantly, PYZ treatment significantly reduced tumor xenograft volume in immunocompromised NOD/SCID/Crl mice without causing apparent toxicity to normal tissues. Taken together, we demonstrate in vitro and in vivo efficacy of PYZ in OSCC. In conclusion, we identified PYZ in HTS assays and demonstrated in vitro and in vivo pre-clinical efficacy of PYZ as a novel anticancer therapeutic candidate in OSCC.

  12. Combining TRAIL and liquiritin exerts synergistic effects against human gastric cancer cells and xenograft in nude mice through potentiating apoptosis and ROS generation.

    PubMed

    Xie, Rui; Gao, Cheng-Cheng; Yang, Xiao-Zhong; Wu, Shang-Nong; Wang, Hong-Gang; Zhang, Jia-Ling; Yan, Wei; Ma, Tian-Heng

    2017-09-01

    Gastric cancer is one of the most factors, leading to cancer-related death worldwide. However, the therapies to prevent gastric cancer are still limited and the emergence of drug resistance leads to development of new anti-cancer drugs and combinational chemotherapy regimens. Our study was aimed to explore the anti-gastric cancer effects of liquiritin (LIQ), a major constituent of Glycyrrhiza Radix, which possesses a variety of pharmacological activities. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially inhibited tumor cells over other normal cells, when used in alone or in combination. The results indicated that LIQ, when applied in single, was moderately effective to suppress proliferation, and migration, as well as to induce apoptosis and reactive oxygen species (ROS) generation of human gastric cancer cell lines, AGS and SNU-216, which are TRAIL-resistant. Significantly, when used in combination, the two drugs functioned synergistically to impede the progression and growth of human gastric cancer cells in vitro and gastric cancer cell xenograft nude mice in vivo. Both intrinsic and extrinsic apoptosis were induced by the two in combination via activating Caspases. And c-Jun N-terminal kinase (JNK) activity was dramatically induced by TRAIL/LIQ. Importantly, TRAIL/LIQ-triggered apoptosis and JNK were dependent on ROS production. The data indicated that application of TRAIL/LIQ in combination had a potential value for clinical use to synergistically prevent human gastric cancer development. Copyright © 2017. Published by Elsevier Masson SAS.

  13. Intraoperative Localization of Insulinoma and Normal Pancreas using Invisible Near-Infrared Fluorescent Light

    PubMed Central

    Winer, Joshua; Choi, Hak Soo; Gibbs-Strauss, Summer L.; Ashitate, Yoshitomo; Colson, Yolonda L.; Frangioni, John V.

    2009-01-01

    Background: Neuroendocrine tumors of the pancreas, such as insulinoma, are difficult to localize and complete resection is essential for cure. Our hypothesis is that a near-infrared (NIR) fluorophore exhibiting uptake in insulinoma could provide high sensitivity detection intraoperatively. Methods: The optical properties of methylene blue (MB) were measured in vitro in 100% serum at 37°C, and in vivo after tissue uptake. MB was injected as a rapid intravenous bolus at doses ranging from 0.25 to 2 mg/kg into wildtype rats and pigs, and into insulinoma-bearing transgenic mice. The FLARE™ imaging system was used to acquire color video and NIR fluorescence images simultaneously, and in real-time. The signal-to-background ratios (SBR) of tissues and tumors were quantified using FLARE™ software. Results: When appropriately diluted, MB exhibits moderate NIR fluorescence emission peaking at 688 nm. At doses ≥ 1 mg/kg, certain normal tissues, such as pancreas, accumulate MB and remain NIR fluorescent for up to 1 hr with an SBR ≥ 1.6. MB spectral properties are maintained after uptake into tissue. Interestingly, insulinoma exhibits even higher uptake for MB than normal pancreas, resulting in insulinoma-to-pancreas ratios of 3.7 and insulinoma-to-muscle ratios of 16.2. MB permitted high-sensitivity, real-time localization of primary, multi-centric, and metastatic insulinoma, and permitted differentiation among tumor, normal pancreas, and other abdominal structures. Conclusion: A single intravenous injection of a clinically available, commonly used NIR fluorophore provides prolonged intraoperative localization of normal pancreas and insulinoma using invisible NIR fluorescent light. PMID:20033320

  14. Stable SET knockdown in head and neck squamous cell carcinoma promotes cell invasion and the mesenchymal-like phenotype in vitro, as well as necrosis, cisplatin sensitivity and lymph node metastasis in xenograft tumor models.

    PubMed

    Sobral, Lays M; Sousa, Lucas O; Coletta, Ricardo D; Cabral, Hamilton; Greene, Lewis J; Tajara, Eloiza H; Gutkind, J Silvio; Curti, Carlos; Leopoldino, Andréia M

    2014-02-20

    SET/I2PP2A is a multifunctional protein that is up-regulated in head and neck squamous cell carcinoma (HNSCC). The action of SET in HNSCC tumorigenicity is unknown. Stable SET knockdown by shRNA (shSET) was established in three HNSCC cell lines: HN12, HN13, and Cal27. Protein expression and phosphorylated protein levels were determined by Western blotting and immunofluorescence, cell migration and invasion were measured by functional analysis, and PP2A activity was determined using a serine/threonine phosphatase assay. A real-time PCR array was used to quantify 84 genes associated with cell motility. Metalloproteinase (MMP) activity was assessed by zymographic and fluorometric assays. HN12shSET xenograft tumors (flank and tongue models) were established in Balb/c nude mice; the xenograft characteristics and cisplatin sensitivity were demonstrated by macroscopic, immunohistochemical, and histological analyses, as well as lymph node metastasis by histology. The HN12shSET cells displayed reduced ERK1/2 and p53 phosphorylation compared with control. ShSET reduced HN12 cell proliferation and increased the sub-G1 population of HN12 and Cal27 cells. Increased PP2A activity was also associated with shSET. The PCR array indicated up-regulation of three mRNAs in HN12 cells: vimentin, matrix metalloproteinase-9 (MMP9) and non-muscle myosin heavy chain IIB. Reduced E-cadherin and pan-cytokeratin, as well as increased vimentin, were also demonstrated as the result of SET knockdown. These changes were accompanied by an increase in MMP-9 and MMP-2 activities, migration and invasion. The HN12shSET subcutaneous xenograft tumors presented a poorly differentiated phenotype, reduced cell proliferation, and cisplatin sensitivity. An orthotopic xenograft tumor model using the HN12shSET cells displayed increased metastatic potential. SET accumulation has important actions in HNSCC. As an oncogene, SET promotes cell proliferation, survival, and resistance to cell death by cisplatin in

  15. Stable SET knockdown in head and neck squamous cell carcinoma promotes cell invasion and the mesenchymal-like phenotype in vitro, as well as necrosis, cisplatin sensitivity and lymph node metastasis in xenograft tumor models

    PubMed Central

    2014-01-01

    Background SET/I2PP2A is a multifunctional protein that is up-regulated in head and neck squamous cell carcinoma (HNSCC). The action of SET in HNSCC tumorigenicity is unknown. Methods Stable SET knockdown by shRNA (shSET) was established in three HNSCC cell lines: HN12, HN13, and Cal27. Protein expression and phosphorylated protein levels were determined by Western blotting and immunofluorescence, cell migration and invasion were measured by functional analysis, and PP2A activity was determined using a serine/threonine phosphatase assay. A real-time PCR array was used to quantify 84 genes associated with cell motility. Metalloproteinase (MMP) activity was assessed by zymographic and fluorometric assays. HN12shSET xenograft tumors (flank and tongue models) were established in Balb/c nude mice; the xenograft characteristics and cisplatin sensitivity were demonstrated by macroscopic, immunohistochemical, and histological analyses, as well as lymph node metastasis by histology. Results The HN12shSET cells displayed reduced ERK1/2 and p53 phosphorylation compared with control. ShSET reduced HN12 cell proliferation and increased the sub-G1 population of HN12 and Cal27 cells. Increased PP2A activity was also associated with shSET. The PCR array indicated up-regulation of three mRNAs in HN12 cells: vimentin, matrix metalloproteinase-9 (MMP9) and non-muscle myosin heavy chain IIB. Reduced E-cadherin and pan-cytokeratin, as well as increased vimentin, were also demonstrated as the result of SET knockdown. These changes were accompanied by an increase in MMP-9 and MMP-2 activities, migration and invasion. The HN12shSET subcutaneous xenograft tumors presented a poorly differentiated phenotype, reduced cell proliferation, and cisplatin sensitivity. An orthotopic xenograft tumor model using the HN12shSET cells displayed increased metastatic potential. Conclusions SET accumulation has important actions in HNSCC. As an oncogene, SET promotes cell proliferation, survival, and

  16. Basal subtype is predictive for response to cetuximab treatment in patient-derived xenografts of squamous cell head and neck cancer.

    PubMed

    Klinghammer, Konrad; Otto, Raik; Raguse, Jan-Dirk; Albers, Andreas E; Tinhofer, Ingeborg; Fichtner, Iduna; Leser, Ulf; Keilholz, Ulrich; Hoffmann, Jens

    2017-09-15

    Cetuximab is the single targeted therapy approved for the treatment of head and neck cancer (HNSCC). Predictive biomarkers have not been established and patient stratification based on molecular tumor profiles has not been possible. Since EGFR pathway activation is pronounced in basal subtype, we hypothesized this activation could be a predictive signature for an EGFR directed treatment. From our patient-derived xenograft platform of HNSCC, 28 models were subjected to Affymetrix gene expression studies on HG U133+ 2.0. Based on the expression of 821 genes, the subtype of each of the 28 models was determined by integrating gene expression profiles through centroid-clustering with previously published gene expression data by Keck et al. The models were treated in groups of 5-6 animals with docetaxel, cetuximab, everolimus, cis- or carboplatin and 5-fluorouracil. Response was evaluated by comparing tumor volume at treatment initiation and after 3 weeks of treatment (RTV). Tumors distributed over the 3 signature-defined subtypes: 5 mesenchymal/inflamed phenotype (MS), 15 basal type (BA), 8 classical type (CL). Cluster analysis revealed a strong correlation between response to cetuximab and the basal subtype. RTV MS 3.32 vs. BA 0.78 (MS vs. BA, unpaired t-test, p 0.0002). Cetuximab responders were distributed as following: 1/5 in MS, 5/8 in CL and 13/15 in the BA group. Activity of classical chemotherapies did not differ between the subtypes. In conclusion basal subtype was associated with response to EGFR directed therapy in head and neck squamous cell cancer patient-derived xenografts. © 2017 UICC.

  17. Efficacy of JAK/STAT pathway inhibition in murine xenograft models of early T-cell precursor (ETP) acute lymphoblastic leukemia.

    PubMed

    Maude, Shannon L; Dolai, Sibasish; Delgado-Martin, Cristina; Vincent, Tiffaney; Robbins, Alissa; Selvanathan, Arthavan; Ryan, Theresa; Hall, Junior; Wood, Andrew C; Tasian, Sarah K; Hunger, Stephen P; Loh, Mignon L; Mullighan, Charles G; Wood, Brent L; Hermiston, Michelle L; Grupp, Stephan A; Lock, Richard B; Teachey, David T

    2015-03-12

    Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed. © 2015 by The American Society of Hematology.

  18. Resveratrol Worsens Survival in SCID Mice With Prostate Cancer Xenografts in a Cell-Line Specific Manner, Through Paradoxical Effects on Oncogenic Pathways

    PubMed Central

    Klink, Joseph C.; Tewari, Alok K.; Masko, Elizabeth M.; Antonelli, Jodi; Febbo, Phillip G.; Cohen, Pinchas; Dewhirst, Mark W.; Pizzo, Salvatore V.; Freedland, Stephen J.

    2013-01-01

    BACKGROUND Resveratrol increases lifespan and decreases the risk of many cancers. We hypothesized resveratrol will slow the growth of human prostate cancer xenografts. METHODS SCID mice were fed Western diet (40% fat, 44% carbohydrate, 16% protein by kcal). One week later, human prostate cancer cells, either LAPC-4 (151 mice) or LNCaP (94 mice) were injected subcutaneously. Three weeks after injection, LAPC-4 mice were randomized to Western diet (control group), Western diet plus resveratrol 50 mg/kg/day, or Western diet plus resveratrol 100 mg/kg/day. The LNCaP mice were randomized to Western diet or Western diet plus resveratrol 50 mg/kg/day. Mice were sacrificed when tumors reached 1,000 mm3. Survival differences among groups were assessed using Cox proportional hazards. Serum insulin and IGF axis were assessed using ELISAs. Gene expression was analyzed using Affymetrix gene arrays. RESULTS Compared to control in the LAPC-4 study, resveratrol was associated with decreased survival (50 mg/kg/day—HR 1.53, P = 0.04; 100 mg/kg/day—HR 1.22, P = 0.32). In the LNCaP study, resveratrol did not change survival (HR 0.77, P = 0.22). In combined analysis of both resveratrol 50 mg/kg/day groups, IGF-1 was decreased (P = 0.05) and IGFBP-2 was increased (P = 0.01). Resveratrol induced different patterns of gene expression changes in each xenograft model, with upregulation of oncogenic pathways E2F3 and beta-catenin in LAPC-4 tumors. CONCLUSION Resveratrol was associated with significantly worse survival with LAPC-4 tumors, but unchanged survival with LNCaP. Based on these preliminary data that resveratrol may be harmful, caution should be advised in using resveratrol for patients until further studies can be conducted. PMID:23192356

  19. Assessing Metabolic Changes in Response to mTOR Inhibition in a Mantle Cell Lymphoma Xenograft Model Using AcidoCEST MRI

    PubMed Central

    Akhenblit, Paul J.; Hanke, Neale T.; Gill, Alexander; Persky, Daniel O.; Howison, Christine M.; Pagel, Mark D.; Baker, Amanda F.

    2016-01-01

    AcidoCEST magnetic resonance imaging (MRI) has previously been shown to measure tumor extracellular pH (pHe) with excellent accuracy and precision. This study investigated the ability of acidoCEST MRI to monitor changes in tumor pHe in response to therapy. To perform this study, we used the Granta 519 human mantle cell lymphoma cell line, which is an aggressive B-cell malignancy that demonstrates activation of the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. We performed in vitro and in vivo studies using the Granta 519 cell line to investigate the efficacy and associated changes induced by the mTOR inhibitor, everolimus (RAD001). AcidoCEST MRI studies showed a statistically significant increase in tumor pHe of 0.10 pH unit within 1 day of initiating treatment, which foreshadowed a decrease in tumor growth of the Granta 519 xenograft model. AcidoCEST MRI then measured a decrease in tumor pHe 7 days after initiating treatment, which foreshadowed a return to normal tumor growth rate. Therefore, this study is a strong example that acidoCEST MRI can be used to measure tumor pHe that may serve as a marker for therapeutic efficacy of anticancer therapies. PMID:27140422

  20. Autophagy induction by leptin contributes to suppression of apoptosis in cancer cells and xenograft model: Involvement of p53/FoxO3A axis

    PubMed Central

    Nepal, Saroj; Kim, Mi Jin; Hong, Jin Tae; Kim, Sang Hyun; Sohn, Dong-Hwan; Lee, Sung Hee; Song, Kyung; Choi, Dong Young; Lee, Eung Seok; Park, Pil-Hoon

    2015-01-01

    Leptin, a hormone mainly produced from adipose tissue, has been shown to induce proliferation of cancer cells. However, the molecular mechanisms underlying leptin-induced tumor progression have not been clearly elucidated. In the present study, we investigated the role of autophagy in leptin-induced cancer cell proliferation using human hepatoma (HepG2) and breast cancer cells (MCF-7), and tumor growth in a xenograft model. Herein, we showed that leptin treatment caused autophagy induction as assessed by increase in expression of autophagy-related genes, including beclin-1, Atg5 and LC3 II, further induction of autophagosome formation and autophagic flux. Interestingly, inhibition of autophagic process by treatment with inhibitors and LC3B gene silencing blocked leptin-induced increase in cell number and suppression of apoptosis, indicating a crucial role of autophagy in leptin-induced tumor progression. Moreover, gene silencing of p53 or FoxO3A prevented leptin-induced LC3 II protein expression, suggesting an involvement of p53/FoxO3A axis in leptin-induced autophagy activation. Leptin administration also accelerated tumor growth in BALB/c nude mice, which was found to be autophagy dependent. Taken together, our results demonstrate that leptin-induced tumor growth is mediated by autophagy induction and autophagic process would be a promising target to regulate development of cancer caused by leptin production. PMID:25704884

  1. Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model

    PubMed Central

    Selvi, Ruthrotha B.; Swaminathan, Amrutha; Chatterjee, Snehajyoti; Shanmugam, Muthu K.; Li, Feng; Ramakrishnan, Gowsica B.; Siveen, Kodappully Sivaraman; Chinnathambi, Arunachalam; Zayed, M. Emam; Alharbi, Sulaiman Ali; Basha, Jeelan; Bhat, Akshay; Vasudevan, Madavan; Dharmarajan, Arunasalam; Sethi, Gautam; Kundu, Tapas K.

    2015-01-01

    Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down-regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing. PMID:26517526

  2. Autophagy induction by leptin contributes to suppression of apoptosis in cancer cells and xenograft model: involvement of p53/FoxO3A axis.

    PubMed

    Nepal, Saroj; Kim, Mi Jin; Hong, Jin Tae; Kim, Sang Hyun; Sohn, Dong-Hwan; Lee, Sung Hee; Song, Kyung; Choi, Dong Young; Lee, Eung Seok; Park, Pil-Hoon

    2015-03-30

    Leptin, a hormone mainly produced from adipose tissue, has been shown to induce proliferation of cancer cells. However, the molecular mechanisms underlying leptin-induced tumor progression have not been clearly elucidated. In the present study, we investigated the role of autophagy in leptin-induced cancer cell proliferation using human hepatoma (HepG2) and breast cancer cells (MCF-7), and tumor growth in a xenograft model. Herein, we showed that leptin treatment caused autophagy induction as assessed by increase in expression of autophagy-related genes, including beclin-1, Atg5 and LC3 II, further induction of autophagosome formation and autophagic flux. Interestingly, inhibition of autophagic process by treatment with inhibitors and LC3B gene silencing blocked leptin-induced increase in cell number and suppression of apoptosis, indicating a crucial role of autophagy in leptin-induced tumor progression. Moreover, gene silencing of p53 or FoxO3A prevented leptin-induced LC3 II protein expression, suggesting an involvement of p53/FoxO3A axis in leptin-induced autophagy activation. Leptin administration also accelerated tumor growth in BALB/c nude mice, which was found to be autophagy dependent. Taken together, our results demonstrate that leptin-induced tumor growth is mediated by autophagy induction and autophagic process would be a promising target to regulate development of cancer caused by leptin production.

  3. Masticadienonic and 3α-OH Masticadienoic Acids Induce Apoptosis and Inhibit Cell Proliferation and Tumor Growth in Prostate Cancer Xenografts in Vivo.

    PubMed

    Sánchez-Monroy, Ma Beatriz; Jacobo-Herrera, Nadia J; Zentella-Dehesa, Alejandro; Hernández-Téllez, Beatriz; Martínez-Vázquez, Mariano

    2017-09-06

    The triterpenes have been constituted as a group of interesting molecules as possible antitumor agents. Despite several of them not presenting a potent cytotoxic activity in vitro against cancer cells, in vivo in xenotransplant tumors studies, they show promising results. Based on the above considerations, we investigated the antitumor activity of both masticadienonic (MDA) and 3α-OH masticadienoic (3α-OH MDA) acids in a mouse prostate cancer xenograft model. Immunohistochemical assays were used to evaluate the decrease in the expression of the Proliferating Cell Nuclear Antigen (PCNA) and the Ki-67 induced by MDA and 3α-OH MDA. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to demonstrate the fragmentation of DNA. Our results showed that the two triterpenes inhibited tumor growth, had anti-proliferative effect in vivo and induced cell death by apoptosis. Collectively, our data suggested that the antitumor mechanism of MDA and 3α-OH MDA involves several molecular targets related to cell proliferation and apoptosis.

  4. Functional and molecular mapping of uncoupling between vascular permeability and loss of vascular maturation in ovarian carcinoma xenografts: the role of stroma cells in tumor angiogenesis.

    PubMed

    Gilad, Assaf A; Israely, Tomer; Dafni, Hagit; Meir, Gila; Cohen, Batya; Neeman, Michal

    2005-11-01

    Maintaining homogeneous perfusion in tissues undergoing remodeling and vascular expansion requires tight orchestration of the signals leading to endothelial sprouting and subsequent recruitment of perivascular contractile cells and vascular maturation. This regulation, however, is frequently disrupted in tumors. We previously demonstrated the role of tumor-associated myofibroblasts in vascularization and exit from dormancy of human ovarian carcinoma xenografts in nude mice. The aim of this work was to determine the contribution of stroma- and tumor cell-derived angiogenic growth factors to the heterogeneity of vascular permeability and maturation in MLS human ovarian carcinoma tumors. We show by RT-PCR and by in situ hybridization that VEGF was expressed by the tumor cells, while angiopoietin-1 and -2 were expressed only by the infiltrating host stroma cells. Vascular maturation was detected in vivo by vasoreactivity to hypercapnia, measured by BOLD contrast MRI and validated by immunostaining of histologic sections to alpha-smooth muscle actin. Vascular permeability was measured in vivo by dynamic contrast-enhanced MRI using albumin-based contrast material and validated in histologic sections by fluorescent staining of the biotinylated contrast material. MRI as well as histologic correlation maps between vascular maturation and vascular permeability revealed a wide range of vascular phenotypes, in which the distribution of vascular maturation and vasoreactivity did not overlap spatially with reduced permeability. The large heterogeneity in the degree of vascular maturation and permeability is consistent with the differential expression pattern of VEGF and angiopoietins during tumor angiogenesis.

  5. CK2 targeted RNAi therapeutic delivered via malignant cell-directed tenfibgen nanocapsule: dose and molecular mechanisms of response in xenograft prostate tumors

    PubMed Central

    Vogel, Rachel I.; Shaughnessy, Daniel P.; Nacusi, Lucas; Korman, Vicci L.; Li, Yingming; Dehm, Scott M.; Zimmerman, Cheryl L.; Niehans, Gloria A.; Unger, Gretchen M.; Trembley, Janeen H.

    2016-01-01

    CK2, a protein serine/threonine kinase, promotes cell proliferation and suppresses cell death. This essential-for-survival signal demonstrates elevated expression and activity in all cancers examined, and is considered an attractive target for cancer therapy. Here, we present data on the efficacy of a tenfibgen (TBG) coated nanocapsule which delivers its cargo of siRNA (siCK2) or single stranded RNA/DNA oligomers (RNAi-CK2) simultaneously targeting CK2α and α′ catalytic subunits. Intravenous administration of TBG-siCK2 or TBG-RNAi-CK2 resulted in significant xenograft tumor reduction at low doses in PC3-LN4 and 22Rv1 models of prostate cancer. Malignant cell uptake and specificity in vivo was verified by FACS analysis and immunofluorescent detection of nanocapsules and PCR detection of released oligomers. Dose response was concordant with CK2αα′ RNA transcript levels and the tumors demonstrated changes in CK2 protein and in markers of proliferation and cell death. Therapeutic response corresponded to expression levels for argonaute and GW proteins, which function in oligomer processing and translational repression. No toxicity was detected in non-tumor tissues or by serum chemistry. Tumor specific delivery of anti-CK2 RNAi via the TBG nanoencapsulation technology warrants further consideration of translational potential. PMID:27557516

  6. Quercetin, Siamois 1 and Siamois 2 induce apoptosis in human breast cancer MDA-mB-435 cells xenograft in vivo.

    PubMed

    Dechsupa, Samarn; Kothan, Suchart; Vergote, Jackie; Leger, Gerard; Martineau, Antoine; Berangeo, Simone; Kosanlavit, Rachain; Moretti, Jean-Luc; Mankhetkorn, Samlee

    2007-01-01

    We sought to investigate the apoptosis-inducing activities of quercetin, Siamois 1, and Siamois 2 against invasive estrogen-receptor negative MDA-MB 435 cells xenografted in athymic nude mice. This study clearly demonstrated that these compounds exhibited apoptosis-inducing activities in cell culture system. Quercetin (20 microg/mL), Siamois 1 (100 microg/mL), and Siamois 2 (200 microg/mL) can induce apoptotic cell death by 40 +/-5%, 44 +/- 14 %, and 31 +/- 13 %, respectively. Two-fold of IC50 of these compounds were clearly found to induce apoptosis in breast tumor tissue which can be determined by 99mTc-Annexin V scintigraphy and histological staining. This is the first report that the apoptosis-inducing effects of quercetin, Siamois 1 and Siamois 2 on the MDA-MB 435 cell in vitro were effectively extrapolated to the in vivo situation. These compounds might be considered as a simple dietary supplement and with further clinical investigation for their use as a nutrition-based intervention in breast cancer treatment.

  7. Lycium barbarum polysaccharides induce apoptosis in human prostate cancer cells and inhibits prostate cancer growth in a xenograft mouse model of human prostate cancer.

    PubMed

    Luo, Qiong; Li, Zhuoneng; Yan, Jun; Zhu, Fan; Xu, Ruo-Jun; Cai, Yi-Zhong

    2009-08-01

    Lycium barbarum polysaccharides (LBPs) are important functional constituents in red-colored fruits of L. barbarum (Guo Qi Zi, a well-known traditional Chinese medicinal plant commonly known as Goji berry or wolfberry). The influence of LBP on human prostate cancer cells was systematically investigated in vitro and in vivo. The in vitro effects of LBP on two cell lines (PC-3 and DU-145) were examined by using trypan blue exclusion staining, single-cell gel electrophoresis, flow cytometry, terminal dUTP nick-end labeling assay, and immunohistochemical assay (assessment of Bcl-2 and Bax expression). The in vivo effect of LBP on PC-3 cells was assessed in the nude mouse xenograft tumor model. The in vitro results showed that LBP can dose- and time-dependently inhibit the growth of both PC-3 and DU-145 cells. LBP caused the breakage of DNA strands of PC-3 and DU-145 cells; the tail frequency and tail length were significantly higher than that of control cells. LBP also markedly induced PC-3 and DU-145 cell apoptosis, with the highest apoptosis rates at 41.5% and 35.5%, respectively. The ratio of Bcl-2/Bax protein expression following LBP treatments decreased significantly with a dose-effect relationship, which suggested that LBP can regulate the expression of Bcl-2 and Bax to induce apoptosis of PC-3 and DU-145 cells. The in vivo experimental results indicate that LBP might significantly inhibit PC-3 tumor growth in nude mice. Both the tumor volume and weight of the LBP treatment group were significantly lower than those of the control group.

  8. Inhibition of Cell Proliferation and Growth of Pancreatic Cancer by Silencing of Carbohydrate Sulfotransferase 15 In Vitro and in a Xenograft Model

    PubMed Central

    Shibazaki, Yuichiro; Yoneyama, Hiroyuki; Fujii, Masato; Hashiguchi, Taishi; Ito, Zensho; Kajihara, Mikio; Misawa, Takeyuki; Homma, Sadamu; Ohkusa, Toshifumi

    2015-01-01

    Chondroitin sulfate E (CS-E), a highly sulfated glycosaminoglycan, is known to promote tumor invasion and metastasis. Because the presence of CS-E is detected in both tumor and stromal cells in pancreatic ductal adenocarcinoma (PDAC), multistage involvement of CS-E in the development of PDAC has been considered. However, its involvement in the early stage of PDAC progression is still not fully understood. In this study, to clarify the direct role of CS-E in tumor, but not stromal, cells of PDAC, we focused on carbohydrate sulfotransferase 15 (CHST15), a specific enzyme that biosynthesizes CS-E, and investigated the effects of the CHST15 siRNA on tumor cell proliferation in vitro and growth in vivo. CHST15 mRNA is highly expressed in the human pancreatic cancer cell lines PANC-1, MIA PaCa-2, Capan-1 and Capan-2. CHST15 siRNA significantly inhibited the expression of CHST15 mRNA in these four cells in vitro. Silencing of the CHST15 gene in the cells was associated with significant reduction of proliferation and up-regulation of the cell cycle inhibitor-related gene p21CIP1/WAF1. In a subcutaneous xenograft tumor model of PANC-1 in nude mice, a single intratumoral injection of CHST15 siRNA almost completely suppressed tumor growth. Reduced CHST15 protein signals associated with tumor necrosis were observed with the treatment with CHST15 siRNA. These results provide evidence of the direct action of CHST15 on the proliferation of pancreatic tumor cells partly through the p21CIP1/WAF1 pathway. Thus, CHST15-CS-E axis-mediated tumor cell proliferation could be a novel therapeutic target in the early stage of PDAC progression. PMID:26642349

  9. Effects of arsenic trioxide under different administration ways on T-cell lymphoma xenografts in nude mice: in vivo and in vitro experiments.

    PubMed

    Gao, Yan; Cai, Qing-Qing; Li, Su; Bu, Qing; Liao, Hong; Zhou, Yin; Huang, Hui-Qiang

    2009-02-01

    In vitro, arsenic trioxide (As(2)O(3)) can inhibit proliferation of many lymphoma cell lines. In clinic, it also can be used to treat many subtypes of lymphoma. But the dosage and administration ways are undetermined yet. In this research, we studied the antitumor effect of As(2)O(3) with different administration ways on T-cell lymphoma and observed the toxicity. Murine T-cell lymphoma cell line EL4 was treated with As(2)O(3) of eight concentrations. Cell proliferation was detected by MTT assay. Cell apoptosis was evaluated by flow cytometry with Annexin-V-FITC/PI staining and observed under electroscope and fluorescent microscope. EL4 cells were inoculated into nude mice to establish lymphoma models. The effect of As(2)O(3) on lymphoma in nude mice was observed. As(2)O(3) inhibited the proliferation of EL4 cells with a 50% inhibition concentration (IC(50)) of 1.28 micromol/L at 72 h (p < 0.05). When treated with the same total dose of As(2)O(3) by 4 mg(kg d)(-1) for seven days or 2 mg(kg d)(-1) for 14 days, the inhibition rates of tumor growth in mice were equivalent (58.8% vs. 55.6%, p = 0.351). Apoptotic cells were increased and apoptotic bodies were observed in xenograft tumor tissues. Acute liver damage is the major toxicity. Shortening the administration course and increasing the daily dosage of As(2)O(3) can be considered as a reasonable model for treating advanced/refractory lymphomas.

  10. Evaluation of an [(18)F]AlF-NOTA Analog of Exendin-4 for Imaging of GLP-1 Receptor in Insulinoma.

    PubMed

    Kiesewetter, Dale O; Guo, Ning; Guo, Jinxia; Gao, Haokao; Zhu, Lei; Ma, Ying; Niu, Gang; Chen, Xiaoyuan

    2012-01-01

    The GLP-1 receptor plays an important role in glucose homeostasis and thus is a very important target for diabetes therapy. The receptor is also overexpressed in insulinoma, a tumor of pancreatic beta-cells. We previously evaluated two fluorine-18-labeled analogs of exendin-4 prepared by conjugation with [(18)F]FBEM (N-[2-(4-[(18)F]fluorobenzamide)ethyl]maleimide). Both compounds demonstrated good tumor uptake, but the synthesis of the radiotracers was time consuming. To overcome this challenge, we developed a NOTA analog and performed radiolabeling using aluminum [(18)F]fluoride complexation. Cys(40)-exendin-4 was conjugated with NOTA mono N-ethylmaleimide. [(18)F]AlF conjugation was conducted and the radiolabeled product purified by preparative HPLC. Dynamic and static PET imaging scans were conducted on nude mice with established INS-1 xenografts. Uptake of tumor and other major organs in static images was quantitated (%ID/g) and comparison with blocking studies was made. PET quantification was also compared with ex vivo biodistribution results. The radiosynthesis provided [(18)F]AlF-NOTA-MAL-cys(40)-exendin-4 in 23.6 ± 2.4 % radiochemical yield (uncorrected, n = 3) after HPLC; the process required about 55 min. The specific activity at time of injection ranged from 19.6 to 31.4 GBq (0.53-0.85 Ci)/µmol. Tumor uptake had reached its maximum (16.09 ± 1.18% ID/g, n = 4) by 5 min and remained nearly constant for the duration of the study. Kidney uptake continued to increase throughout the entire one hour time course. Pre-injection of exendin-4 caused a marked reduction in tissue uptake with the major exception of liver and kidneys, in which uptake was not affected. HPLC analysis of the radioactive components in extracts of the tumor and plasma showed primarily parent compound at 60 min post-injection, whereas extracts of kidney and urine contained exclusively one polar radioactive component. The radiotracer is prepared in a simple one-step procedure and obtained

  11. The Novel Organic Arsenical Darinaparsin Induces MAPK-Mediated and SHP1-Dependent Cell Death in T-cell Lymphoma and Hodgkin’s Lymphoma Cells and Human Xenograft Models

    PubMed Central

    Ravi, Dashnamoorthy; Bhalla, Savita; Gartenhaus, Ronald B; Crombie, Jennifer; Kandela, Irawati; Sharma, Jaya; Mazar, Andrew; Evens, Andrew M.

    2014-01-01

    Purpose Darinaparsin (Zio-101) is a novel organic arsenical compound with encouraging clinical activity in relapsed/refractory T-cell lymphoma (TCL) and Hodgkin lymphoma (HL), however little is known regarding its mechanism of action. Experimental Design TCL cell lines (Jurkat, Hut78, and HH) and HL cell lines (L428, L540, and L1236) were examined for in vitro cell death by MTT assay and Annexin-V based flow cytometry. Jurkat and L540-derived xenografts in SCID mice were examined for in vivo tumor inhibition and survival. Biological effects of darinaparsin on MAPK pathway were investigated using pharmacological inhibitors, RNA interference (RNAi) and transient transfection for overexpression for SHP1 and MEK. Results Darinaparsin treatment resulted in dose-dependent cytotoxicity and apoptosis in all TCL and HL cell lines. Additionally, darinaparsin had more rapid, higher, and sustained intracellular arsenic levels compared with arsenic trioxide via mass spectrometry. In vivo experiments with Jurkat (TCL) and L540 (HL)-derived lymphoma xenografts showed significant inhibition of tumor growth and improved survival in darinaparsin-treated SCID mice. Biologically, darinaparsin caused phosphorylation of ERK (and relevant downstream substrates) primarily by decreasing the inhibitory SHP1 phosphatase and co-immunoprecipitation showed significant ERK/SHP1 interaction. Furthermore, ERK shRNA knockdown or constitutive overexpression of SHP1 resulted in increased apoptosis, while co-treatment with pharmacologic MEK inhibitors resulted in synergistic cell death. Conversely, SHP1 blockade (via pharmacologic inhibition or RNAi) as well as MEK constitutive activation decreased darinaparsin-related cell death. Conclusions Altogether, these data show that darinaparsin is highly active in HL and TCL and its activity is dependent primarily on MAPK mechanisms. PMID:25316819

  12. Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma

    PubMed Central

    2013-01-01

    Background The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model. Methods NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 μg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively. Results The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated. Conclusions The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies. PMID:24305507

  13. Robust and cost effective expansion of human regulatory T cells highly functional in a xenograft model of graft-versus-host disease.

    PubMed

    Chakraborty, Rikhia; Mahendravada, Aruna; Perna, Serena K; Rooney, Cliona M; Heslop, Helen E; Vera, Juan F; Savoldo, Barbara; Dotti, Gianpietro

    2013-04-01

    The low frequency of naturally occurring regulatory T cells (nTregs) in peripheral blood and the suboptimal protocols available for their ex vivo expansion limit the development of clinical trials based on the adoptive transfer of these cells. We have, therefore, generated a simplified, robust and cost-effective platform for the large-scale expansion of nTregs using a gas permeable static culture flask (G-Rex) in compliance with Good Manufacturing Practice. More than 10(9) putative Tregs co-expressing CD25 and CD4 molecules (92 ± 5%) and FoxP3 (69 ± 19%) were obtained within 21 days of culture. Expanded Tregs showed potent regulatory activity in vitro (80 ± 13% inhibition of CD8(+) cell division) and in vivo (suppression or delay of graft-versus-host disease in a xenograft mouse model) indicating that the cost-effective and simplified production of nTregs we propose will facilitate the implementation of clinical trials based on their adoptive transfer.

  14. Selective small molecule Stat3 inhibitor reduces breast cancer tumor-initiating cells and improves recurrence free survival in a human-xenograft model.

    PubMed

    Dave, Bhuvanesh; Landis, Melissa D; Tweardy, David J; Chang, Jenny C; Dobrolecki, Lacey E; Wu, Meng-Fen; Zhang, Xiaomei; Westbrook, Thomas F; Hilsenbeck, Susan G; Liu, Dan; Lewis, Michael T

    2012-01-01

    Metastasis and disease relapse are hypothesized to result from tumor initiating cells (TICs). Previously, we have defined a CD44+/CD24-/low mammosphere-forming tumorigenic 493-gene signature in breast cancer. Stat3 was identified as a critical node in self-renewal based on an ongoing lentiviral shRNA screen being conducted in two breast cancer cell lines SUM159 and BT549. In corroborating work, targeting the SH2 domain of Stat3 with a novel small molecule decreased the percentage of cells expressing TIC markers (CD44+/CD24-/low and ALDH+) and mammosphere formation in p-Stat3 overexpressing human breast cancer xenografts in SCID-beige mice. Importantly, we observed a four-fold improvement in the 30-day recurrence-free survival relative to docetaxel alone with the addition of the Stat3 inhibitor in the chemoresistant tumor model. Thus, these findings provide a strong impetus for the development of selective Stat3 inhibitors in order to improve survival in patients with p-Stat3 overexpressing tumors.

  15. Selective Small Molecule Stat3 Inhibitor Reduces Breast Cancer Tumor-Initiating Cells and Improves Recurrence Free Survival in a Human-Xenograft Model

    PubMed Central

    Dave, Bhuvanesh; Landis, Melissa D.; Dobrolecki, Lacey E.; Wu, Meng-Fen; Zhang, Xiaomei; Westbrook, Thomas F.; Hilsenbeck, Susan G.; Liu, Dan; Lewis, Michael T.; Tweardy, David J.; Chang, Jenny C.

    2012-01-01

    Metastasis and disease relapse are hypothesized to result from tumor initiating cells (TICs). Previously, we have defined a CD44+/CD24−/low mammosphere-forming tumorigenic 493-gene signature in breast cancer. Stat3 was identified as a critical node in self-renewal based on an ongoing lentiviral shRNA screen being conducted in two breast cancer cell lines SUM159 and BT549. In corroborating work, targeting the SH2 domain of Stat3 with a novel small molecule decreased the percentage of cells expressing TIC markers (CD44+/CD24−/low and ALDH+) and mammosphere formation in p-Stat3 overexpressing human breast cancer xenografts in SCID-beige mice. Importantly, we observed a four-fold improvement in the 30-day recurrence-free survival relative to docetaxel alone with the addition of the Stat3 inhibitor in the chemoresistant tumor model. Thus, these findings provide a strong impetus for the development of selective Stat3 inhibitors in order to improve survival in patients with p-Stat3 overexpressing tumors. PMID:22879872

  16. Long-term efficiency of mesenchymal stromal cell-mediated CD-MSC/5FC therapy in human melanoma xenograft model.

    PubMed

    Kucerova, L; Skolekova, S; Demkova, L; Bohovic, R; Matuskova, M

    2014-10-01

    Mesenchymal stromal cells (MSC) can be exploited as cellular delivery vehicles for the enzymes converting non-toxic prodrugs to toxic substances. Because of their inherent chemoresistance, they exert potent bystander and antitumor effect. Here we show that the human adipose tissue-derived MSC expressing fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) in combination with 5-fluorocytosine (5FC) mediated a long-term tumor-free survival in the 83.3% of tumor-bearing animals. CD-MSC/5FC treatment induced cytotoxicity against model human melanoma cells EGFP-A375. Only 4% of the therapeutic CD-MSC cells eliminated >98.5% of the tumor cells in vitro. Long-term tumor-free survival was confirmed in 15 out of the 18 animals. However, repeatedly used CD-MSC/5FC therapeutic regimen generated more aggressive and metastatic variant of the melanoma cells EGFP-A375/Rel3. These cells derived from the refractory xenotransplants exhibited increased resistance to the CD-MSC/5FC treatment, altered cell adhesion, migration, tumorigenic and metastatic properties. However, long-term curative effect was achieved by the augmentation of the CD-MSC/5FC regimen along with the inhibition of c-Met/hepatocyte growth factor signaling axis in this aggressive melanoma derivative. In summary, the CD-MSC/5FC regimen can be regarded as a very effective antitumor approach to achieve long-term tumor-free survival as demonstrated on a mouse model of aggressive human melanoma xenografts.

  17. EGFRvIII-Specific Chimeric Antigen Receptor T Cells Migrate to and Kill Tumor Deposits Infiltrating the Brain Parenchyma in an Invasive Xenograft Model of Glioblastoma

    PubMed Central

    Miao, Hongsheng; Choi, Bryan D.; Suryadevara, Carter M.; Sanchez-Perez, Luis; Yang, Shicheng; De Leon, Gabriel; Sayour, Elias J.; McLendon, Roger; Herndon, James E.; Healy, Patrick; Archer, Gary E.; Bigner, Darell D.; Johnson, Laura A.; Sampson, John H.

    2014-01-01

    Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and is uniformly lethal. T-cell-based immunotherapy offers a promising platform for treatment given its potential to specifically target tumor tissue while sparing the normal brain. However, the diffuse and infiltrative nature of these tumors in the brain parenchyma may pose an exceptional hurdle to successful immunotherapy in patients. Areas of invasive tumor are thought to reside behind an intact blood brain barrier, isolating them from effective immunosurveillance and thereby predisposing the development of "immunologically silent" tumor peninsulas. Therefore, it remains unclear if adoptively transferred T cells can migrate to and mediate regression in areas of invasive GBM. One barrier has been the lack of a preclinical mouse model that accurately recapitulates the growth patterns of human GBM in vivo. Here, we demonstrate that D-270 MG xenografts exhibit the classical features of GBM and produce the diffuse and invasive tumors seen in patients. Using this model, we designed experiments to assess whether T cells expressing third-generation chimeric antigen receptors (CARs) targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, would localize to and treat invasive intracerebral GBM. EGFRvIII-targeted CAR (EGFRvIII+ CAR) T cells demonstrated in vitro EGFRvIII antigen-specific recognition and reactivity to the D-270 MG cell line, which naturally expresses EGFRvIII. Moreover, when administered systemically, EGFRvIII+ CAR T cells localized to areas of invasive tumor, suppressed tumor growth, and enhanced survival of mice with established intracranial D-270 MG tumors. Together, these data demonstrate that systemically administered T cells are capable of migrating to the invasive edges of GBM to mediate antitumor efficacy and tumor regression. PMID:24722266

  18. Isoliquiritigenin Induces Apoptosis and Inhibits Xenograft Tumor Growth of Human Lung Cancer Cells by Targeting Both Wild Type and L858R/T790M Mutant EGFR*

    PubMed Central

    Jung, Sung Keun; Lee, Mee-Hyun; Lim, Do Young; Kim, Jong Eun; Singh, Puja; Lee, Sung-Young; Jeong, Chul-Ho; Lim, Tae-Gyu; Chen, Hanyong; Chi, Young-In; Kundu, Joydeb Kumar; Lee, Nam Hyouck; Lee, Charles C.; Cho, Yong-Yeon; Bode, Ann M.; Lee, Ki Won; Dong, Zigang

    2014-01-01

    Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR. PMID:25368326

  19. Isoliquiritigenin induces apoptosis and inhibits xenograft tumor growth of human lung cancer cells by targeting both wild type and L858R/T790M mutant EGFR.

    PubMed

    Jung, Sung Keun; Lee, Mee-Hyun; Lim, Do Young; Kim, Jong Eun; Singh, Puja; Lee, Sung-Young; Jeong, Chul-Ho; Lim, Tae-Gyu; Chen, Hanyong; Chi, Young-In; Kundu, Joydeb Kumar; Lee, Nam Hyouck; Lee, Charles C; Cho, Yong-Yeon; Bode, Ann M; Lee, Ki Won; Dong, Zigang

    2014-12-26

    Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Additive effects of ulinastatin and docetaxel on growth of breast cancer xenograft in nude mice and expression of PGE2, IL-10, and IL-2 in primary breast cancer cells.

    PubMed

    Zhong, Biao; Shen, Hongyan; Sun, Xin; Wang, Hong; Zhang, Yonghua; Sun, Zhijun

    2012-05-01

    Ulinastatin is a broad-spectrum enzyme inhibitor extracted from urine. Previous data from our group suggested that ulinastatin could significantly inhibit proliferation of human breast MDA-MB-231 cells, growth of tumor xenograft in nude mice, and expression of interleukin (IL)-6 and IL-8. In the present study, we investigated whether there is an additive effect of ulinastatin and docetaxel on growth of breast cancer xenografts in nude mice and its possible mechanisms. Nude mice and primary human breast cancer cells were treated with phosphate buffered saline (PBS), ulinastatin, docetaxel, or ulinastatin plus docetaxel, respectively. Their effects on xenograft growth; expressions of cyclooxygenase-2 (COX2), prostaglandin E2 receptor 2 (EP2), IL-10, and IL-2; and secretion of prostaglandin E2 (PGE2) were examined using variety of methods, including semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent (ELISA) assay, and immunohistochemistry SP method. The treatment with ulinastatin, docetaxel, or ulinastatin plus docetaxel could significantly (1) inhibit COX2 and IL-10 expression in primary tumor cells at both mRNA and protein levels, (2) reduce PGE2 secretion in culture supernatant (p<0.05), (3) inhibit COX2, EP2, and IL-10 protein levels in primary xenograft of nude mice, and (4) increase IL-2 expression (p<0.05) in primary xenografts of nude mice. In addition, ulinastatin and docetaxel had additive effects. We suggest that ulinastatin had similar effects of docetaxel and can enhance docetaxel's anticancer effects possibly by inhibiting COX2 expression, reducing PGE2 and EP2 expression and their binding, upregulating IL-2, and downregulating IL-10.

  1. Lower Succinyl-CoA:3-ketoacid-CoA Transferase (SCOT) and ATP Citrate Lyase In Pancreatic Islets of A Rat Model of Type 2 Diabetes: Knockdown of SCOT Inhibits Insulin Release In Rat Insulinoma Cells

    PubMed Central

    Hasan, Noaman M.; Longacre, Melissa J.; Seed Ahmed, Mohammed; Kendrick, Mindy A.; Gu, Harvest; Ostenson, Claes-Goran; Fukao, Toshiyuki; MacDonald, Michael J.

    2010-01-01

    Succinyl-CoA:3-ketoacid-CoA transferase (SCOT) is a mitochondrial enzyme that catalyzes the reversible transfer of coenzyme-A from acetoacetyl-CoA to succinate to form acetoacetate and succinyl-CoA. mRNAs of SCOT and ATP citrate lyase were decreased 55% and 58% and enzyme activities were decreased >70% in pancreatic islets of the GK rat, a model of type 2 diabetes. INS-1 832/13 cells were transfected with shRNAs targeting SCOT mRNA to generate cell lines with reduced SCOT activity. Two cell lines with > 70% knockdown of SCOT activity showed > 70% reduction in glucose- or methyl succinate-plus-β-hydroxybutyrate-stimulated insulin release. Less inhibition of insulin release was observed with two cell lines with less knockdown of SCOT. Previous studies showed knockdown of ATP citrate lyase in INS-1 832/13 cells does not lower insulin release. The results further support work which suggests mitochondrial pathways involving SCOT that supply acetoacetate for export to the cytosol are important for insulin secretion. PMID:20460097

  2. Linarin sensitizes tumor necrosis factor-related apoptosis (TRAIL)-induced ligand-triggered apoptosis in human glioma cells and in xenograft nude mice.

    PubMed

    Xu, Zan-Feng; Sun, Xiao-Ke; Lan, Ying; Han, Chao; Zhang, Yong-Dong; Chen, Gang

    2017-09-22

    Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is reported as a promising anti-cancer therapeutic agent. Nevertheless, a variety of cancer cells, including human malignant glioma cells, are resistant to TRAIL treatment, indicating that it is necessary to find effective strategies to overcome the TRAIL resistance. Linarin (LIN), a natural flavonoid compound in Flos Chrysanthemi Indici (FCI), has been exhibited to exert various pharmacological activities, including anti-cancer. Here in our study, we found that non-cytotoxic doses of LIN (5μM) dramatically potentiated TRAIL (80ng/ml)-induced cytotoxicity (52.36±1.58%) and apoptosis (68.50±1.23%) using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and flow cytometry assays, respectively, in human glioma cells of U87MG. Apoptosis was evidenced by enhanced cleavage of Caspase-8/-9/-3 and poly (ADP-ribose) polymerase (PARP), and reduced anti-apoptotic proteins, including B-cell leukemia/lymphoma 2 (Bcl-2), mantle cell lymphoma (Mcl)-1, and Survivin. Moreover, both intrinsic and extrinsic apoptosis pathways were included in apoptosis induced by LIN and TRAIL co-treatment, along with high release of Cyto-c into cytoplasm and enhancement of fas-associated protein with death domain (FADD), death-inducing signaling complex (DISC), death receptor 4 (DR) 4 and DR5, respectively. Reactive oxygen species (ROS) generation, up to 39.86±2.32%, was also highly triggered by TRAIL and LIN combinational treatment, which was accompanied with high phosphorylation of c-Jun-N-terminal kinase (JNK). In vivo, TRAIL and LIN double treatment significantly reduced the tumor growth using xenograft tumor model through inducing apoptosis. We demonstrated that combining LIN with TRAIL treatments might be effective against TRAIL-resistant glioma cells through inducing apoptosis regulated by ROS generation. Copyright © 2017. Published by Elsevier Masson SAS.

  3. Lidocaine Induces Apoptosis and Suppresses Tumor Growth in Human Hepatocellular Carcinoma Cells In Vitro and in a Xenograft Model In Vivo.

    PubMed

    Xing, Wei; Chen, Dong-Tai; Pan, Jia-Hao; Chen, Yong-Hua; Yan, Yan; Li, Qiang; Xue, Rui-Feng; Yuan, Yun-Fei; Zeng, Wei-An

    2017-05-01

    Recent epidemiologic studies have focused on the potential beneficial effects of regional anesthetics, and the differences in cancer prognosis may be the result of anesthetics on cancer biologic behavior. However, the function and underlying mechanisms of lidocaine in hepatocellular carcinoma both in vitro and in vivo have been poorly studied. Human HepG2 cells were treated with lidocaine. Cell viability, colony formation, cell cycle, and apoptosis were assessed. The effects of lidocaine on apoptosis-related and mitogen-activated protein kinase protein expression were evaluated by Western blot analysis. The antitumor activity of lidocaine in hepatocellular carcinoma with or without cisplatin was investigated with in vitro experiments and also with animal experiments. Lidocaine inhibited the growth of HepG2 cells in a dose- and time-dependent manner. The authors also found that lidocaine arrested cells in the G0/G1 phase of the cell cycle (63.7 ± 1.7% vs. 72.4 ± 3.2%; P = 0.0143) and induced apoptosis (1.7 ± 0.3% vs. 5.0 ± 0.7%; P = 0.0009). Lidocaine may exert these functions by causing an increase in Bax protein and activated caspase-3 and a corresponding decrease in Bcl-2 protein through the extracellular signal-regulated kinase 1/2 and p38 pathways. More importantly, for the first time, xenograft experiments (n = 8 per group) indicated that lidocaine suppressed tumor development (P < 0.0001; lidocaine vs. control) and enhanced the sensitivity of cisplatin (P = 0.0008; lidocaine plus cisplatin vs. cisplatin). The authors' findings suggest that lidocaine may exert potent antitumor activity in hepatocellular carcinoma. Furthermore, combining lidocaine with cisplatin may be a novel treatment option for hepatocellular carcinoma.

  4. Cathepsin B Contributes to Autophagy-related 7 (Atg7)-induced Nod-like Receptor 3 (NLRP3)-dependent Proinflammatory Response and Aggravates Lipotoxicity in Rat Insulinoma Cell Line

    PubMed Central

    Li, Shali; Du, Leilei; Zhang, Lu; Hu, Yue; Xia, Wenchun; Wu, Jia; Zhu, Jing; Chen, Lingling; Zhu, Fengqi; Li, Chunxian; Yang, SiJun

    2013-01-01

    Impairment of glucose-stimulated insulin secretion caused by the lipotoxicity of palmitate was found in β-cells. Recent studies have indicated that defects in autophagy contribute to pathogenesis in type 2 diabetes. Here, we report that autophagy-related 7 (Atg7) induced excessive autophagic activation in INS-1(823/13) cells exposed to saturated fatty acids. Atg7-induced cathepsin B (CTSB) overexpression resulted in an unexpected significant increase in proinflammatory chemokine and cytokine production levels of IL-1β, monocyte chemotactic protein-1, IL-6, and TNF-α. Inhibition of receptor-interacting protein did not affect the inflammatory response, ruling out involvement of necrosis. CTSB siRNA suppressed the inflammatory response but did not affect apoptosis significantly, suggesting that CTSB was a molecular linker between autophagy and the proinflammatory response. Blocking caspase-3 suppressed apoptosis but did not affect the inflammatory response, suggesting that CTSB induced inflammatory effects independently of apoptosis. Silencing of Nod-like receptor 3 (NLRP3) completely abolished both IL-1β secretion and the down-regulation effects of Atg7-induced CTSB overexpression on glucose-stimulated insulin secretion impairment, thus identifying the NLRP3 inflammasome as an autophagy-responsive element in the pancreatic INS-1(823/13) cell line. Combined together, our results indicate that CTSB contributed to the Atg7-induced NLRP3-dependent proinflammatory response, resulting in aggravation of lipotoxicity, independently of apoptosis in the pancreatic INS-1(823/13) cell line. PMID:23986436

  5. Mangosenone F, A Furanoxanthone from Garciana mangostana, Induces Reactive Oxygen Species-Mediated Apoptosis in Lung Cancer Cells and Decreases Xenograft Tumor Growth.

    PubMed

    Seo, Kyung Hye; Ryu, Hyung Won; Park, Mi Jin; Park, Ki Hun; Kim, Jin Hyo; Lee, Mi-Ja; Kang, Hyeon Jung; Kim, Sun Lim; Lee, Jin Hwan; Seo, Woo Duck

    2015-11-01

    Mangosenone F (MSF), a natural xanthone, was isolated form Carcinia mangotana, and a few studies have reported its glycosidase inhibitor effect. In this study we investigated the anti lung cancer effect of MSF both in vitro and in vivo. MSF inhibited cancer cell cytotoxicity and induced and induced apoptosis via reactive oxygen species (ROS) generation in NCI-H460. MSF treatment also showed in pronounced release of apoptogenic cytochrome c from the mitochondria to the cytosol, downregulation of Bcl-2 and Bcl-xL, and upregulation of Bax, suggesting that caspase-mediated pathways were involved in MSF-induced apoptosis. ROS activation of the mitogen-activated protein kinase signaling pathway was shown to play a predominant role in the apoptosis mechanism of MSF. Compared with cisplatin treatment, MSF treatment showed significantly increased inhibition of the growth of NCI-H460 cells xenografted in nude mice. Together, these results indicate the potential of MSF as a candidate natural anticancer drug by promoting ROS production. Copyright © 2015 John Wiley & Sons, Ltd.

  6. Gene and microRNA modulation upon trabectedin treatment in a human intrahepatic cholangiocarcinoma paired patient derived xenograft and cell line

    PubMed Central

    Peraldo Neia, Caterina; Cavalloni, Giuliana; Chiorino, Giovanna; Ostano, Paola; Aglietta, Massimo; Leone, Francesco

    2016-01-01

    Intrahepatic cholangiocarcinoma (ICC) is an aggressive and lethal malignancy with limited therapeutic options. Trabectedin has a high antitumor activity in preclinical models of biliary tract carcinoma (BTC), being a promising alternative treatment. Here, we studied the effect of trabectedin at transcriptomic level on an ICC patient derived xenograft (PDX) and on the derived cell line, MT-CHC01. Further, putative targets of trabectedin were explored in the in vitro model. In vitro, trabectedin inhibited genes involved in protein modification, neurogenesis, migration, and motility; it induced the expression of genes involved in keratinization, tissues development, and apoptotic processes. In the PDX model, trabectedin affected ECM-receptor interaction, focal adhesion, complement and coagulation cascades, Hedgehog, MAPK, EGFR signaling via PIP3 pathway, and apoptosis. Among down-regulated genes, we selected SYK and LGALS1; their silencing caused a significantly reduction of migration, but did not affect proliferation in in vitro models. In MT-CHC01 cells, 24 microRNAs were deregulated upon drug treatment, while only 5 microRNAs were perturbed by trabectedin in PDX. The target prediction analysis showed that SYK and LGALS1 are putative targets of up-regulated microRNAs. In conclusion, we described that trabectedin affected genes and microRNAs involved in tumor progression and metastatic processes, reflecting data previously obtained at macroscopically level; in particular, we identified SYK and LGALS1 as new putative targets of trabectedin. PMID:27902465

  7. The Neuropeptide Y Y1 Receptor: A Diagnostic Marker? Expression in MCF-7 Breast Cancer Cells Is Down-Regulated by Antiestrogens In Vitro and in Xenografts

    PubMed Central

    Memminger, Martin; Keller, Max; Lopuch, Miroslaw; Pop, Nathalie; Bernhardt, Günther; von Angerer, Erwin; Buschauer, Armin

    2012-01-01

    The neuropeptide Y (NPY) Y1 receptor (Y1R) has been suggested as a tumor marker for in vivo imaging and as a therapeutic target. In view of the assumed link between estrogen receptor (ER) and Y1R in mammary carcinoma and with respect to the development of new diagnostic tools, we investigated the Y1R protein expression in human MCF-7 cell variants differing in ER content and sensitivity against antiestrogens. ER and Y1R expression were quantified by radioligand binding using [3H]-17β-estradiol and the Y1R selective antagonist [3H]-UR-MK114, respectively. The latter was used for cellular binding studies and for autoradiography of MCF-7 xenografts. The fluorescent ligands Cy5-pNPY (universal Y1R, Y2R and Y5R agonist) and UR-MK22 (selective Y1R antagonist), as well as the selective antagonists BIBP3226 (Y1R), BIIE0246 (Y2R) and CGP71683 (Y5R) were used to identify the NPY receptor subtype(s) by confocal microscopy. Y1R functionality was determined by mobilization of intracellular Ca2+. Sensitivity of MCF-7 cells against antiestrogen 4-hydroxytamoxifen correlated directly with the ER content. The exclusive expression of Y1Rs was confirmed by confocal microscopy. The Y1R protein was up-regulated (100%) by 17β-estradiol (EC50 20 pM) and the predominant role of ERα was demonstrated by using the ERα-selective agonist “propylpyrazole triol”. 17β-Estradiol-induced over-expression of functional Y1R protein was reverted by the antiestrogen fulvestrant (IC50 5 nM) in vitro. Furthermore, tamoxifen treatment of nude mice resulted in an almost total loss of Y1Rs in MCF-7 xenografts. In conclusion, the value of the Y1R as a target for therapy and imaging in breast cancer patients may be compromised due to Y1R down-regulation induced by hormonal (antiestrogen) treatment. PMID:23236424

  8. Progenitor cell dose determines the pace and completeness of engraftment in a xenograft model for cord blood transplantation

    PubMed Central

    Liu, Congxiao; Chen, Benny J.; DeOliveira, Divinomar; Sempowski, Gregory D.; Chao, Nelson J.

    2010-01-01

    Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma–null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34+ human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks), human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses, as documented by the presence of CD4+ CD8+ T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation, human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses, the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. PMID:20833978

  9. Neuronal Subtype and Satellite Cell Tropism Are Determinants of Varicella-Zoster Virus Virulence in Human Dorsal Root Ganglia Xenografts In Vivo

    PubMed Central

    Zerboni, Leigh; Arvin, Ann

    2015-01-01

    Varicella zoster virus (VZV), a human alphaherpesvirus, causes varicella during primary infection. VZV reactivation from neuronal latency may cause herpes zoster, post herpetic neuralgia (PHN) and other neurologic syndromes. To investigate VZV neuropathogenesis, we developed a model using human dorsal root ganglia (DRG) xenografts in immunodeficient (SCID) mice. The SCID DRG model provides an opportunity to examine characteristics of VZV infection that occur in the context of the specialized architecture of DRG, in which nerve cell bodies are ensheathed by satellite glial cells (SGC) which support neuronal homeostasis. We hypothesized that VZV exhibits neuron-subtype specific tropism and that VZV tropism for SGC contributes to VZV-related ganglionopathy. Based on quantitative analyses of viral and cell protein expression in DRG tissue sections, we demonstrated that, whereas DRG neurons had an immature neuronal phenotype prior to implantation, subtype heterogeneity was observed within 20 weeks and SGC retained the capacity to maintain neuronal homeostasis longterm. Profiling VZV protein expression in DRG neurons showed that VZV enters peripherin+ nociceptive and RT97+ mechanoreceptive neurons by both axonal transport and contiguous spread from SGC, but replication in RT97+ neurons is blocked. Restriction occurs even when the SGC surrounding the neuronal cell body were infected and after entry and ORF61 expression, but before IE62 or IE63 protein expression. Notably, although contiguous VZV spread with loss of SGC support would be predicted to affect survival of both nociceptive and mechanoreceptive neurons, RT97+ neurons showed selective loss relative to peripherin+ neurons at later times in DRG infection. Profiling cell factors that were upregulated in VZV-infected DRG indicated that VZV infection induced marked pro-inflammatory responses, as well as proteins of the interferon pathway and neuroprotective responses. These neuropathologic changes observed in sensory

  10. Human mammary fibroblasts stimulate invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts.

    PubMed

    Olsen, Charlotta J; Moreira, José; Lukanidin, Eugene M; Ambartsumian, Noona S

    2010-08-19

    Tumour phenotype is regulated in a complex fashion as a result of interactions between malignant cells and the tumour stroma. Fibroblasts are the most abundant and perhaps most active part of the tumour stroma. A better understanding of the changes that occur in fibroblasts in response to the presence of malignant cells may lead to the development of new strategies for cancer treatment. We explored the effects of fibroblasts on the growth and invasion of mammary carcinoma tumour cells in vitro and in vivo. In order to analyse secreted factors that affect invasive abilities of breast cancer cells we co-cultured human mammary fibroblasts (HMF3s) and cancer cells (MCF7S1) in three-dimensional (3D) growth conditions devoid of heterogeneous cell-cell contact. To study the possible influence of fibroblasts on MCF7S1 cancer cell growth in vivo we co-injected HMF3s and MCF7S1 cells in Balb/c nu/nu mice. In 3D co-culture both HMF3s and MCF7S1 cells demonstrated enhanced invasion into a Matrigel matrix. This was correlated with enhanced expression of the metastasis promoting S100A4 protein in fibroblasts, stimulation of the matrix metalloproteinase (MMP)-2 activity, and enhanced secretion of a range of different cytokines. Orthotopic injection of oestrogen-dependent MCF7S1 cancer cells together with fibroblasts showed stimulation of tumour growth in mice without an external oestrogen supply. The resulting tumours were characterized by increased development of extracellular matrix, as well as an increase of murine S100A4 concentration and activity of MMP-2 in the tumour interstitial fluid. Stimulation of the invasive phenotype of tumour cells in 3D co-cultures with fibroblasts could be correlated with increased production of S100A4 and MMP-2. We propose that enhanced development of mouse host-derived tumour stroma in a MCF7S1 co-injection xenograft model leads to oestrogen independency and is triggered by the initial presence of human fibroblasts.

  11. Epithelial requirement for in vitro proliferation and xenograft growth and metastasis of MDA-MB-468 human breast cancer cells: oncogenic rather than tumor-suppressive role of E-cadherin.

    PubMed

    Hugo, H J; Gunasinghe, N P A D; Hollier, B G; Tanaka, T; Blick, T; Toh, A; Hill, P; Gilles, C; Waltham, M; Thompson, E W

    2017-07-27

    Epithelial-to-mesenchymal transition (EMT) is associated with downregulated E-cadherin and frequently with decreased proliferation. Proliferation may be restored in secondary metastases by mesenchymal-to-epithelial transition (MET). We tested whether E-cadherin maintains epithelial proliferation in MDA-MB-468 breast cancer cells, facilitating metastatic colonization in severe combined immunodeficiency (SCID) mice. EMT/MET markers were assessed in xenograft tumors by immunohistochemistry. Stable E-cadherin manipulation was effected by transfection and verified by Western blotting, immunocytochemistry, and quantitative polymerase chain reaction (qPCR). Effects of E-cadherin manipulation on proliferation and chemomigration were assessed in vitro by performing sulforhodamine B assays and Transwell assays, respectively. Invasion was assessed by Matrigel outgrowth; growth in vivo was assessed in SCID mice; and EMT status was assessed by qPCR. Hypoxic response of E-cadherin knockdown cell lines was assessed by qPCR after hypoxic culture. Repeated measures analysis of variance (ANOVA), one- and two-way ANOVA with posttests, and paired Student's t tests were performed to determine significance (p < 0.05). EMT occurred at the necrotic interface of MDA-MB-468 xenografts in regions of hypoxia. Extratumoral deposits (vascular and lymphatic inclusions, local and axillary nodes, and lung metastases) strongly expressed E-cadherin. MDA-MB-468 cells overexpressing E-cadherin were more proliferative and less migratory in vitro, whereas E-cadherin knockdown (short hairpin CDH1 [shCDH1]) cells were more migratory and invasive, less proliferative, and took longer to form tumors. shCDH1-MDA-MB-468 xenografts did not contain the hypoxia-induced necrotic areas observed in wild-type (WT) and shSCR-MDA-MB-468 tumors, but they did not exhibit an impaired hypoxic response in vitro. Although vimentin expression was not stimulated by E-cadherin knockdown in 2D or 3D cultures, xenografts of

  12. Embryonic mouse STO cell-derived xenografts express hepatocytic functions in the livers of nonimmunosuppressed adult rats.

    PubMed

    Zhang, Mingjun; Joseph, Brigid; Gupta, Sanjeev; Guest, I; Xu, Meng; Sell, Stewart; Son, Kyung-Hwa; Koch, Katherine S; Leffert, Hyam L

    2005-02-01

    Cells derived from embryonic mouse STO cell lines differentiate into hepatocytes when transplanted into the livers of nonimmunosuppressed dipeptidylpeptidase IV (DPPIV)-negative F344 rats. Within 1 day after intrasplenic injection, donor cells moved rapidly into the liver and were found in intravascular and perivascular sites; by 1 month, they were intrasinusoidal and also integrated into hepatic plates with approximately 2% efficiency and formed conjoint bile canaliculi. Neither donor cell proliferation nor host inflammatory responses were observed during this time. Detection of intrahepatic mouse COX1 mitochondrial DNA and mouse albumin mRNA in recipient rats indicated survival and differentiation of donor cells for at least 3 months. Mouse COX1 targets were also detected intrahepatically 4-9 weeks after STO cell injection into nonimmunosuppressed wild-type rats. In contrast to STO-transplanted rats, mouse DNA or RNA was not detectable in untreated or mock-transplanted rats or in rats injected with donor cell DNA. In cultured STO donor cells, DPPIV and glucose-6-phosphatase activities were observed in small clusters; in contrast, mouse major histocompatibility complex class I H-2Kq, H-2Dq, and H-2Lq and class II I-Aq markers were undetectable in vitro before or after interferon gamma treatment. Together with H-2K allele typing, which confirmed the Swiss mouse origin of the donor cells, these observations indicate that mouse-derived STO cell lines can differentiate along hepatocytic lineage and engraft into rat liver across major histocompatibility barriers.

  13. Sequence and functional expression in Xenopus oocytes of a human insulinoma and islet potassium channel

    SciTech Connect

    Philipson, L.H.; Hice, R.E.; Schaefer, K.; LaMendola, J.; Bell, G.I.; Nelson, D.J.; Steiner, D.F. )

    1991-01-01

    Regulation of insulin secretion involves the coordinated control of ion channels in the {beta}-cell membrane. The authors have isolated and characterized cDNA and genomic clones encoding a voltage-dependent K{sup +} channel isoform expressed in human islets and in a human insulinoma. This K{sup +} channel isoform, designated hPCN1, with a deduced amino acid sequence of 613 residues is related to the Shaker family of Drosophila K{sup +} channels. hPCN1 is homologous to two other human K{sup +} channel isoforms. They have isolated, hPCN2 and hPCN3, with 55% and 65% amino acid sequence identity, respectively. The electrophysiological characteristics of hPCN1 were determined after microinjuection of synthetic RNA into Xenopus oocytes. Two-microelectrode voltage-clamp recordings of oocytes injected with hPCN1 RNA revealed a voltage-dependent outward K{sup +} current that inactivated slowly with time. Outward currents were inhibited by 4-aminopyridine with a K{sub i} less that 0.01 mM and were relatively insensitive to tetraethylammonium ion or Ba{sup 2+}. A delayed rectifier K{sup +} channel such as hPCN1 could restore the resting membrane potential of {beta} cells after depolarization and thereby contribute to the regulation of insulin secretion.

  14. Anticancer activity of taraxerol acetate in human glioblastoma cells and a mouse xenograft model via induction of autophagy and apoptotic cell death, cell cycle arrest and inhibition of cell migration.

    PubMed

    Hong, Jing-Fang; Song, Ying-Fang; Liu, Zheng; Zheng, Zhao-Cong; Chen, Hong-Jie; Wang, Shou-Sen

    2016-06-01

    The aim of the present study was to investigate the in vitro and in vivo anticancer and apoptotic effects of taraxerol acetate in U87 human glioblastoma cells. The effects on cell cycle phase distribution, cell cycle-associated proteins, autophagy, DNA fragmentation and cell migration were assessed. Cell viability was determined using the MTT assay, and phase contrast and fluorescence microscopy was utilized to determine the viability and apoptotic morphological features of the U87 cells. Flow cytometry using propidium iodide and Annexin V-fluorescein isothiocyanate demonstrated the effect of taraxerol acetate on the cell cycle phase distribution and apoptosis induction. Western blot analysis was performed to investigate the effect of the taraxerol acetate on cell cycle‑associated proteins and autophagy‑linked LC3B‑II proteins. The results demonstrated that taraxerol acetate induced dose‑ and time‑dependent cytotoxic effects in the U87 cells. Apoptotic induction following taraxerol acetate treatment was observed and the percentage of apoptotic cells increased from 7.3% in the control cells, to 16.1, 44.1 and 76.7% in the 10, 50 and 150 µM taraxerol acetate‑treated cells, respectively. Furthermore, taraxerol acetate treatment led to sub‑G1 cell cycle arrest with a corresponding decrease in the number of S‑phase cells. DNA fragments were observed as a result of the gel electrophoresis experiment following taraxerol acetate treatment. To investigate the inhibitory effects of taraxerol acetate on the migration of U87 cell, a wound healing assay was conducted. The number of cells that migrated to the scratched area decreased significantly following treatment with taraxerol acetate. In addition, taraxerol acetate inhibited tumor growth in a mouse xenograft model. Administration of 0.25 and 0.75 µg/g taraxerol acetate reduced the tumor weight from 1.2 g in the phosphate‑buffered saline (PBS)‑treated group (control) to 0.81 and 0.42

  15. Cryoprotectant Delivery and Removal from Murine Insulinomas at Vitrification-Relevant Concentrations

    PubMed Central

    Mukherjee, Indra Neil; Song, Ying C.; Sambanis, Athanassios

    2009-01-01

    Development of optimal cryopreservation protocols requires delivery and removal of cryoprotective agents (CPAs) in such a way that negative osmotic and cytotoxic effects on cells are minimized. This is especially true for vitrification, where high CPA concentrations are employed. In this study, we report on the determination of cell membrane permeability parameters for water (Lp) and solute (Ps), and on the design and experimental verification of CPA addition and removal protocols at vitrification-relevant concentrations for a murine insulinoma cell line, βTC-tet cells. Using membrane permeability values and osmotic tolerance limits, mathematical modeling and computer simulations were used to design CPA addition and removal protocols at high concentrations. The cytotoxic effects of CPAs were also evaluated. Cells were able to tolerate the addition and removal of 2.5 M dimethyl sulfoxide (DMSO) and 2.5 M 1,2 propanediol (PD) in single steps, but required multi-step addition and removal with 3.0 M DMSO, 3.0 M PD, and a vitrification-relevant concentration of 3.0 M DMSO+3.0M PD. Cytotoxicity studies revealed that βTC-tet cells were able to tolerate the presence of single component 6.0 M DMSO and 6.0 M PD and to a lesser extent 3.0 M DMSO+3.0 M PD. These results determine the time and concentration domain of CPA exposure that cells can tolerate and are essential for designing cryopreservation protocols for free cells as well as cells in engineered tissues. PMID:17533114

  16. Development of (111)In-labeled exendin(9-39) derivatives for single-photon emission computed tomography imaging of insulinoma.

    PubMed

    Kimura, Hiroyuki; Matsuda, Hirokazu; Ogawa, Yu; Fujimoto, Hiroyuki; Toyoda, Kentaro; Fujita, Naotaka; Arimitsu, Kenji; Hamamatsu, Keita; Yagi, Yusuke; Ono, Masahiro; Inagaki, Nobuya; Saji, Hideo

    2017-02-15

    Insulinoma is a tumor derived from pancreatic β-cells, and the resulting hyperinsulinemia leads to characteristic hypoglycemia. Recent studies have reported the frequent overexpression of glucagon-like peptide-1 receptor (GLP-1R) in human insulinomas, suggesting that the binding of a radiolabeled compound to GLP-1R is useful for the imaging of such tumors. Exendin(9-39), a fragment peptide of exendin-3 and -4, binds GLP-1R with high affinity and acts as an antagonist. Accordingly, radiolabeled exendin(9-39) derivatives have also been investigated as insulinoma imaging probes that might be less likely to induce hypoglycemia. In this study, we synthesized a novel indium-111 ((111)In)-benzyl-diethylenetriaminepentaacetic acid ((111)In-BnDTPA)-conjugated exendin(9-39), (111)In-BnDTPA-exendin(9-39), and evaluated its utility as a probe for the SPECT imaging of insulinoma. natIn-BnDTPA-exendin(9-39) exhibited a high affinity for GLP-1R (IC50=2.5nM), stability in plasma, and a specific activity that improved following reactions with a solvent and solubilizer. Regarding the in vivo biodistribution of (111)In-BnDTPA-exendin(9-39) in INS-1 tumor-bearing mice, high uptake levels were observed in tumors (14.6%ID/g at 15min), with corresponding high tumor-to-blood (T/B), tumor-to-muscle (T/M), and tumor-to-pancreas (T/P) ratios (T/B=2.55, T/M=22.7, T/P=2.7 at 1h). The pre-administration of excess nonradioactive exendin(9-39) significantly reduced accumulation in both the tumor and pancreas (76% and 68% inhibition, respectively) at 1h after (111)In-BnDTPA-exendin(9-39) injection, indicating that the GLP-1R mediated a majority of (111)In-BnDTPA-exendin(9-39) uptake in the tumor and pancreas. Finally, (111)In-BnDTPA-exendin(9-39) SPECT/CT studies in mice yielded clear images of tumors at 30min post-injection. These results suggest that (111)In-BnDTPA-exendin(9-39) could be a useful SPECT molecular imaging probe for the detection and exact localization of insulinomas. Copyright

  17. Endostatin enhances antitumor effect of tumor antigen-pulsed dendritic cell therapy in mouse xenograft model of lung carcinoma

    PubMed Central

    Liang, Jing; Liu, Xiaolin; Xie, Qi; Chen, Guoling; Li, Xingyu; Jia, Yanrui; Yin, Beibei; Qu, Xun; Li, Yan

    2016-01-01

    Objective To investigate the antitumor effect of endostatin combined with tumor antigen-pulsed dendritic cell (DC)-T cell therapy on lung cancer. Methods Transplanted Lewis lung cancer (LLC) models of C57BL/6 mice were established by subcutaneous injection of LLC cells in left extremity axillary. Tumor antigen-pulsed DC-T cells from spleen cells and bone of mice were cultured in vitro. Tumor-bearing mice were randomly divided into three groups, including DC-T+endostatin group, DC-T group, and phosphate-buffered saline (PBS) control group. Microvessel density (MVD) of tumor tissue in tumor-bearing mice was determined by immunohistochemistry (IHC). The expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by Western blotting and IHC staining. The proportions of CD8+ T cells, mature dendritic cells (mDC), tumor-associated macrophages [TAM (M1/M2)], and myeloid-derived suppressor cells (MDSC) in suspended cells of tumor tissue were determined by flow cytometry. The expressions of interleukin (IL)-6, IL-10, IL-17, transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ) in suspended cells of tumor tissue were detected by enzyme-linked immune sorbent assay (ELISA). Results DC-T cells combined with endostatin remarkably suppressed tumor growth. MVD of mice in DC-T+endostatin group was significantly lower than that of the control group and DC-T monotherapy group. The expressions of VEGF, IL-6 and IL-17 in tumors were markedly decreased, but IFN-γ and HIF-1α increased after treating with DC-T cells combined with endostatin, compared to control group and DC-T group. In the DC-T+endostatin group, the proportions of MDSC and TAM (M2 type) were significantly decreased, mDC and TAM (M1 type) were up-regulated, and CD8+ T cells were recruited to infiltrate tumors, in contrast to PBS control and DC-T monotherapy. DC-T cells combined with endostatin potently reduced the expressions of IL-6, IL-10, TGF-β and

  18. Sorafenib Inhibits Lymphoma Xenografts by Targeting MAPK/ERK and AKT Pathways in Tumor and Vascular Cells

    PubMed Central

    Carlo-Stella, Carmelo; Locatelli, Silvia L.; Giacomini, Arianna; Cleris, Loredana; Saba, Elena; Righi, Marco; Guidetti, Anna; Gianni, Alessandro M.

    2013-01-01

    The anti-lymphoma activity and mechanism(s) of action of the multikinase inhibitor sorafenib were investigated using a panel of lymphoma cell lines, including SU-DHL-4V, Granta-519, HD-MyZ, and KMS-11 cell lines. In vitro, sorafenib significantly decreased cell proliferation and phosphorylation levels of MAPK and PI3K/Akt pathways while increased apoptotic cell death. In vivo, sorafenib treatment resulted in a cytostatic rather than cytotoxic effect on tumor cell growth associated with a limited inhibition of tumor volumes. However, sorafenib induced an average 50% reduction of tumor vessel density and a 2-fold increase of necrotic areas. Upon sorafenib treatment, endothelial and tumor cells from SU-DHL-4V, Granta-519, and KMS-11 nodules showed a potent inhibition of either phospho-ERK or phospho-AKT, whereas a concomitant inhibition of phospho-ERK and phospho-AKT was only observed in HD-MyZ nodules. In conclusion, sorafenib affects the growth of lymphoid cell lines by triggering antiangiogenic mechanism(s) and directly targeting tumor cells. PMID:23620775

  19. 5-Aza-2'-deoxycytidine suppresses human renal carcinoma cell growth in a xenograft model via up-regulation of the connexin 32 gene.

    PubMed

    Hagiwara, H; Sato, H; Ohde, Y; Takano, Y; Seki, T; Ariga, T; Hokaiwado, N; Asamoto, M; Shirai, T; Nagashima, Y; Yano, T

    2008-04-01

    The connexin (Cx) 32 gene, a member of the gap junction gene family, acts as a tumour suppressor gene in human renal cell carcinoma (RCC) and is down-regulated by the hypermethylation of CpG islands in a promoter region of the Cx gene. The current study investigated whether the restoration of Cx32 silenced by hypermethylation in RCC by a DNA demethylating agent could be an effective treatment against RCC. Using nude mice bearing Caki-1 cells (a human metastatic RCC cell line), the effects of 5-aza-2'-deoxycytidine (5-aza-CdR), a DNA demethylase inhibitor, on Cx32 mRNA expression and tumour growth were examined by RT-PCR, and by measuring tumour weight and volume. Cx32 expression in Caki-1 tumours was inhibited by Cx32 short interfering (si) RNA, and the effect of siRNA on 5-aza-CdR-dependent suppression of tumour growth in nude mice was evaluated. 5-aza-CdR treatment inhibited the growth of Caki-1 cells in nude mice by 70% and increased 7-fold the level of Cx32 mRNA. The intratumour injection of Cx32 siRNA almost totally inhibited the expression of Cx32 mRNA and significantly reduced the suppression of tumour growth in 5-aza-CdR-treated nude mice. 5-aza-CdR suppressed the growth of Caki-1 tumours in a xenograft model, by restoring Cx32 expression. This finding suggests that treatment with 5-aza-CdR could be a new effective therapy against human metastatic RCC and that Cx32 could be a potential target for the treatment of RCC.

  20. Progesterone receptor membrane component 1 promotes survival of human breast cancer cells and the growth of xenograft tumors

    PubMed Central

    Clark, Nicole C.; Friel, Anne M.; Pru, Cindy A.; Zhang, Ling; Shioda, Toshi; Rueda, Bo R.; Peluso, John J.; Pru, James K.

    2016-01-01

    ABSTRACT Triple negative breast cancers (TNBCs) are highly aggressive and grow in response to sex steroid hormones despite lacking expression of the classical estrogen (E2) and progesterone (P4) receptors. Since P4 receptor membrane component 1 (PGRMC1) is expressed in breast cancer tumors and is known to mediate P4-induced cell survival, this study was designed to determine the expression of PGRMC1 in TNBC tumors and the involvement of PGRMC1 in regulating proliferation and survival of TNBC cells in vitro and the growth of TNBC tumors in vivo. For the latter studies, the MDA-MB-231 (MDA) cell line derived from TNBC was used. These cells express PGRMC1 but lack expression of the classical P4 receptor. A lentiviral-based shRNA approach was used to generate a stably transfected PGRMC1-deplete MDA line for comparison to the PGRMC1-intact MDA line. The present studies demonstrate that PGRMC1: 1) is expressed in TNBC cells; 2) mediates the ability of P4 to suppress TNBC cell mitosis in vitro; 3) is required for P4 to reduce the apoptotic effects of doxorubicin in vitro; and 4) facilitates TNBC tumor formation and growth in vivo. Taken together, these findings indicate that PGRMC1 plays an important role in regulating the growth and survival of TNBC cells in vitro and ultimately in the formation and development of these tumors in vivo. Thus, PGRMC1 may be a therapeutic target for TNBCs. PMID:26785864

  1. Progesterone receptor membrane component 1 promotes survival of human breast cancer cells and the growth of xenograft tumors.

    PubMed

    Clark, Nicole C; Friel, Anne M; Pru, Cindy A; Zhang, Ling; Shioda, Toshi; Rueda, Bo R; Peluso, John J; Pru, James K

    2016-01-01

    Triple negative breast cancers (TNBCs) are highly aggressive and grow in response to sex steroid hormones despite lacking expression of the classical estrogen (E2) and progesterone (P4) receptors. Since P4 receptor membrane component 1 (PGRMC1) is expressed in breast cancer tumors and is known to mediate P4-induced cell survival, this study was designed to determine the expression of PGRMC1 in TNBC tumors and the involvement of PGRMC1 in regulating proliferation and survival of TNBC cells in vitro and the growth of TNBC tumors in vivo. For the latter studies, the MDA-MB-231 (MDA) cell line derived from TNBC was used. These cells express PGRMC1 but lack expression of the classical P4 receptor. A lentiviral-based shRNA approach was used to generate a stably transfected PGRMC1-deplete MDA line for comparison to the PGRMC1-intact MDA line. The present studies demonstrate that PGRMC1: 1) is expressed in TNBC cells; 2) mediates the ability of P4 to suppress TNBC cell mitosis in vitro; 3) is required for P4 to reduce the apoptotic effects of doxorubicin in vitro; and 4) facilitates TNBC tumor formation and growth in vivo. Taken together, these findings indicate that PGRMC1 plays an important role in regulating the growth and survival of TNBC cells in vitro and ultimately in the formation and development of these tumors in vivo. Thus, PGRMC1 may be a therapeutic target for TNBCs.

  2. [Video-laparoscopic excision of pancreatic insulinoma. Experience with 3 cases].

    PubMed

    Pugliese, Raffaele; Boniardi, Marco; Sansonna, Fabio; Maggioni, Dario; Scandroglio, Ildo; Costanzi, Andrea; Rapetti, Rosangela; Oppizzi, Giuseppe; Loli, Paola

    2008-01-01

    Laparoscopic treatment of lesions of the distal pancreas has gained favour worldwide in the last decade. The objective of this study was to analyze 3 cases of insulinoma successfully treated with the laparoscopic approach. From 2000 to 2007 in our institution 3 patients with insulinoma of the left pancreas were treated with a laparoscopic approach. The insulinoma was diagnosed by helical CT scan, Two cases were treated by left pancreatectomy and one by enucleation. The resections were achieved by laparoscopy with no conversion to laparotomy. There were no intraoperative complications. Average blood loss was 180 mi (range: 150-350). Mean operative time was 232 minutes (range: 225-240). Morbidity consisted in one mild pancreatic fistula after left pancreatectomy that was healed by conservative treatment after 24 days. The mean hospital stay was 13 days (range: 10-20). During the follow-up insulinoma symptoms have disappeared in all patients. This study confirms the feasibility of laparoscopic resection for insulinoma. Operative times were quite acceptable and the conversion rate was nil. Times to oral intake and walking were shorter than after open surgery, as was the mean postoperative hospital stay.

  3. Synergistic and attenuated effect of HSS in combination treatment with docetaxel plus cisplatin in human non-small-cell lung SPC-A-1 tumor xenograft.

    PubMed

    Jia, Yuping; Zhou, Dongshun; Jia, Qingwen; Ying, Yong; Chen, Shuntai

    2016-04-01

    Platinum based combination regimens are first-line treatment option in treatment of non-small cell lung cancer (NSCLC) but the clinical utility has been limited because of their toxicities. Many reports indicated that patients with tumors can benefit from adjuvant chemotherapy drugs. The aim of this study was to confirm adjuvant chemotherapy of HSS with docetaxel plus cisplatin (DP) against NSCLC by evaluating antitumor activity and attenuated effect. In vivo SPC-A-1 xenograft model was established to evaluate antitumor activity and toxicity of HSS along or combination with DP. Evaluation indexes include the relative tumor proliferation rate, tumor growth inhibition rate, body weight, food consumption, hematological and biochemical analysis. HSS treatment showed inhibited tumor growth and increased tumor inhibition of DP treatment at doses of 250 mg/kg and 500 mg/kg. No significant toxicity was found in HSS-treated mice, but significant toxicity was found in DP-treated mice. HSS combination with DP could reduce toxicity of DP treatment by improving body weight and food consumption, and increasing the number of WBC and PLT, decreasing the level of ALT, AST and BUN. HSS combined with DP treatment has additive effect which contributes to enhance tumor growth inhibition of DP treatment and attenuated effect which contributes to reduce toxicity of DP treatment. These findings indicate potential benefit for use of HSS adjuvant chemotherapy for NSCLC treatment. Copyright © 2016. Published by Elsevier Masson SAS.

  4. An in vivo swine study for xeno-grafts of calcium sulfate-based bone grafts with human dental pulp stem cells (hDPSCs).

    PubMed

    Kuo, Tzong-fu; Lee, Sheng-Yang; Wu, Hong-Da; Poma, Malosi; Wu, Yu-Wei; Yang, Jen-Chang

    2015-05-01

    The purpose of this in vivo study was to evaluate the effect of human dental pulp stem cells (hDPSCs) on various resorbable calcium sulfate/calcium phosphate bone grafts in bone regeneration. Granular particles of calcium sulfate dehydrate (CSD), α-calcium sulfate hemihydrate/amorphous calcium phosphate (α-CSH/ACP), and CSD/β-tricalcium phosphates (β-TCP) were prepared for in vitro dissolution and implantation test. The chemical compositions of specimen residues after dissolution test were characterized by XRD. The ratios of new bone formation for implanted grafts/hDPSCs were evaluated using mandible bony defect model of Lanyu pig. All the graft systems exhibited a similar two-stage dissolution behavior and phase transformation of poor crystalline HAp. Eight weeks post-operation, the addition of hDPSCs to various graft systems showed statistically significant increasing in the ratio of new bone formation (p<0.05). Null hypothesis of hDPSCs showing no scaffold dependence in bone regeneration was rejected. The results suggest that the addition of hDPSCs to calcium sulfate based xenografts could enhance the bone regeneration in the bony defect. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Generation of human/rat xenograft animal model for the study of human donor stem cell behaviors in vivo

    PubMed Central

    Sun, Yan; Xiao, Dong; Pan, Xing-Hua; Zhang, Ruo-Shuang; Cui, Guang-Hui; Chen, Xi-Gu

    2007-01-01

    AIM: To accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed. METHODS: Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients. RESULTS: Of 29 live-born recipients, 19 had the presence of human CD45+ cells in peripheral blood (PB) detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues (i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin) examined at the time of tissue collection, as confirmed by detecting human β2-microglobulin expression using immunohistochemistry. In this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specific differentiation into CK18-positive human cells in chimeric liver and CD45-positive human cells in chimeric spleen and thymus of recipients. CONCLUSION: Taken together, these findings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the

  6. Targeting autophagy overcomes Enzalutamide resistance in castration-resistant prostate cancer cells and improves therapeutic response in a xenograft model

    PubMed Central

    Nguyen, H G; Yang, J C; Kung, H-J; Shi, X-B; Tilki, D; Lara, P N; DeVere White, R W; Gao, A C; Evans, C P

    2014-01-01

    Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Androgen deprivation or treatment with androgen receptor (AR) signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or bicalutamide induced autophagy in androgen-dependent and in castration-resistant CaP (castration-resistant prostate cancer (CRPC)) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased light chain 3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays, we showed that inhibiting autophagy with clomipramine (CMI), chloroquine or metformin increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMP-dependent protein kinase (AMPK) activation and the suppression of mammalian target of rapamycin (mTOR) through Raptor phosphorylation (Serine 792). Furthermore, small interfering RNA targeting AMPK significantly inhibited autophagy and promoted cell death in CaP cells acutely or chronically exposed to ENZA or androgen deprivation, suggesting that autophagy is an important survival mechanism in CRPC. Lastly, in vivo studies with mice orthotopically implanted with ENZA-resistant cells demonstrated that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduced tumor growth when compared with control groups (P<0.005). In conclusion, autophagy is as an important mechanism of resistance to ARSI in CRPC. Antiandrogen-induced autophagy is mediated through the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in vitro and in vivo, implying the therapeutics potential of autophagy inhibitors

  7. Recombinant interleukin-2 significantly augments activity of rituximab in human tumor xenograft models of B-cell non-Hodgkin lymphoma.

    PubMed

    Lopes de Menezes, Daniel E; Denis-Mize, Kimberly; Tang, Yan; Ye, Helen; Kunich, John C; Garrett, Evelyn N; Peng, Jing; Cousens, Lawrence S; Gelb, Arnold B; Heise, Carla; Wilson, Susan E; Jallal, Bahija; Aukerman, Sharon L

    2007-01-01

    Recombinant interleukin-2 (rIL-2) is a pleiotropic cytokine that activates select immune effector cell responses associated with antitumor activity, including antibody-dependent cellular cytotoxicity (ADCC). Rituximab is an anti-CD20 monoclonal antibody that activates ADCC in non-Hodgkin lymphoma (NHL). The ability of rIL-2 to augment rituximab-dependent tumor responses was investigated. The efficacy of rIL-2 in combination with rituximab was evaluated in 2 NHL tumor xenograft models: the CD20hi, rituximab-sensitive, low-grade Daudi model and the CD20lo, aggressive, rituximab-resistant Namalwa model. Combination of rIL-2 plus rituximab was synergistic in a rituximab-sensitive Daudi tumor model, as evidenced by significant tumor regressions and increased time to tumor progression, compared with rIL-2 and rituximab single agents. In contrast, rituximab-resistant Namalwa tumors were responsive to single-agent rIL-2 and showed an increased response when combined with rituximab. Using in vitro killing assays, rIL-2 was shown to enhance activity of rituximab by activating ADCC and lymphokine-activated killer activity. Additionally, the activity of rIL-2 plus rituximab F(ab')2 was similar to that of rIL-2 alone, indicating a critical role for immunoglobulin G1 Fc-FcgammaR-effector responses in mediating ADCC. Antiproliferative and apoptotic tumor responses, along with an influx of immune effector cells, were observed by immunohistochemistry. Collectively, the data suggest that rIL-2 mediates potent tumoricidal activity against NHL tumors, in part, through activation and trafficking of monocytes and natural killer cells to tumors. These data support the mechanistic and therapeutic rationale for combination of rIL-2 with rituximab in NHL clinical trials and for single-agent rIL-2 in rituximab-resistant NHL patients.

  8. DICER governs characteristics of glioma stem cells and the resulting tumors in xenograft mouse models of glioblastoma

    PubMed Central

    Alamsahebpour, Amir; Burrell, Kelly; Li, Mira; Karabork, Merve; Ekinci, Can; Koch, Elizabeth; Solaroglu, Ihsan; Chang, Jeffery T.; Wouters, Bradly; Aldape, Kenneth; Zadeh, Gelareh

    2016-01-01

    The RNAse III endonuclease DICER is a key regulator of microRNA (miRNA) biogenesis and is frequently decreased in a variety of malignancies. We characterized the role of DICER in glioblastoma (GB), specifically demonstrating its effects on the ability of glioma stem-like cells (GSCs) to form tumors in a mouse model of GB. DICER silencing in GSCs reduced their stem cell characteristics, while tumors arising from these cells were more aggressive, larger in volume, and displayed a higher proliferation index and lineage differentiation. The resulting tumors, however, were more sensitive to radiation treatment. Our results demonstrate that DICER silencing enhances the tumorigenic potential of GSCs, providing a platform for analysis of specific relevant miRNAs and development of potentially novel therapies against GB. PMID:27421140

  9. Bryostatin 1 down-regulates mdr1 and potentiates vincristine cytotoxicity in diffuse large cell lymphoma xenografts.

    PubMed

    Al-Katib, A M; Smith, M R; Kamanda, W S; Pettit, G R; Hamdan, M; Mohamed, A N; Chelladurai, B; Mohammad, R M

    1998-05-01

    The down-regulation of multidrug resistance (mdr1) gene expression as detected by competitive reverse transcription-PCR and the antitumor activity of bryostatin 1 (Bryo1) are investigated in a newly established cell line from a patient with relapsed diffuse large cell lymphoma (DLCL). The cell line (WSU-DLCL2) grows in liquid culture and forms s.c. tumors in mice with severe combined immune deficiency. WSU-DLCL2 is a mature B-cell line (IgG lambda) that is negative for EBV nuclear antigen, expresses the multidrug resistance phenotype, and has t(14;18)(q32;q21) plus other chromosomal aberrations. Exposure of the WSU-DLCL2 cells to Bryo1 in culture reversed the multidrug resistance phenotype within 24 h. A functional assay revealed a 4-fold increase in [3H]vincristine accumulation in Bryo1-treated cells compared with control. Vincristine (VCR), doxorubicin, Bryo1, and 1-beta-D-arabinofuranosylcytosine showed no clinically significant activity when given alone to WSU-DLCL2-bearing severe combined immune deficiency mice. However, when given 24 h before each cytotoxic agent, Bryo1 substantially increased the antitumor activity of VCR but not 1-beta-D-arabinofuranosylcytosine. There was a statistically significant (P < 0.001) decrease in the expression of P-glycoprotein in WSU-DLCL2 tumors taken from Bryo1-treated animals compared with untreated controls. In vivo, a competitive reverse transcription-PCR assay revealed decreased mdr1 RNA expression 24 h after Bryo1 treatment. These findings taken together indicate that Bryo1-induced down-regulation of mdr1 might be one mechanism by which Bryo1 potentiates VCR activity. The sequential use of both agents resulted in clinically significant antitumor activity in this lymphoma model.

  10. Hypersomnia as the first presentation in a patient with insulinoma: A case report and review of the literature

    PubMed Central

    Pu, Jiali; Zhang, Baorong; Yin, Xinzhen

    2016-01-01

    Insulinoma is a rare neuroendocrine tumor. Hypersomnia as the first presentation in a patient with insulinoma is even more rare and may be easy to misdiagnose. We are herein reporting a case of insulinoma initially presenting with prolonged sleep time and extreme difficulty in waking. The abovementioned symptoms occurred every 2–3 months. Over the last 2 months, the attacks had become more frequent and severe. On computed tomography examination, a 12×9-mm cystic nodule was detected in the uncinate process of the pancreas, which was pathologically diagnosed as insulinoma. Since resection, the symptom of hypersomnia has not occurred again. The aim of the present report was to raise awareness among physicians to consider insulinoma in the differential diagnosis of hypersomnia in patients without other known diseases. PMID:27699047

  11. Methylselenol, a selenium metabolite, inhibits colon cancer cell growth and cancer xenografts in C57BL/6 mice

    USDA-ARS?s Scientific Manuscript database

    Data indicate that methylselenol is a critical selenium (Se) metabolite for anticancer activity in vivo but its role in colon cancer prevention remains to be characterized. This study tested the hypothesis that methylselenol inhibits the growth of colon cancer cells and tumors. We found that submicr...

  12. Effect of dietary selenium and cancer cell xenograft on peripheral T and B lymphocytes in adult nude mice

    USDA-ARS?s Scientific Manuscript database

    Selenium (Se) is known to regulate tumorigenesis and immunity at nutritional and supranutritional levels. Because the immune system provides critical defenses against cancer and the athymic, immune-deficient NU/J nude mice are known to gradually develop CD8+ and CD4+ T cells, we asked whether B and ...

  13. Gypenosides suppress growth of human oral cancer SAS cells in vitro and in a murine xenograft model: the role of apoptosis mediated by caspase-dependent and caspase-independent pathways.

    PubMed

    Lu, Kung-Wen; Chen, Jung-Chou; Lai, Tung-Yuan; Yang, Jai-Sing; Weng, Shu-Wen; Ma, Yi-Shih; Lin, Hui-Yi; Wu, Rick Sai-Chuan; Wu, King-Chuen; Wood, W Gibson; Chung, Jing-Gung

    2012-06-01

    Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino. The authors investigated the effects of Gyp on cell morphology, viability, cell cycle distribution, and induction of apoptosis in human oral cancer SAS cells and the determination of murine SAS xenograft model in vivo. Flow cytometry was used to quantify the percentage of viable cells; cell cycle distribution; sub-G1 phase (apoptosis); caspase-3, -8, and -9 activity; reactive oxygen species (ROS) production, intracellular Ca(2+) determination; and the level of mitochondrial membrane potential (ΔΨ(m)). Western blotting was used to examine levels of apoptosis-associated proteins, and confocal laser microscopy was used to examine the translocation of proteins in cells. Gyp induced morphological changes, decreased the percentage of viable cells, caused G0/G1 phase arrest, and triggered apoptotic cell death in SAS cells. Cell cycle arrest induced by Gyp was associated with apoptosis. The production of ROS, increased intracellular Ca(2+) levels, and the depolarization of ΔΨ(m) were observed. Gyp increased levels of the proapoptotic protein Bax but inhibited the levels of the antiapoptotic proteins Bcl-2 and Bcl-xl. Gyp also stimulated the release of cytochrome c and Endo G. Translocation of GADD153 to the nucleus was stimulated by Gyp. Gyp in vivo attenuated the size and volume of solid tumors in a murine xenograft model of oral cancer. Gyp-induced cell death occurs through caspase-dependent and caspase-independent apoptotic signaling pathways, and the compound reduced tumor size in a xenograft nu/nu mouse model of oral cancer.

  14. Focal hepatic glycogenosis associated with metastatic insulinoma presenting as mass lesions.

    PubMed

    Vyas, Monika; Zhang, Xuchen; Morrow, Jon S; Jain, Dhanpat; Salem, Ronald R; West, A Brian

    2016-01-01

    One of the important functions of the liver is glycogen storage. Most processes associated with increased hepatic glycogen, or glycogenoses, are metabolic and affect the entire liver leading to diffuse glycogenosis. We present a case in which the liver contained multiple small pale nodules that on initial assessment were recognized to be composed of glycogenated hepatocytes. Most of the known causes of hepatic glycogenosis were not pertinent to this case. After cutting many deeper levels and obtaining additional sections, small foci of insulinoma were revealed in the center of each of these lesions. The glycogenosis surrounding the foci of insulinoma can be best explained as a local effect of insulin on the hepatocytes, a phenomenon that has been previously described in primate models, but not in human subjects. Here, we report the first case of metastatic insulinoma causing local hepatic glycogenosis.

  15. [INVASIVE TECHNIQUES AND INTRAOPERATIVE ECHOGRAPHY IN THE LOCALIZATION OF INSULINOMAS; A CASE REPORT].

    PubMed

    Herrera-Martínez, Aura D; Padillo-Cuenca, José C; Calañas Continente, Alfonso; Bahamondes-Opazo, Rodrigo; Muñoz-Jiménez, Concepción; Gálvez Moreno, María A

    2015-07-01

    The insulinoma is the most common pancreatic neuroendocrine tumor. Surgery is curative in most cases, an appropriate preoperative localization allows a minimal invasive surgical technique for keeping the exo and endocrine function of the pancreas. Some authors suggest the use of invasive localization techniques just in cases with non-identified tumor lesion, others recommend their routinely use. We describe a case with clinical and biochemical diagnosis of insulinoma, conventional image studies revealed a tumor image in the pancreas which corresponded to a lipoma, the intraoperative ultrasound allowed the localization of the real tumor, but body-tail pancreatectomy was performed due to pancreatic necrosis in relation with the duration of the surgery. The systematic use of invasive localization techniques as the intra-arterial calcium stimulation and the pancreatic intraoperative ultrasound would allow a better localization of insulinoma for avoiding complications and associated morbidity.

  16. Soy isoflavone extracts stimulate the growth of nude mouse xenografts bearing estrogen-dependent human breast cancer cells (MCF-7)☆

    PubMed Central

    Wu, Qian; Yang, Ye; Yu, Jing; Jin, Nianzu

    2012-01-01

    We explored the effects of different lifetime exposures to soy isoflavone extracts on the growth of estrogen-dependent human breast cancer cells (MCF-7) implanted into athymic mice of different ovarian statuses. The athymic mice, ovariectomized or not, were implanted with MCF-7 cells. Mice were fed with low, moderate and high doses of soy isoflavone extract, at dietary concentrations of 6.25, 12.5 and 25 g/kg, in different reproductive models, respectively. The expression of ki-67 was detected by immunohistochemistry. pS2 expression in tumors was analyzed by real-time PCR. Estrogen level in the serum was measured by chemiluminescence enzyme immunoassay. Total genistein and daidzein levels in serum and urine were determined by liquid chromatography-electrospray tandem mass spectrometry (LC-ES/MS/MS). In Group A, on week 4, nude mice were exposed to different doses of soy iosflavone extracts. In Group B, the experimental diets were given to the nude mice following ovariectomy and tumor implantation. In both groups, 6.25 and 12.5 g/kg soy isoflavone extracts stimulated the growth of MCF-7 xenografts, increased pS2 expression, proliferation and estrogen level in serum. In both Group B (postmenopausal mouse model) and Group C (premenopausal mouse model), soy isoflavone extracts at doses of 6.25 and 12.5 g/kg showed stimulatory effects on the growth of MCF-7 tumors. In conclusion, administration of soy isoflavone extracts at doses of 6.25 and 12.5 g/kg during adolescence or later in life stimulated tumor growth in both menopausal and postmenopausal mouse models. PMID:23554729

  17. Function of the Blood-Brain Barrier and Restriction of Drug Delivery to Invasive Glioma Cells: Findings in an Orthotopic Rat Xenograft Model of Glioma

    PubMed Central

    Agarwal, Sagar; Manchanda, Pooja; Vogelbaum, Michael A.; Ohlfest, John R.

    2013-01-01

    Despite aggressive treatment with radiation and chemotherapy, recurrence of glioblastoma multiforme (GBM) is inevitable. The objective of this study was to show that the blood-brain barrier (BBB), through a combination of tight junctions and active efflux transporters in the brain microvasculature, can significantly restrict delivery of molecularly targeted agents to invasive glioma cells. Transgenic mice lacking P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) were used to study efflux of erlotinib at the BBB. A U87 rat xenograft model of GBM was used to investigate the regional distribution of erlotinib to the tumor, and brain regions surrounding the tumor. The effect of concurrent administration of elacridar on regional tumor distribution of erlotinib was evaluated. We show that erlotinib transport across an intact BBB is significantly restricted due to P-gp- and Bcrp-mediated efflux transport. We then show that the BBB is sufficiently intact in areas of brain adjacent to the tumor core to significantly restrict erlotinib delivery. Inhibition of P-gp and Bcrp by the dual inhibitor elacridar dramatically increased erlotinib delivery to the tumor core, rim, and normal brain. These results provide conclusive evidence of the impact that active efflux at the BBB has on the delivery of molecularly targeted therapy to different tumor regions in glioma. These data also support the possibility that the repeated failure of clinical trials of new drugs for gliomas may be in part due to a failure to achieve effective concentrations in invasive tumor cells that reside behind an intact BBB. PMID:23014761

  18. Hwanggeumchal sorghum Induces Cell Cycle Arrest, and Suppresses Tumor Growth and Metastasis through Jak2/STAT Pathways in Breast Cancer Xenografts

    PubMed Central

    Lim, Eun Joung; Joung, Youn Hee; Hong, Dae Young; Park, Eui U.; Park, Seung Hwa; Choi, Soo Keun; Moon, Eon-Soo; Cho, Byung Wook; Park, Kyung Do; Lee, Hak Kyo; Kim, Myong-Jo; Park, Dong-Sik; Yang, Young Mok

    2012-01-01

    Background Cancer is one of the highly virulent diseases known to humankind with a high mortality rate. Breast cancer is the most common cancer in women worldwide. Sorghum is a principal cereal food in many parts of the world, and is critical in folk medicine of Asia and Africa. In the present study, we analyzed the effects of HSE in metastatic breast cancer. Methodology/Principal Findings Preliminary studies conducted on MDA-MB 231 and MCF-7 xenograft models showed tumor growth suppression by HSE. Western blotting studies conducted both in vivo and in vitro to check the effect of HSE in Jak/STAT pathways. Anti-metastatic effects of HSE were confirmed using both MDA-MB 231 and MCF-7 metastatic animal models. These studies showed that HSE can modulate Jak/STAT pathways, and it hindered the STAT5b/IGF-1R and STAT3/VEGF pathways not only by down-regulating the expression of these signal molecules and but also by preventing their phosphorylation. The expression of angiogenic factors like VEGF, VEGF-R2 and cell cycle regulators like cyclin D, cyclin E, and pRb were found down-regulated by HSE. In addition, it also targets Brk, p53, and HIF-1α for anti-cancer effects. HSE induced G1 phase arrest and migration inhibition in MDA-MB 231 cells. The metastasis of breast cancer to the lungs also found blocked by HSE in the metastatic animal model. Conclusions/Significance Usage of HS as a dietary supplement is an inexpensive natural cancer therapy, without any side effects. We strongly recommend the use of HS as an edible therapeutic agent as it possesses tumor suppression, migration inhibition, and anti-metastatic effects on breast cancer. PMID:22792362

  19. Impact of bevacizumab in combination with erlotinib on EGFR-mutated non-small cell lung cancer xenograft models with T790M mutation or MET amplification.

    PubMed

    Furugaki, Koh; Fukumura, Junko; Iwai, Toshiki; Yorozu, Keigo; Kurasawa, Mitsue; Yanagisawa, Mieko; Moriya, Yoichiro; Yamamoto, Kaname; Suda, Kenichi; Mizuuchi, Hiroshi; Mitsudomi, Tetsuya; Harada, Naoki

    2016-02-15

    Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non-small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression-free survival in patients with EGFR-mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR-mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR-mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug-cessation period. In the HCC827-EPR model (DEL+T790M) and HCC827-vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI-H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL-resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation- or MET amplification-positive tumors as long as their growth remained significantly suppressed by ERL. © 2015 UICC.

  20. Chronic moderate ethanol intake differentially regulates vitamin D hydroxylases gene expression in kidneys and xenografted breast cancer cells in female mice.

    PubMed

    García-Quiroz, Janice; García-Becerra, Rocío; Lara-Sotelo, Galia; Avila, Euclides; López, Sofía; Santos-Martínez, Nancy; Halhali, Ali; Ordaz-Rosado, David; Barrera, David; Olmos-Ortiz, Andrea; Ibarra-Sánchez, María J; Esparza-López, José; Larrea, Fernando; Díaz, Lorenza

    2017-10-01

    Factors affecting vitamin D metabolism may preclude anti-carcinogenic effects of its active metabolite calcitriol. Chronic ethanol consumption is an etiological factor for breast cancer that affects vitamin D metabolism; however, the mechanisms underlying this causal association have not been fully clarified. Using a murine model, we examined the effects of chronic moderate ethanol intake on tumoral and renal CYP27B1 and CYP24A1 gene expression, the enzymes involved in calcitriol synthesis and inactivation, respectively. Ethanol (5% w/v) was administered to 25-hydroxyvitamin D3-treated or control mice during one month. Afterwards, human breast cancer cells were xenografted and treatments continued another month. Ethanol intake decreased renal Cyp27b1 while increased tumoral CYP24A1 gene expression.Treatment with 25-hydroxyvitamin D3 significantly stimulated CYP27B1 in tumors of non-alcohol-drinking mice, while increased both renal and tumoral CYP24A1. Coadministration of ethanol and 25-hydroxyvitamin D3 reduced in 60% renal 25-hydroxyvitamin D3-dependent Cyp24a1 upregulation (P<0.05). We found 5 folds higher basal Cyp27b1 than Cyp24a1 gene expression in kidneys, whereas this relation was inverted in tumors, showing 5 folds more CYP24A1 than CYP27B1. Tumor expression of the calcitriol target cathelicidin increased only in 25-hydroxyvitamin D3-treated non-ethanol drinking animals (P<0.05). Mean final body weight was higher in 25-hydroxyvitamin D3 treated groups (P<0.001). Overall, these results suggest that moderate ethanol intake decreases renal and tumoral 25-hydroxyvitamin D3 bioconversion into calcitriol, while favors degradation of both vitamin D metabolites in breast cancer cells. The latter may partially explain why alcohol consumption is associated with vitamin D deficiency and increased breast cancer risk and progression. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Characterization of triple-phase computed tomography in dogs with pancreatic insulinoma

    PubMed Central

    FUKUSHIMA, Kenjiro; FUJIWARA, Reina; YAMAMOTO, Kie; KANEMOTO, Hideyuki; OHNO, Koichi; TSUBOI, Masaya; UCHIDA, Kazuyuki; MATSUKI, Naoaki; NISHIMURA, Ryohei; TSUJIMOTO, Hajime

    2015-01-01

    Little information is available regarding triple-phase computed tomography (CT) of canine pancreatic insulinoma. A few case reports with small numbers of cases have indicated that hyper-attenuation in the arterial phase was a common finding on multi-phasic CT in dogs with insulinoma. Our purpose was to clarify the characteristic findings of dogs with insulinoma on triple-phase CT. Nine dogs with insulinoma that underwent triple-phase CT were included in the present study. Attenuation patterns in the arterial phase indicated hypo-attenuation in 4 cases and hyper-attenuation in 2 cases. In the remaining 3 cases, 1 case showed hypo-attenuation and 1 case showed hyper-attenuation in the pancreatic phase, and 1 case presented hyper-attenuation in the later phase. Altogether, 5 cases showed hypo and 4 cases showed hyper-attenuation in at least one phase. The enhancement pattern was homogenous in 7 cases and heterogeneous in 2 cases. Tumor margins were well-defined in 5 cases and ill-defined in 4 cases. Capsule formation was present in 5 cases and absent in 4 cases. In conclusion, it is important to note that hypo-attenuation was as common as hyper-attenuation in dogs with insulinoma in triple-phase CT in at least one phase. Additionally, mass lesions were most conspicuous not only in the arterial phase but in the pancreatic and later phases in some cases. Therefore, it is important to perform triple-phase CT and notice about variable findings for the detection of canine pancreatic insulinoma. PMID:26118410

  2. Alkylator-Induced and Patient-Derived Xenograft Mouse Models of Therapy-Related Myeloid Neoplasms Model Clinical Disease and Suggest the Presence of Multiple Cell Subpopulations with Leukemia Stem Cell Activity

    PubMed Central

    Johnson, Carl; Gratzinger, Dita; Majeti, Ravindra

    2016-01-01

    Acute myeloid leukemia (AML) is a heterogeneous group of aggressive bone marrow cancers arising from transformed hematopoietic stem and progenitor cells (HSPC). Therapy-related AML and MDS (t-AML/MDS) comprise a subset of AML cases occurring after exposure to alkylating chemotherapy and/or radiation and are associated with a very poor prognosis. Less is known about the pathogenesis and disease-initiating/leukemia stem cell (LSC) subpopulations of t-AML/MDS compared to their de novo counterparts. Here, we report the development of mouse models of t-AML/MDS. First, we modeled alkylator-induced t-AML/MDS by exposing wild type adult mice to N-ethyl-N-nitrosurea (ENU), resulting in several models of AML and MDS that have clinical and pathologic characteristics consistent with human t-AML/MDS including cytopenia, myelodysplasia, and shortened overall survival. These models were limited by their inability to transplant clinically aggressive disease. Second, we established three patient-derived xenograft models of human t-AML. These models led to rapidly fatal disease in recipient immunodeficient xenografted mice. LSC activity was identified in multiple HSPC subpopulations suggesting there is no canonical LSC immunophenotype in human t-AML. Overall, we report several new t-AML/MDS mouse models that could potentially be used to further define disease pathogenesis and test novel therapeutics. PMID:27428079

  3. 5-Aminolevulinic acid-mediated sonodynamic therapy reverses macrophage and dendritic cell passivity in murine melanoma xenografts.

    PubMed

    Wang, Shan; Hu, Zheng; Wang, Xiaolong; Gu, Chuanwen; Gao, Zhongxiuzi; Cao, Wenwu; Zheng, Jinhua

    2014-09-01

    Sonodynamic therapy (SDT) uses a combination of sonosensitizing drugs and low-intensity therapeutic ultrasound to cause apoptosis and autophagy of tumor cells. However, its effects on the tumor microenvironment, especially on the immune state, remain unknown. In this study, we investigated the transformation of macrophages and dendritic cells (DCs) in the tumor microenvironment during 5-aminolevulinic acid (5-ALA)-mediated SDT in mice transplanted with B16F10 melanomas. Tumor growth and mouse weight were measured. Hematoxylin-eosin staining was used to evaluate tumor morphology to quantify the anti-tumor efficacy of 5-ALA-mediated SDT. We investigated anti-tumor immunity in the tumor microenvironment by immunocytochemical staining of CD68, CD163, CD80, CD86, tumor necrosis factor α (TNF-α), interleukin 10 (IL-10) and interferon γ (IFN-γ). Tumor growth was restrained by 5-ALA-mediated SDT in B16F10 melanoma-bearing mice. CD68 levels increased and CD163 decreased, indicating that M2 macrophages were converted to the M1 phenotype in the tumor. The increase in CD80 and CD86 showed that DCs in the tumor microenvironment tend to mature after SDT treatment. The cytokines INF-γ, TNF-α and IL-10 significantly increased in SDT. Application of low-intensity therapeutic ultrasound alone also led to similar trends in our study, but combined treatment with 5-ALA yielded a change. The original stabilized immune state in the tumor microenvironment can be interrupted by low-intensity therapeutic ultrasound combined with 5-ALA, which enhanced the pro-inflammatory response and reversed the passive properties of macrophages and dendritic cells.

  4. Cancer-associated fibroblasts in a human HEp-2 established laryngeal xenografted tumor are not derived from cancer cells through epithelial-mesenchymal transition, phenotypically activated but karyotypically normal.

    PubMed

    Wang, Mei; Wu, Chun-Ping; Pan, Jun-Yan; Zheng, Wen-Wei; Cao, Xiao-Juan; Fan, Guo-Kang

    2015-01-01

    Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression and even initiation. However, the origins of CAFs in various cancer types remain controversial, and one of the important hypothesized origins is through epithelial-mesenchymal transition (EMT) from cancer cells. In this study, we investigated whether the HEp-2 laryngeal cancer cells are able to generate CAFs via EMT during tumor formation, which is now still unknown. The laryngeal xenografted tumor model was established by inoculating the HEp-2 laryngeal cancer cell line in nude mice. Primary cultured CAFs from the tumor nodules and matched normal fibroblasts (NFs) from the adjacent connective tissues were subcultured, purified, and verified by immunofluorescence. Migration, invasion, and proliferation potentials were compared between the CAFs and NFs. A co-culture of CAFs with HEp-2 cells and a co-injection of CAFs with HEp-2 cells in nude mice were performed to examine the cancer-promoting potential of CAFs to further verify their identity. Karyotypic analyses of the CAFs, NFs, and HEp-2 cells were conducted. A co-culture of NFs with HEp-2 cells was also performed to examine the expression of activated markers of CAFs. A pathological examination confirmed that the laryngeal xenografted tumor model was successfully established, containing abundant CAFs. Immunocytochemical staining verified the purities and identities of the CAFs and NFs. Although the CAFs manifested higher migration, invasion, proliferation, and cancer-promoting capacities compared with the NFs, an analysis of chromosomes revealed that both the CAFs and NFs showed typical normal mouse karyotypes. In addition, the NFs co-cultured with HEp-2 cells did not show induced expressions of activated markers of CAFs. Our findings reveal that the CAFs in the HEp-2 established laryngeal xenografted tumor are not of laryngeal cancer origin but of mouse origin, indicating that the HEp-2 laryngeal cancer cells cannot generate their

  5. Cancer-Associated Fibroblasts in a Human HEp-2 Established Laryngeal Xenografted Tumor Are Not Derived from Cancer Cells through Epithelial-Mesenchymal Transition, Phenotypically Activated but Karyotypically Normal