Pancreatic PANC-1 Cells Research Articles

5‐Fluorouracil and Gemcitabine Alter Nuclear Transport

The GTPase, Ran, is a major regulator of nuclear transport and disruption of its established gradient disrupts nuclear export and leads to cell death through the accumulation of pro‐apoptotic factors. The chemotherapy 5‐Fluorouracil (5‐FU) increases nuclear pore permeability causing Ran to leak into the cytoplasm, disrupting the gradient and in turn nuclear export, leading to cell death of HeLa cervical cancer cells. Our previous work demonstrated that combining 5‐FU with topoisomerase inhibitors can have a greater total impact on disruption of the Ran gradient and accumulates proteins in the nucleus. Gemcitabine (GEM) is widely used when treating pancreatic cancer; yet a resistance is common. However, combination of GEM with nuclear export inhibitors can help overcome this problem. Therefore, we sought to determine if combination of 5‐FU and GEM could impact nuclear export to help overcome GEM resistance. Our initial studies found that 5‐FU+GEM further decreased nuclear Ran in HeLa cells and lead to increased cell death in combination over GEM alone. Therefore, we transitioned to exploring the effects of 5‐FU+GEM on disrupting the Ran gradient in PANC1 pancreatic cells. Both 5‐FU and GEM individually and in combination decreased the nuclear Ran levels by 15%, 17%, and 23% respectively. This disruption of nuclear export may help overcome GEM resistance in PANC1 cells. Specifically, our ongoing work is exploring the impact of 5FU+GEM on the nuclear retention of the tumor suppressors p27 and p21 as well as cell viability to identify a possible mechanism for the impact of the 5‐FU+GEM combination on apoptosis. Our work helps support a possible mechanism for overcoming GEM resistance in Panc1 cells and identifies a potential combination treatment with 5‐FU for use against pancreatic cancer.

Effects and Mechanisms of Tripartite Motif Containing 14 Downregulation on Proliferation, Migration, and Invasion of Cancerous Pancreatic PANC-1 Cells

This study aimed to investigate the effect and mechanism of TRIM14 downregulation on the apoptosis, migration, and invasion of cancerous pancreatic PANC-1 cells. PANC-1 cells cultured in vitrowere classified to a control (normal culture), negative (neutral siRNA transfection), and siTRIM14 group (TRIM14 siRNA transfection). RT-PCR was adopted to test TRIM14 mRNA expression. Cellular proliferation was determined by CCK-8, and transwell chamber invasion and apoptosis by flow cytometry. AKT signaling pathway related proteins CyclinD1, MMP-2, Bcl-2, and AKT phosphorylation, and TRIMI14 protein expression, were determined by western blotting. Compared with the control group, TRIMI14 expression, cellular proliferation ability, infiltration, transfer AKT phosphorylation, and TRIMI14, CyclinD1, MMP-2, and Bcl-2 protein expression were greatly reduced in siTRIM14 cells, and the apoptotic ability was significantly enhanced (P < 0.05). However, no striking differences were detected between the negative and control groups (P > 0.05). Downregulating TRIM14 expression can inhibit the proliferation, invasion, and migration of PANC-1 cells, and promote apoptosis. The mechanism may be associated with the inhibition of AKT signaling pathway activation.

Anti proliferative and apoptotic effects on pancreatic cancer cell lines indicate new roles for ANGPTL8 (Betatrophin).

Despite considerable advances, the treatment of pancreatic cancer (PC) still requires much effort. Unusual regulation of the Wnt and apoptotic signaling pathways is widespread in cancer incidence. For instance, the WIF1 (Wnt inhibitory factor 1) gene is down-regulated in many cancers. The purpose of this study was to determine the effects of recombinant Betatrophin, a recently discovered hormone, on MiaPaca-II and Panc-1 pancreatic cell lines. Various concentrations of Betatrophin were added to MiaPaca-II and Panc-1 pancreatic cell lines during periods of 24 , 48, and 72 h. The MTT assay was applied to investigate cell proliferation after treatment. The rate of apoptotic cells was assessed using double-staining flow cytometry, and the expression levels of the WIF1 gene and Bcl2 protein was observed by real-time PCR and western blotting, respectively. The findings of this study suggest that Betatrophin has an anti-proliferative effect on both MiaPaca-II and Panc-1 cell lines, in line with the up-regulation of WIF1 as a tumor suppressor gene. Moreover, the induction of apoptosis by ANGPTL8 occurred by the down-regulation of Bcl2. Thus, Betatrophin can be proposed as a potential therapeutic drug for treating pancreatic cancer.

Abstract 134: A dual mTOR-PI3K inhibitor DCB-HDG2-57 with Hedgehog signaling pathway antagonist activity

Abstract The Hedgehog (Hh) signaling pathway is a critical regulator of embryonic patterning, and aberrant Hh pathway activation has been implicated in a diverse spectrum of cancers. Therefore, components of the Shh pathway (such as Shh, SMO, and GLI1/2) are viable therapeutic targets for anti-tumor strategy. Smo antagonists such as GDC-0449 and NVP-LDE225 have received FDA approval for treating basal cell carcinoma. However, acquired resistance has emerged as a challenge to targeted therapeutics and may limit their anti-cancer efficacy. Studies have linked the hyperactive phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling pathway in a variety of human cancers and drug resistance condition. The synergistic role of PI3K/AKT/mTOR in Hh signaling in embryonic development and Hh-dependent tumors has been reported. Both the PI3K/Akt/mTOR and Hh pathway also play important role in maintaining the stem cell-like properties of cancer stem cell (CSCs). We report here the development of a potent dual mTOR-PI3K inhibitor designated DCB-HDG2-57 with Hh signaling pathway antagonist activity. In vitro biochemical assays showed that DCB-HDG2-57 inhibited recombinant pI3Kα and mTOR kinase with IC50 of 27.2 and 133 nM, respectively. DCB-HDG2-57 is also highly active in cellular assays, as evidenced by inhibition of phosphorylation of cellular downstream targets of PI3K-mTOR kinases ribosomal protein S6 kinase (S6K1, Ser240/24) in the colon (LS-180) and pancreatic cancer (MiaPaCa2 and PANC1) cell lines. Interestingly, DCB-HDG2-57 demonstrated Hh signaling pathway antagonist activity in a 293 cell-based Gli-luciferase inhibition assay upon agonist treatment, and retains inhibition activity against the Smo wild-type and D473H mutant co-transfection with IC50 of 261.8 and 327.7 nM, respectively. In addition, DCB-HDG2-57 can significantly decrease the cancer stem cell liked side population of PANC1 pancreatic cell, and inhibited the migration of E3LZ10.7 pancreatic cancer cell. DCB-HDG2-57 demonstrated in vivo tumor growth inhibition activity in LS-180 colon cancer xenograft model and reduced the Gli-1 RNA abundance in the tumor. Taken together, our data demonstrate that combined pharmacological blockade of mTOR-PI3K and Hh pathway of DCB-HDG2-57 can provide a therapeutic strategy for targeting ligand-dependent Hh cancer. Citation Format: Ying-Shuan Lee, Mann-Yan Kuo, Chia Wei Liu, Yu-Wen Tseng, Win Yin Wei, Shian-Yi Chiou, Her Sheng Lin, Y.-Y. Lu, Chu-Bin Liao, Shao-Zheng Pen. A dual mTOR-PI3K inhibitor DCB-HDG2-57 with Hedgehog signaling pathway antagonist activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 134. doi:10.1158/1538-7445.AM2017-134

Novel Hyaluronic Acid Conjugates for Dual Nuclear Imaging and Therapy in CD44-Expressing Tumors in Mice In Vivo

Hyaluronic acid, a natural CD44 receptor ligand, has attracted attention in the past years as a macromolecular delivery of anticancer agents to cancer. At the same time, the clinical applications of Gemcitabine (Gem) have been hindered by its short biological half-life, high dose and development of drug resistance. This work reports the synthesis of a hyaluronic acid (HA) conjugate for nuclear imaging, and in vivo Gem delivery to CD44-expressing solid tumors in mice. HA was individually conjugated, via amide coupling, to Gem (HA-Gem), 4'-(aminomethyl)fluorescein hydrochloride (HA-4'-AMF) or tris(hydroxypyridinone) amine (HA-THP) for cancer therapy, in vitro tracking or single photon emission computed tomography/computed tomography (SPECT/CT) imaging, respectively. Gem conjugation to HA was directly confirmed by nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC) and UV-visible spectrometry, or indirectly by a nucleoside transporter inhibition study. Gem conjugation to HA improved its plasma stability, reduced blood hemolysis and resulted in delayed cytotoxicity in vitro. Uptake inhibition studies in colon CT26 and pancreatic PANC-1 cells, by flow cytometry, revealed that uptake of fluorescent HA conjugate is CD44 receptor and macropinocytosis-dependent. Gamma scintigraphy and SPECT/CT imaging confirmed the relatively prolonged blood circulation profile and uptake in CT26 (1.5 % ID/gm) and PANC-1 (1 % ID/gm) subcutaneous tumors at 24 h after intravenous injection in mice. Four injections of HA-Gem at ~15 mg/kg, over a 28-day period, resulted in significant delay in CT26 tumor growth and prolonged mice survival compared to the free drug. This study reports for the first time dual nuclear imaging and drug delivery (Gem) of HA conjugates to solid tumors in mice. The conjugates show great potential in targeting, imaging and killing of CD44-over expressing cells in vivo. This work is likely to open new avenues for the application of HA-based macromolecules in the field of image-guided delivery in oncology.

A novel nuclear Src and p300 signaling axis controls migratory and invasive behavior in pancreatic cancer.

The presence of Src in the nuclear compartment has been previously reported, although its significance has remained largely unknown. We sought to delineate the functions of the nuclear pool of Src within the context of malignant progression. Active Src is localized within the nuclei of human pancreatic cancer cells and mouse fibroblasts over-expressing c-Src where it is associated with p300. Nuclear Src additionally promotes the tyrosine phosphorylation of p300 in pancreatic cancer Panc-1 cells. Src, together with p300, is associated with the high-mobility group AT-hook (HMGA)2 and SET and MYND domain-containing protein (SMYD)3 gene promoters and regulates their expression in a Src-dependent manner. These nuclear Src-dependent events correlate with anchorage-independent soft-agar growth and the migratory properties in both pancreatic Panc-1 cells and mouse fibroblasts over-expressing Src. Moreover, analyses of human pancreatic ductal adenocarcinoma (PDAC) tumor tissues detected the association of nuclear Src with the HMGA2 and SMYD3 gene promoters. Our findings for the first time show the critical importance of nuclear Src and p300 function in the migratory properties of pancreatic cancer cells. Further, data together identify a previously unknown role of nuclear Src in the regulation of gene expression in association with p300 within the context of cells harboring activated or over-expressing Src. This novel mechanism of nuclear Src-p300 axis in PDAC invasiveness and metastasis may provide an opportunity for developing more effective early clinical interventions for this lethal disease.Active Src is complexed with and phosphorylates p300 in the nucleus, and the complex is bound to HMGA2 and SMYD3 genes, thereby regulating their expression to promote pancreatic tumor cell migration and invasiveness.

Efficient “green” encapsulation of a highly hydrophilic anticancer drug in metal–organic framework nanoparticles

Metal–organic frameworks (MOFs) are coordination polymers of interest for biomedical applications. Of particular importance, nanoparticles made of iron(III) trimesate (MIL-100, MIL standing for Material Institut Lavoisier) (nanoMOFs) can be conveniently synthesised under mild and green conditions. They were shown to be biodegradable, biocompatible and efficient to encapsulate a variety of active molecules. We have addressed here the challenges to encapsulate a highly hydrophilic anticancer prodrug, phosphated gemcitabin (Gem-MP) known for its instability and inability to bypass cell membranes. MIL-100 nanoMOFs acted as efficient “nanosponges”, soaking Gem-MP from its aqueous solution with almost perfect efficiency (>98%). Maximal loadings reached ∼30 wt% reflecting the strong interaction between the drug and the iron trimesate matrices. Neither degradation nor loss of crystalline structure was observed after the loading process. Storage of the loaded nanoMOFs in water did not result in drug release over three days. However, Gem-MP was released in media containing phosphates, as a consequence to particle degradation. Drug-loaded nanoMOFs were effective against pancreatic PANC-1 cells, in contrast to free drug and empty nanoMOFs. However, an efflux phenomenon could contribute to reduce the efficacy of the nanocarriers. Size optimization and surface modification of the nanoMOFs are expected to further improve these findings.

Abstract 2620: 3D multicellular pancreatic cancer spheroids as drug screening tool for pharmacological evaluation of EHT 5372 and other Mirk/DYRK1B inhibitors

Abstract The dual-specificity tyrosine phosphorylated and regulated kinase Mirk/DYRK1B belongs to an evolutionary conserved family of kinases involved in the control of growth and development. DYRK1B has low level of expression in most normal cell types but is amplified or over-expressed in a number of human cancers. DYRK1B regulates the exit of cancer cells from quiescence through regulating cyclin D turnover and p27kip1 stabilization, thus participating as substantial actor in the control of cancer cell cycle progression. Diaxonhit has developed a novel class of DYRK inhibitors with potent in vitro efficacy. Among them, EHT 5372 reduces tumor growth in a PANC-1 xenograft model and reduces ascites spheroids to single cells and induces their apoptosis. Three-dimensional (3D) multicellular spheroids are symmetrical cellular aggregates that model an in vitro system of intermediate complexity between monolayer cultures and tumors in vivo. Here, we have conducted genome-wide analysis of transcriptional changes between normal (monolayer, 2D) and 3D culture conditions of pancreatic PANC-1 cells using Diaxonhit's GWSATM platform to identify tumor-relevant genes and pathways. The PANC-1 multicellular spheroid model was first characterized based on phenotypical and functional properties and we then analyzed the pharmacological response of spheroids to EHT 5372 and other DYRK inhibitors treatment. A strong up-regulation of DYRK1B was evidenced and confirmed at the protein level. Stemness-related markers were investigated and some were also found much higher in the spheroids than within the monolayer cultures and further increased in a culture time-dependent manner. Stem-like cancer cells may be the cause of therapy-resistance and relapse in patients and sphere forming ability is one of properties of this quiescent cancer-initiating cells. As DYRK1B contributes to G0 arrest to maintain the viability of quiescent cancer cells, pharmacological DYRK inhibition would reduce the capacity of cells to enter into quiescence and sensitize cancer cells to conventional chemotherapeutic agents or radiation. Molecular and physiological consequences of inhibiting DYRK1B with EHT 5372 are now being investigated along with genes expression changes in stemness-related markers and in biological pathways involved in tumor cell growth regulation. A molecular characterization of pancreatic cancer cell spheroids is currently undertaken to determine the utility of the 3D assay as a surrogate tool enriched in cancer stem cells. Spheroids also enable the study of quiescence, chemoresistance and metastasis. DYRK1B inhibitors screening in 3D models is a powerful approach that can help focus on compounds active on these crucial mechanisms of cancer cell biology. The most active compounds identified in this program are likely good candidates for in vivo xenograft studies. Citation Format: Anne-Sophie CASAGRANDE, Florence BACHELOT, Emeline THROO, Florence MAHE, Bertrand LEBLOND, Thierry BESSON, Matthew PANDO, Laurent DESIRE. 3D multicellular pancreatic cancer spheroids as drug screening tool for pharmacological evaluation of EHT 5372 and other Mirk/DYRK1B inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2620. doi:10.1158/1538-7445.AM2014-2620

Abstract 2953: Inhibition of calmodulin enhances TRA-8-induced apoptosis in pancreatic cancer cells .

Abstract Current chemotherapy and radiotherapy are not curative for pancreatic cancer. Tumor necrosis factor-related apoptosis-inducing ligand or agonist antibodies for death receptor 4/5 have been shown to be a promising cancer therapy. TRA-8, a humanized activating antibody for the death receptor 5 (DR5), is a potent apoptosis-inducing reagent for cancer cells. However, several pancreatic cell lines are resistant to TRA-8-induced apoptosis. We have previously demonstrated that calmodulin (CaM) antagonists enhance the Fas death receptor-mediated apoptosis pathway. Therefore, we investigated whether inhibition of CaM might affect DR5-induced apoptotic signaling in pancreatic cancer cells. We found that CaM antagonists, trifluoperazine (TFP) and tamoxifen (TMX), dose dependently enhanced TRA-8-induced apoptosis of the TRA-8-resistant PANC-1 pancreatic cells. Consistently, TFP and TMX increased TRA-8-induced activation of caspase 8, caspase 9 and caspase 3. Pretreatment of PANC-1 with Z-IETD-FMK, the caspase-8 inhibitor, decreased TFP-enhanced apoptosis by 2 fold, and markedly decreased caspase 3 activation. Immunoprecipitation analysis of the DR5-induced death inducing signaling complex (DISC) identified the interaction of CaM with DR5. Activation of DR-5 by TRA-8 increased the recruitment of CaM into the DISC, which was inhibited by TFP. In addition, TFP decreased the recruitment of the adaptor protein, FADD, and other DISC proteins that regulate cell survival, including Src and the caspase-8 inhibitory protein FLIP. Collectively, these results support an important role for CaM in DR5-induced DISC formation, thus regulating DR5-mediated survival and apoptotic signaling in pancreatic cancer cells. In summary, we have demonstrated that CaM antagonists sensitize pancreatic cancer cell to TRA 8-induced apoptosis, and that CaM binding to DR5 regulates DR5-induced DISC formation. Defining the function of CaM in regulating DR5-induced signaling pathways may lead to novel therapeutic strategies including the use of CaM antagonists with TRA-8 as combination therapy for pancreatic cancer. Citation Format: Kaiyu Yuan, Tong Zhou, Jay McDonald, Yabing Chen. Inhibition of calmodulin enhances TRA-8-induced apoptosis in pancreatic cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2953. doi:10.1158/1538-7445.AM2013-2953

Selective Induction of Apoptosis and Autophagy Through Treatment With Dandelion Root Extract in Human Pancreatic Cancer Cells

Pancreatic cancer has a 100% mortality rate; the aim of this study is to evaluate the efficacy of dandelion root extract (DRE) in inducing apoptosis and autophagy in aggressive and resistant pancreatic cancer cells. The effect of DRE was evaluated using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay. Apoptotic cell death was confirmed by nuclear condensation by Hoechst staining and externalization of phosphatidylserine to the outer leaflet of the plasma membrane by Annexin-V binding assay. Loss of mitochondrial membrane potential was observed using the JC-1 (5,5',6, 6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) dye. The induction of autophagy was detected using a monodansylcadaverine assay and this was confirmed by immunofluorescence for light chain 3-II. BxPC-3 and PANC-1 pancreatic cells were sensitive to aqueous DRE. This extract induces selective apoptosis in a dose- and time-dependent manner. Dandelion root extract caused the collapse of the mitochondrial membrane potential, leading to prodeath autophagy. Normal human fibroblasts were resistant at similar doses. We demonstrate that DRE has the potential to induce apoptosis and autophagy in human pancreatic cancer cells with no significant effect on noncancerous cells. This will provide a basis on which further research in cancer treatment through DRE can be executed.

Abstract 1470: Enhancing radiation therapy by simultaneous inhibition of thioredoxin- and glutathione-dependent metabolism

Abstract Cancer cells have been shown to be in a constant state of metabolic oxidative stress, concurrent with an elevated level of reactive oxygen species (ROS). In order to facilitate survival in the face of increased steady-sate levels of ROS, cancer cells rely on glutathione (GSH) and thioredoxin (Trx) dependent metabolism. We have recently demonstrated that the simultaneous inhibition of GSH using Buthionine sulphoximine [(BSO), a GHS synthesis inhibitor] and thioredoxin reductase using auronofin [(Au), a thioredoxin reductase inhibitor] effectively chemo-radio-sensitized lung cancer cells via a mechanism involving oxidative stress. In this work we extend this observation to demonstrate that Au 2 mg/kg and BSO 450 mg/kg, given prior to ionizing radiation (6 Gy x 2 doses) can significantly delay tumor growth rate for mice treated with radiation alone in H292 lung cancer xenografts. In addition, we demonstrate through clonogenic survival assays that the combination of Au (250-500 nM) and BSO (100 μM) radio-sensitizes two breast cancer cell lines in vitro, and more importantly the combination of Au and BSO given by intraperitoneal injection prior to radiation decreases the percent of breast cancer stem/progenitor cells in vivo. The rate limiting substrate for GSH synthesis is cysteine, whose level is controlled by the xc- cystine/glutamate antiporter. Sulfasazine (SSZ) has been shown to be a potent inhibitor of the xc- cystine/glutamate antiporter. We demonstrate that SSZ (0.1-0.5 mM) decreases total GSH (up to 90%) levels in a dose dependent manner in both Mia PaCa-2 and PANC-1 human pancreatic cell lines. The combination of Au (250 nM for 3 hours) with SSZ (0.2 mM for 24 hours) demonstrates an 80% and 30% decrease in clonogenic cell survival in MiaPaCa-2 and PANC-1 pancreatic cells that is inhibited by treatment with the thiol antioxidant, 15 mM N-acetylcysteine. These results indicate that inhibition of hydroperoxide metabolism using pharmacologically relevant agents to simultaneously disrupt GSH and Trx system, can sensitize multiple cancer cell lines to radiation. These results support the hypothesis that standard of care radiotherapy protocols could be enhanced using SSZ, Au, and/or BSO. Supported by The Iowa Center for Research by Undergraduates, R21CA139182, R01CA133114 and a Breast Cancer Research Group Seed Grand from the Holden Comprehensive Cancer Center. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1470. doi:1538-7445.AM2012-1470

Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique

Endogenous ribonucleotides and deoxyribonucleotides play a critical role in cell function, and determination of their levels is of fundamental importance in understanding key cellular processes involved in energy metabolism and molecular and biochemical signaling pathways. In this study, we determined the respective ribonucleotide and deoxyribonucleotide pool sizes in different human cell lines using a simple sample preparation method and LC/MS/MS. This assay was used to determine alterations in deoxyribonucleotide pools in human pancreatic PANC1 cells in response to hypoxia and to treatment with either hydroxyurea or aphidicolin. The levels of all deoxyribonucleotide metabolites decreased with hypoxia treatment, except for dUMP, which increased by two-fold. This LC/MS/MS assay is simple, fast, and sensitive, and it represents a significant advance over previously published methodologies.

Abstract 2532: In vitro cytotoxicity, and pharmacokinetics, tissue distribution, and metabolism of the protein kinase D inhibitors, kb-NB142-70 and kb-NB184-43, in mice bearing human cancer xenografts

Abstract Introduction: Protein kinase D (PKD) contributes to prostate and pancreatic cancer cell growth and survival. Therefore, PKD inhibitors are potential anticancer agents. kb-NB142-70 (NB142) and its methoxy-analogue, kb-NB184-43 (NB184), inhibit PKD in vitro. Methods: The in vitro inhibition of growth of Pc 3 prostate cells and Panc-1 pancreatic cells by NB142 and NB184 was evaluated by MTT assay. We evaluated the pharmacokinetics of NB142 in SCID mice bearing Pc 3 human prostate cancer xenografts and of NB184 in SCID mice bearing Panc-1 cancer xenografts. NB142 or NB184 was administered i.v. at 25 mg/kg. Three mice per time point were euthanized at 5, 15, 30, 60, 120, 240, 360, 960 and 1440 min after dosing. Plasma, tissues and urine were collected, and concentrations of NB142 and NB184 were quantitated by HPLC-UV. Metabolites in plasma and urine were characterized by LC-MS/MS. Results: The IC50s of NB142 and NB184 were 21.0 µM and 24.3 µM, respectively, against Pc 3; and 33.7 µM and 27.4 µM, respectively, against Panc-1. After dosing NB142, the plasma NB142 Cmax (at 5 min) was 36.9 µM, and the NB142 Pc 3 tumor Cmax was 11.8 µM. NB142 was not detected in plasma, liver, kidney, spleen, or heart beyond 30 min. After dosing NB184, the plasma, liver, and kidney NB184 Cmax, (at 5 min) were 61.9 µM, 42.6 µM, and 94.8 µM, respectively. The NB184 Cmax in Panc-1 tumors (at 15 min) was 8.0 µM. Areas under the concentration vs time curves of NB142 and NB184 in plasma were 409 and 782 nmol·min/ml, respectively. The plasma half-lives of NB142 and NB184 were 6 and 14 min, respectively. The major metabolite of NB142 was a glucuronide. Between 0-6 h, urinary excretion of NB142 and metabolites accounted for 2.5 % and 7.6%, respectively, of the administered dose. NB184 underwent oxidation and glucuronidation, and more than 10 metabolites were observed. NB142 was not detected in plasma after administration of NB184. Between 0-16 h, urinary excretion of NB184 and metabolites accounted for 2.1 % and 5.3%, respectively, of the administered dose. Conclusions: Although the inclusion of a methoxy group in NB184 increased its plasma half-life approximately 3-fold compared to NB142, metabolic and urinary elimination of both compounds was relatively rapid. Peak tumor concentrations of NB142 and NB184 were of the same order of magnitude as the concentrations required in vitro for cytotoxicity. (Support: P30-CA47904 and P01-CA078039) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2532.

Ptf1a and RBP-J cooperate in activating Pdx1 gene expression through binding to Area III

Pancreatic and duodenal homeobox factor 1 ( Pdx1) has been demonstrated to play a crucial role in pancreas development and in maintenance of mature β-cell function. However, it remains to be elucidated how Pdx1 gene expression is regulated in non-β cells during pancreas development. Pdx1 and Ptf1a are expressed in pancreatic progenitor cells, which give rise to all three types of pancreatic tissue. In addition, Ptf1a has been shown to bind the mammalian Suppressor of Hairless (RBP-J) within the PTF1 complex. Furthermore, loss-of-function approaches have revealed that all three factors are essential for early pancreas development. We therefore hypothesized that Ptf1a and RBP-J regulate expression of the Pdx1 gene in pancreatic precursors. Reporter gene analyses showed that Ptf1a transactivated Pdx1 promoter in pancreatic Panc-1 cells, which was enhanced by RBP-J. Deletion/mutation analyses of the Pdx1 promoter and electrophoretic gel-mobility shift assays identified the Ptf1a binding site in the well-conserved regulatory sequence domain, termed Area III, which was also confirmed by the chromatin immunoprecipitation assay. Furthermore, adenovirus-mediated overexpression of Ptf1a, together with RBP-J, markedly increased Pdx1 protein levels in pancreatic AR42J-B13 cells. Our data suggest a novel transcriptional network, where Ptf1a and RBP-J cooperatively regulate Pdx1 gene expression through binding to Area III.

Two New Cytotoxic Linderazulenes from a Deep-Sea Gorgonian of the Genus Paramuricea

The known compound linderazulene (1) and two new linderazulenes (2, 3) were isolated from a deep-sea gorgonian Paramuricea sp. The structures of 2 and 3 were determined through spectroscopic methods. Compounds 1-3 show moderate in vitro cytotoxicity against the P388 murine leukemia cell line with IC(50)'s of 18.8, 2.7, and 15.6 microg/mL, respectively. Compound 2 showed moderate activity against the PANC-1 pancreatic cell line with an IC(50) of 18.7 microg/mL.

Transforming Growth Factor-β (TGF-β) Type I Receptor/ALK5-dependent Activation of the GADD45β Gene Mediates the Induction of Biglycan Expression by TGF-β

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.