KP4 Cells Research Articles

Silencing of keratin 17 by lentivirus-mediated short hairpin RNA inhibits the proliferation of PANC-1 human pancreatic cancer cells.

Keratin 17 (KRT17) has been demonstrated to be a potential biological marker for the prediction of prognosis in particular types of cancer. The aim of the present study was to investigate the molecular mechanisms underlying the function of KRT17 in the pancreatic cancer (PAC) cell line PANC-1 and the potential of KRT17 as a therapeutic target for PAC. KRT17 expression levels were analyzed using quantitative PCR and compared with histological data using bioinformatics tools in PAC samples and three human PAC cell lines. Cell proliferation was determined using an MTT assay, in addition to cell cycle distribution and apoptosis analysis using flow cytometry, colony formation assay using Giemsa staining and cell motility analysis using a Transwell migration assay. Tumor growth was evaluated in vivo in nude mice. The expression levels of a number of signaling molecules were measured to establish the potential mechanism by which silencing KRT17 expression affected PAC PANC-1 cells. Increased levels of KRT17 expression were observed in human PAC compared with normal tissues, as well as in three human PAC cell lines (MIA PaCa-2, PANC-1 and KP-3 cells) compared with the H6c7 human immortal pancreatic duct epithelial cell line. High expression levels of KRT17 in PAC samples were associated with poor overall survival (P=0.036) and disease-free survival (P=0.017). Lentivirus-mediated KRT17 silencing inhibited cell proliferation, colony formation and migration, but promoted apoptosis and resulted in cell cycle arrest in the G0/G1 phase in PANC-1 cells. In addition, KRT17 knockdown inhibited in vivo tumor growth. KRT17 knockdown induced dysregulation of ERK1/2 and upregulation of the pro-apoptotic Bcl-2 protein Bad. In conclusion, the present study demonstrated that elevated KRT17 levels are positively associated with pancreatic cancer progression; KRT17 knockdown suppressed cell growth, colony formation, migration and tumor growth, and induced apoptosis and cell cycle arrest, affecting ERK1/2/Bad signaling. Therefore, the results of the present study suggested that KRT17 may be a potential target for the treatment of pancreatic cancer.

Abstract 1864: Pharmacologic inhibition of NAMPT sensitizes pancreatic cancer cells to the antineoplastic effects of metformin

Abstract The use of the anti-diabetic drug metformin has been correlated with a reduced cancer incidence, suggesting an unexpected anti-neoplastic activity for this compound. Since metformin treatment is safe and economical, there is considerable interest in exploring its anticancer activity in patients. Pancreatic cancer (pancreatic ductal adenocarcinoma: PDAC) is one of the most aggressive neoplastic diseases, for which there is no treatment significantly increasing patient's survival. Metformin use was associated with reduced pancreatic cancer incidence or better survival in diabetics. In vitro, metformin decreases cell survival and growth of pancreatic cancer cells and appears to target tumor-initiating cells. In vivo, metformin decreases growth of human pancreatic cancer cell lines xenografts in mice. However, clinical trials using metformin failed to decrease pancreatic cancer progression in patients, raising important questions about molecular mechanisms that protect tumor cells from the antineoplastic activities of metformin. We discovered a new mechanism of resistance to the anti-oncogenic properties of metformin in PDAC cells through up-regulation of NAMPT and increase of NAD+ synthesis. Using an inhibitor specific to NAMPT, FK866, we sensitized PDAC cells to the effects of metformin in vitro. In vivo, FK866 increased the efficiency of metformin treatment on KP4 cells xenografts. As both metformin and FK866 clinical trials have failed to efficiently treat cancer, the combination of these two compounds may be a promising strategy to treat pancreatic cancer and maybe other malignancies. Citation Format: Gerardo Ferbeyre, Maxime Parisotto, Marie-Camille Rowell, Véronique Bourdeau, Andreea R. Schmitzer. Pharmacologic inhibition of NAMPT sensitizes pancreatic cancer cells to the antineoplastic effects of metformin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1864.

Chemical profiles and anticancer effects of saponin fractions of different polarity from the leaves of Panax notoginseng

AimTo evaluate the chemical profiles and cytotoxic effects among the total saponin fraction (TSF), 25% ethanol fraction (25EF), 50% ethanol fraction (50EF), and 85% ethanol fraction (85EF) prepared by macroporous resin from the leaves of Panax notoginseng. MethodThe simultaneous determination of thirteen main saponins, as well as the chemical profiles of saponin fractions of different polarity, was made by HPLC-DAD and LC-ESI-MSn analysis. The cytotoxic effects were determined against KP4 cells (human pancreatic cancer), NCI-H727 cells (human lung cancer), HepG2 cells (human hepatocellular cancer), and SGC-7901 cells (human gastric adenocarcinoma). ResultsChemical analysis indicated that 85EF possessed the most abundant cytotoxic protopanaxadiol saponins, including the marker saponins F2, 20(R)-Rg3, 20(S)-Rg3, and Rh2. The MTT assay showed that 85EF also had the strongest cytotoxic effects among the four fractions. 25EF showed no anti-proliferative effects, while 50EF and TSF exhibited weak anti-proliferative activity. ConclusionFrom the aspect of comprehensive utilization of resources, 85EF, enriched with low polarity PPD group saponins, is a new alternative source of anticancer saponins, and a promising botanical preparation for further anticancer studies.

Abstract C284: IRE1 and PERK as targets of cellular adaptation and survival to hypoxia.

Abstract Hypoxia is an important and unique feature of the solid tumour microenvironment. Hypoxia promotes resistance to radiotherapy, aggressive disease phenotypes, and poor patient prognosis. Consequently, therapies directed against hypoxic cells are needed to improve patient outcome. Hypoxic stress leads to the activation of several adaptive pathways including the unfolded protein response (UPR). The UPR is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling pathways initiated by the ER-membrane resident proteins PERK, IRE1 and ATF6. Previous work using genetic models has implicated the PERK pathway, as well as downstream components of the IRE1 pathway (XBP1) as important mediators of hypoxia tolerance and tumor response to therapy. Here we employed short hairpin RNAs (shRNAs) and recently developed small-molecule inhibitors of IRE1 (4μ8c) and PERK (GSK-compound 39) to investigate the relative importance of these two pathways in promoting hypoxia tolerance and their potential as therapeutic targets. We tested the pathway activity of PERK and IRE1 by quantitative PCR and western blotting. Hypoxia tolerance was measured by in vitro clonogenic assays. First, knockdown experiments using two different shRNAs showed that IRE1 is essential for survival of HCT116 colorectal cancer cells during mild hypoxia while PERK was essential in both normoxia and hypoxia. Interestingly, the IRE1 knockdown cells had a significantly altered glycolytic metabolism, a known mechanism of hypoxia adaptation. Using our small-molecule IRE1 inhibitor, we show that 4μ8c is a potent and non-toxic inhibitor of IRE1 RNase activation in response to both hypoxia and other ER stress-inducing agents. This compound effectively inhibited IRE1 induced activation of the downstream target genes in both HCT116 colorectal cancer and KP4 pancreatic cancer cell lines under hypoxia. However, despite potent inhibition of IRE1 activation and the, 4μ8c had no effect on cell proliferation or clonogenic survival of HCT116 and KP4 cells during exposure to hypoxia or anoxia. Similarly, 4μ8c inhibition of IRE1 did not sensitize cells to other ER stress inducing agents. In contrast, genetic inactivation of PERK signaling or treatment with the highly selective and potent PERK inhibitor GSK-compound 39 substantially reduced tolerance to hypoxia, anoxia and other ER stress inducing agents. Our results demonstrate that although hypoxia potently activates both PERK and IRE1 arms of the UPR pathway, the PERK arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress. Furthermore, our results suggest that newly developed and orally available PERK inhibitors such as GSK-compound 39 are selectively toxic to hypoxic cells, and thus warrant further development for use in combination therapies with radiation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C284. Citation Format: Dan Cojocari, Ravi Vellanki, Brandon Sit, Marianne Koritzinsky, Bradly G. Wouters. IRE1 and PERK as targets of cellular adaptation and survival to hypoxia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C284.

New small molecule inhibitors of UPR activation demonstrate that PERK, but not IRE1α signaling is essential for promoting adaptation and survival to hypoxia

Background and purposeThe unfolded protein response (UPR) is activated in response to hypoxia-induced stress in the endoplasmic reticulum (ER) and consists of three distinct signaling arms. Here we explore the potential of targeting two of these arms with new potent small-molecule inhibitors designed against IRE1α and PERK. MethodsWe utilized shRNAs and small-molecule inhibitors of IRE1α (4μ8c) and PERK (GSK-compound 39). XBP1 splicing and DNAJB9 mRNA was measured by qPCR and was used to monitor IRE1α activity. PERK activity was monitored by immunoblotting eIF2α phosphorylation and qPCR of DDIT3 mRNA. Hypoxia tolerance was measured using proliferation and clonogenic cell survival assays of cells exposed to mild or severe hypoxia in the presence of the inhibitors. ResultsUsing knockdown experiments we show that PERK is essential for survival of KP4 cells while knockdown of IRE1α dramatically decreases the proliferation and survival of HCT116 during hypoxia. Further, we show that in response to both hypoxia and other ER stress-inducing agents both 4μ8c and the PERK inhibitor are selective and potent inhibitors of IRE1α and PERK activation, respectively. However, despite potent inhibition of IRE1α activation, 4μ8c had no effect on cell proliferation or clonogenic survival of cells exposed to hypoxia. This was in contrast to the inactivation of PERK signaling with the PERK inhibitor, which reduced tolerance to hypoxia and other ER stress inducing agents. ConclusionsOur results demonstrate that IRE1α but not its splicing activity is important for hypoxic cell survival. The PERK signaling arm is uniquely important for promoting adaptation and survival during hypoxia-induced ER stress and should be the focus of future therapeutic efforts.

Abstract 3042: LY2801653: An orally bioavailable MET kinase inhibitor with inhibitory activity against the oncoproteins ROS1 and MKNK1/2.

Abstract Under normal conditions MET receptor activation, internalization, and degradation is a highly controlled process regulated by the binding of its ligand, HGF. In many cancers however, MET becomes highly dysregulated, and MET overexpression, with or without gene amplification, is associated with a poorer prognosis. Aberrant activation of MET can also occur through mutation, or increased autocrine or paracrine HGF production. MET activation leads to increased oncogenic activities including cell migration, proliferation, invasion, survival, tumor neovascularization and decreased apoptosis. Several inhibitors of MET, including type I and type II kinase inhibitors, are currently being investigated in clinical trials. LY2801653 is a type II MET kinase inhibitor being investigated in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037). In addition to MET, LY2801653 also inhibits ROS1 kinase, and the serine/threonine kinases MKNK1/2, with cell-based IC50 of 23nM and 7nM respectively. ROS1 is an orphan receptor that can be constitutively activated as a fusion protein either by genomic microdeletion or translocation. ROS1 fusion proteins are found in glioblastoma, NSCLC and cholangiocarcinoma. MKNK1/2 are kinases downstream of ERK and p38 and are capable of regulating translation through the direct phosphorylation of eIF4E. Increased MKNK1/2-dependent phosphorylation of the translation factor eIF4E is observed in head and neck squamous cell carcinoma and is correlated with a poor prognosis. This study evaluated the relative inhibitory effects of LY2801653, Crizotinib, XL184, XL880, and PF4217903 in a variety of cell lines against MET, ROS1, MKNK1/2, ERK, and other downstream molecules. In HCC78, a ROS1-fusion protein bearing cell line, and in NIH3T3 cells stably transfected with the FIG-ROS(s) fusion, LY2801653, XL184 and XL880 were more potent inhibitors of ROS1 compared with Crizotinib. Moreover, LY2801653 and Crizotinib were both efficacious in NIH3T3 FIG-ROS(s) xenografts, although LY2801653 was more potent in reducing p-ROS1 in these tumors than Crizotinib. In NCI-H2122 and Hs746T cells (mutated MET), A2780 cells (MET-negative), KP-4 cells (MET autocrine), H441 cells (HGF-independent; MET positive), HCT116 (mutated KRAS), and the cholangiocarcinoma cell lines EGI-1 and TFK-1, LY2801653 demonstrated more potent inhibitory activity against MKNK1/2 versus treatment with XL184 and XL880, while Crizotinib had no observed inhibitory activity against MKNK1/2. LY2801653 also showed inhibitory activity against MKNK1/2 in MET-amplified MKN45 cells. These findings suggests there may be clinical merits to testing LY2801653 in tumor types associated with increased MKNK1/2 activity or ROS1 fusion proteins alone, or in conjunction with aberrant MET activity, such as colorectal carcinoma, head and neck squamous cell carcinoma, and cholangiocarcinoma. Citation Format: Sau-Chi B. Yan, Megan N. Thobe, Bruce W. Konicek, Victoria L. Peek, Suzane L. Um, Richard A. Walgren, Huaxing Pei, Timothy P. Burkholder, Jeremy R. Graff. LY2801653: An orally bioavailable MET kinase inhibitor with inhibitory activity against the oncoproteins ROS1 and MKNK1/2. [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 3042. doi:10.1158/1538-7445.AM2013-3042

Abstract 2087: Detection of KRAS mutations in circulating tumor cells (CTCs) and in formalin-fixed, paraffin-embedded (FFPE) tissue using castPCR method

Abstract CTCs are tumor cells in circulation in the blood and are believed to be shed from both the primary and metastatic tumors. CTC counts are prognostic of survival in metastatic breast, colorectal, and prostate cancers. It is postulated that these cells are genetically similar to cells comprising the patient's tumor(s). The presence of these cells in blood provides a minimally invasive source of nucleic acid for serial monitoring of tumors for disease progression and emergence of drug treatment resistance without the risks of traditional solid tumor biopsy. Activating mutations in the KRAS gene are frequently found in human cancers (up to 30% of cancer cases), and are present in a large portion of non-small cell lung cancer (NSCLC), pancreatic, and colorectal cancers. Reported mutations are mostly single point mutations in codons 12, 13 and 61, with the majority of mutations present in codon 12. Importantly, KRAS mutation status can be predictive of response to treatment, and may be used to determine the best therapeutic strategy. Therefore, it is critical to develop a robust method for detecting mutations from a limited number of enriched tumor cells (e.g. CTCs or fine needle aspirate) in a high background of normal cells. Our studies utilize competitive allele-specific PCR (castPCR) for detecting mutant KRAS from human cancer cells spiked into and retrieved from blood, and from fixed cells embedded in paraffin. This technology is reportedly capable of detecting one mutant allele molecule in 10 million wild-type molecules. KRAS is mutated in human NSCLC H441 cells (G12V) and in KP-4 pancreatic cells (G12D). After spiking H441 and KP-4 cells into human blood, cells were captured/enriched and enumerated by the Veridex CellTracks™ system. Both CXC and Profile kits (Veridex) were used. Recovered cells were then lysed overnight with Arcturus® PicoPure® DNA extraction lysis buffer, and the resulting DNA was analyzed for mutant KRAS with the castPCR TaqMan® Mutation Detection Assay (Applied Biosystems) specific for the mutation found in H441 cells (G12V). Under these conditions, we detected KRAS mutation in H441 cells with robust sensitivity (20 spiked cells into 7.5ml of blood; KP-4 cells were negative for the G12V mutation). To further examine if this method could be used for detecting mutations in human tissue, FFPE tumor cells were analyzed. The WaxFreeTM DNA kit (TrimGen) was used to extract DNA from FFPE cells, and castPCR was performed. H441 mutant KRAS was detected from as little as ∼200 pg DNA (100-fold dilution of DNA). Ongoing studies involve correlating mutation status between CTCs and tumor FFPE tissue. These data will aid in clinical biomarker development used in clinical trials and improve methods to determine the most beneficial treatment options for patients. Importantly, this approach may enable patients to be serially monitored for tumor mutation status with blood draws. 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 2087. doi:1538-7445.AM2012-2087

Ultra-violet irradiation induces apoptosis via mitochondrial pathway in pancreatic cancer cells

Pancreatic cancer is a highly lethal disease and gemcitabine is considered to be the standard of care for the treatment of advanced pancreatic cancer. However, the outcome of the patients treated with gemcitabine is still unstatisfactory and further development of new treatments is required. We recently found that short wavelength ultra-violet (UV-C) suppresses cell proliferation with downregulation of epidermal growth factor receptor (EGFR) in human pancreatic cancer cells, but not in normal pancreatic epithelial (PE) cells. In this study, we investigated the effect of UV-C on apoptosis in several cell lines derived from the pancreas. UV-C induced poly(ADP-ribose) polymerase (PARP) cleavage, which is a marker of cells undergoing apoptosis, in Panc1, MiaPaca2, KP3 and BxPC3 pancreatic cancer cells, but not in PE cells. We also observed similar effects in Hoechst 33258 staining, which shows DNA fragmentation. While p53, a tumor suppressor protein, plays a critical role in UV-C-induced cell damage, we did not observe the correlation between the sensitivity to UV-C and p53 status. Thapsigargin, an agent that promotes endoplasmic reticulum (ER) stress by depletion of lumenal calcium stores, as well as cis-diamineplatinum (II) dichloride, a classical anti-cancer drug that causes DNA damage, induced PARP cleavage even in PE cells. Moreover, UV-C-induced apoptosis in Panc1 and KP3 cells was associated with the release of cytochrome c, indicating that it was mediated via mitochondrial pathway. Taken together, UV-C has a potent anti-cancer effect on pancreatic cancer cells without adverse effect on normal cells and it could be useful for the treatment of human pancreatic cancers.

UVC Radiation Induces Downregulation of EGF Receptor via Phosphorylation at Serine 1046/1047 in Human Pancreatic Cancer Cells

Epidermal growth factor receptor (EGFR) is overexpressed in human pancreatic cancer and is one of the clinical targets in its treatment. In the present study we investigated the mechanism underlying ultraviolet C (UVC)-radiation-induced cell growth inhibition and downregulation of EGFR in human pancreatic cancer cells (Panc1 and KP3). The cell proliferation assay indicated that Panc1 and KP3 cells were more sensitive to UVC radiation, and their growth was significantly inhibited compared to cells of the normal human pancreatic epithelial cell line, PE. Although EGFR levels was extremely low in PE cells, EGFR were highly overexpressed in Panc1 and KP3 cells, and UVC radiation downregulated the expression of EGFR in a time-dependent manner and induced phosphorylation of EGFR at Ser1046/1047 (S1046/7) in Panc1 and KP3 cells. UVC radiation induced activation of p38 mitogen-activated protein kinase (MAPK), and EGFR phosphorylation at S1046/7 induced by UVC radiation was markedly attenuated by the inhibition of p38 MAPK. Moreover, fluorescence microscopy revealed that p38 MAPK activated by UVC radiation triggered EGFR internalization and that this was not correlated with c-Cbl, an ubiquitin ligase, which plays an important role in EGF-induced EGFR downregulation. Taken together, our results suggest that in pancreatic cancer cells UVC radiation induced desensitization of the cells to EGFR stimuli via phosphorylation of EGFR at S1046/7 by activation of p38 MAPK, independent of c-Cbl.

The CD40‐CD154 interaction would correlate with proliferation and immune escape in pancreatic ductal adenocarcinoma

CD40 and CD154 are associated with lymphocyte signaling pathways and they are also expressed in some malignant neoplasms, but the significance in pancreatic cancer is unknown. Eighty pancreatic cancer specimens were stained immunohistochemically, and the results were correlated with the patients' clinicopathologic features. Subsequently, in vitro analysis of CD40-CD154 signaling was performed. Immunohistochemical analysis of tumor cells showed that 29 patients (36.3%) were positive for CD40, and 17 patients (21.3%) had very high CD154 expression. The survival of patients who had very high CD154 expression was significantly better than that of others (P = 0.0198). Univariate and multivariate analysis revealed that very high CD154 expression in cancer cells was not an independent, favorable prognostic factor (risk ratio, 0.493; P = 0.0224). On in vitro proliferation assay, the growth of PK-45P and KP-4 cells was blocked by CD40 and CD154 blocking antibodies. Moreover, on in vitro cytokine assay, Th-2 cytokines from PK-45P and SUIT-2 were blocked by CD40 or CD154 blocking antibody. These results suggest that the CD40-CD154 interaction would correlate with cell proliferation and secretion of cytokines in PDAC cells, and CD154 overexpression could be a favorable prognostic factor in PDAC patients.

HSP90 inhibitors induce desensitization of EGF receptor via p38 MAPK-mediated phosphorylation at Ser1046/1047 in human pancreatic cancer cells

Heat shock protein (HSP) 90 is known to be a molecular chaperone whose association is required for the stability and function of oncogenic protein including epidermal growth factor receptor (EGFR) that promotes cancer cell growth. Therefore, HSP90 is a promising target for therapy against cancer including in the pancreas, some of which are highly dependent on EGFR. We investigated the effects of HSP90 inhibitors on cytotoxicity and desensitization of EGFR in human pancreatic cancer cells (KP3, BxPc3 and AsPc1). 17-allylamino-17-demethoxy-geldanamycin (17-AAG), an inhibitor of HSP90, caused de-sensitization of EGFR in a time-dependent manner, concurrently inducing phosphorylation of EGFR at Ser1046/1047 (Ser1046/7), a site which plays an important role in EGFR desensitization in these pancreatic cancer cells. We also found similar effects in KP3 cells treated with other HSP90 inhibitors, geldanamycin and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG). In KP3 cells, 17-AAG induced activation of either p44/p42 mitogen-activated protein kinase (MAPK) or p38 MAPK. Interestingly, whereas the inhibition of p44/p42 MAPK attenuated neither phosphorylation of EGFR at Ser1046/7 nor desensitization of EGFR, the phosphorylation at Ser1046/7 induced by 17-AAG was markedly attenuated by the inhibition of p38 MAPK, indicating that p38 MAPK induced this phosphorylation. Moreover, the inhibition of p38 MAPK significantly attenuated 17-AAG-induced EGFR desensitization. These results strongly suggest that EGFR phosphorylation at Ser1046/7 via activation of p38 MAPK induced by HSP90 inhibitors plays a pivotal role in EGFR desensitization in human pancreatic cancer cells.

Insulin-like growth factor-I receptor in proliferation and motility of pancreatic cancer

To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed. Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4) were cultured in media with 10 mL/L fetal bovine serum. Western blotting analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Picropodophyllin (PPP), a specific inhibitor of IGF-IR, LY294002, a specific inhibitor of phosphatidylinositol 3 kinase (PI3K), and PD98059, a specific inhibitor of mitogen-activated protein kinase, were added to the media. After 72 h, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay was performed to analyze cell proliferation. A wound assay was performed to analyze cell motility with hematoxylin and eosin (HE) staining 48 h after addition of each inhibitor. All cell lines clearly expressed not only IGF-IR but also phosphorylated IGF-IR. PPP significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 36.9% +/- 2.4% (mean +/- SD), 30.9% +/- 5.5%, 23.8% +/- 3.9%, 37.1% +/- 5.3%, 10.4% +/- 4.5%, 52.5% +/- 4.5% and 22.6% +/- 0.4%, at 2 micromol/L, respectively (P < 0.05). LY294002 significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 44.4% +/- 7.6%, 32.9% +/- 8.2%, 53.9% +/- 8.0%, 52.8% +/- 4.0%, 32.3% +/- 4.2%, 51.8% +/- 4.5%, and 30.6% +/- 9.4%, at 50 micromol/L, respectively (P < 0.05). PD98059 did not significantly suppress cell proliferation. PPP at 2 micromol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 3.0% +/- 0.2%, 0%, 0%, 2.0% +/- 0.1%, 5.0% +/- 0.2%, 3.0% +/- 0.1%, and 5.0% +/- 0.2%, respectively (P < 0.05). LY294002 at 50 micromol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 to 3.0% +/- 0.2%, 0%, 3.0% +/- 0.2%, 0%, 0%, 0% and 3% +/- 0.1%, respectively (P < 0.05). PD980509 at 20 micromol/L did not suppress motility. Cells were observed by microscopy to analyze the morphological changes induced by the inhibitors. Cells in medium treated with 2 micromol/L PPP or 50 micromol/L LY294002 had pyknotic nuclei, whereas those in medium with 20 micromol/L PD98059 did not show apoptosis. IGF-IR and PI3K are good candidates for molecular therapy of pancreatic cancer.

Tranilast strongly sensitizes pancreatic cancer cells to gemcitabine via decreasing protein expression of ribonucleotide reductase 1

Gemcitabine (Gem) is a dFdC analogue with activity against several solid tumors. Gem is intracellularly phosphorylated by dCK, leading to the production of the metabolite dFdCDP. dFdCDP exhibits the cytotoxic effect by inactivating ribonucleotide reductase larger subunit 1 (RRM1), which is a rate limiting enzyme for de novo DNA synthesis. To date, RRM1 expression is believed to determine sensitivity to Gem in pancreatic and non-small cell lung cancer. In the present study, we found that an anti-allergic drug, tranilast strongly enhanced the sensitivity of pancreatic cancer cell line KP4 to Gem. In growth inhibition assay, 100 microM of tranilast plus 1 microM of Gem more strongly suppressed the growth of KP4 at 12.7-fold in IC50 than single Gem treatment, while this compound no longer affected the sensitivity to other drugs such as 5-fluorouracil, irinotecan or paclitaxel. FACS and TUNEL analysis demonstrated the increased apoptotic population in KP4 cells under tranilast plus Gem, compared with single Gem treatment. In Western blot analysis, tranilast treatment decreased RRM1 expression at protein level with dose-dependency in KP4 cells. Proteasome inhibitor MG132 disturbed the reduction of RRM1 expression in tranilast treated KP4 cells, indicating protein degradation by the activated proteasome. Transfection using siRNA against RRM1 increased the sensitivity of KP4 to Gem, suggesting that RRM1 suppression is an important step in increasing Gem efficacy. Finally, we demonstrated that tranilast reduced RRM1 protein and increased Gem efficacy in 4 other pancreatic cell lines. In a future, a novel chemotherapeutic strategy by Gem along with tranilast might improve Gem efficacy against pancreatic cancer.

Homeobox Gene CDX2 Inhibits Human Pancreatic Cancer Cell Proliferation by Down-Regulating Cyclin D1 Transcriptional Activity

Homeobox gene caudal related homeobox gene 2 (CDX2) is an intestine-specific tumor suppressor gene. This study is intended to investigate the effect of CDX2 expression on cell proliferation and cyclin D1 expression in pancreatic cancer cells. Four pancreatic ductal adenocarcinoma cell lines (PancQGO-1, BxPC-3, MIAPaCa-2, CFPAC-1), 1 islet carcinoma cell line (QGP-1), and 1 adenosquamous carcinoma cell line (KP-3) were analyzed for CDX1 and CDX2 expression using real-time reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of pancreatic cancer cells was analyzed using WST-1 assay after CDX2 transfection. Luciferase assay was performed to examine the effects of CDX2 on cyclin D1 transcriptional activity. CDX2 was expressed at a significantly higher level in QGP-1 cells than in KP-3 cells. Moreover, CDX2 was expressed at a middle level in 4 pancreatic ductal adenocarcinoma cells. Cell proliferation and cyclin D1 mRNA level were inhibited significantly after CDX2 transfection in pancreatic cancer cells. Furthermore, CDX2 inhibited exogenous nuclear factor kappaB-p65-induced luciferase gene expression in a dose-dependent manner. In addition, CDX2 inhibited pGL2HIVD1kappaB2-luciferase activity. CDX2 might play a role in inhibiting cell proliferation and repressing cyclin D1 transcriptional activity through the proximal nuclear factor kappaB binding site in pancreatic cancer cells.

MetMAb, the One-Armed 5D5 Anti-c-Met Antibody, Inhibits Orthotopic Pancreatic Tumor Growth and Improves Survival

The hepatocyte growth factor (HGF) and its receptor, c-Met, have been implicated in driving proliferation, invasion, and poor prognosis in pancreatic cancer. Here, we investigated the expression of HGF and c-Met in primary pancreatic cancers and described in vitro and in vivo models in which MetMAb, a monovalent antibody against c-Met, was evaluated. First, expression of HGF and MET mRNA was analyzed in 59 primary pancreatic cancers and 51 normal samples, showing that both factors are highly expressed in pancreatic cancer. We next examined HGF responsiveness in pancreatic cancer lines to select lines that proliferate in response to HGF. Based on these studies, two lines were selected for further in vivo model development: BxPC-3 (c-Met(+), HGF(-)) and KP4 (c-Met(+), HGF(+)) cells. As BxPC-3 cells are responsive to exogenous HGF, s.c. tumor xenografts were grown in a paracrine manner with purified human HGF provided by osmotic pumps, wherein MetMAb treatment significantly inhibited tumor growth. KP4 cells are autocrine for HGF and c-Met, and MetMAb strongly inhibited s.c. tumor growth. To better model pancreatic cancer and to enable long-term survival studies, an orthotopic model of KP4 was established. MetMAb significantly inhibited orthotopic KP4 tumor growth in 4-week studies monitored by ultrasound and also improved survival in 90-day studies. MetMAb significantly reduced c-Met phosphorylation in orthotopic KP4 tumors with a concomitant decrease in Ki-67 staining. These data suggest that the HGF/c-Met axis plays an important role in the progression of pancreatic cancer and that targeting c-Met therein may have therapeutic value.

Down-Regulation of Molecular Chaperone 78-kd Glucose-Regulated Protein/Immunoglobulin-Binding Protein Expression Involved in Enhancement of Human RS Cell Mutability

Enhancement of cell mutability via extracellular materials of cancer cells is a crucial event leading to the development of cancers; however, the activation process of mutability is still not well understood. In this study, to identify the regulatory mechanism of cell mutability, we investigated mutability modulated in response to human pancreatic cancer cell-conditioned medium and identified the candidates for cellular molecules involved in the mutability modulation. To test the mutation-modulating effects of the conditioned medium, human RS cells were cultured with medium derived by culturing human pancreatic cancer KP-4 cells, followed by irradiation with UV (mainly 254 nm in wavelength). Mutations were detected by phenotypic ouabain resistance and genetic base substitution of K-ras codon 12. Messenger RNA differential display was used to identify genes that were differentially expressed between conditioned medium-treated and mock-treated RSa cells. The influence of 78-kd glucose-regulated protein/immunoglobulin-binding protein (GRP78/BiP) expression on mutability was assessed by the down-regulation of GRP78/BiP using antisense oligonucleotides or antisense complementary DNA. The UV-induced mutagenicity in RS cells was strengthened by preculture with KP-4 cell-conditioned medium. Messenger RNA differential display revealed that GRP78/BiP expression was suppressed in RS cells after treatment of the conditioned medium. Furthermore, the level of UV-induced mutations was elevated significantly in GRP78/BiP down-regulated cells. Culture of human RS cells with pancreatic cancer KP-4 cell-conditioned medium resulted in increased UV mutagenicity, possibly via the down-regulation of GRP78/BiP.

Generation of Inhibitory DNA Aptamers Against Human Hepatocyte Growth Factor

Hepatocyte growth factor (HGF), a multifunctional cytokine, can act on many cell types. It is involved in cancer growth and metastasis by enhancing the motility of cancer cells and stimulating angiogenesis. The development of effective inhibitors for HGF is an important issue in cancer therapy. In this study, we isolated DNA aptamers against human HGF using the systematic evolution of ligands by exponential enrichment method. The selected DNA aptamers had a highly conserved consensus sequence, and could be divided into two major classes (classes I and II). The consensus motif of classes I and II might contribute to the formation of a hairpin loop structure and a G-quartet structure, respectively. These DNA aptamers bound to human HGF with high affinity and specificity. The dissociation constants of typical aptamers H38-15 and H38-21, representative of the two classes, were calculated to be approximately 20 nM. H38-15 and H38-21 inhibited the biological activities of HGF including the stimulation of scattering, migration, and invasion of pancreatic cancer KP-3 cells. Furthermore, both aptamers inhibited HGF-induced tube formation by human umbilical vein endothelial cells. These results suggested that the isolated DNA aptamers will be useful as therapeutic and diagnostic reagents for cancers.

Mitochondrial signal lacking manganese superoxide dismutase failed to prevent cell death by reoxygenation following hypoxia in a human pancreatic cancer cell line, KP4.

One of the major characteristics of tumor is the presence of a hypoxic cell population, which is caused by abnormal distribution of blood vessels. Manganese superoxide dismutase (MnSOD) is a nuclear-encoded mitochondrial enzyme, which scavenges superoxide generated from the electron-transport chain in mitochondria. We examined whether MnSOD protects against hypoxia/reoxygenation (H/R)-induced oxidative stress using a human pancreas carcinoma-originated cell line, KP4. We also examined whether MnSOD is necessarily present in mitochondria to have a function. Normal human MnSOD and MnSOD without a mitochondrial targeting signal were transfected to KP4 cells, and reactive oxygen species, nitric oxide, lipid peroxidation, and apoptosis were examined as a function of time in air following 1 day of hypoxia as a H/R model. Our results showed H/R caused no increase in nitric oxide, but resulted in increases in reactive oxygen species, 4-hydroxy-2-nonenal protein adducts, and apoptosis. Authentic MnSOD protected against these processes and cell death, but MnSOD lacking a mitochondrial targeting signal could not. These results suggest that only when MnSOD is located in mitochondria is it efficient in protecting against cellular injuries by H/R, and they also indicate that mitochondria are primary sites of H/R-induced cellular oxidative injuries.

NK4, a four-kringle antagonist of HGF, inhibits spreading and invasion of human pancreatic cancer cells.

Because of the highly aggressive behaviour, i.e. invasive, disseminative and metastatic properties, the outcome for patients with pancreatic cancer is morbid. A better understanding and interference with the malignant behaviour of pancreatic cancer may provide new directions for treatment. We report here the induction of highly motile and invasive properties in human pancreatic cancer cells by hepatocyte growth factor (HGF) and blockage of these properties by NK4, a newly identified antagonist for HGF. In all of eight human pancreatic cancer cell lines we used (AsPC-1, BxPC-3, H-48N, KP-1N, KP-2, KP-3, MIA PaCa-2 and SUIT-2 cells), the c-Met/HGF receptor was expressed at varying levels. Although weak mitogenic activity of HGF was seen only in SUIT-2 and KP-3 cells, HGF strongly stimulated migration and invasion of these pancreatic cancer cells, except for BxPC-3 and MIA PaCa-2 cells. In contrast, migration and invasion potently induced by HGF in KP-1N, KP-3 and SUIT-2 cells were inhibited by NK4. The invasion of SUIT-2 cells was also potently stimulated with the influence of cocultured pancreatic fibroblasts and by ascitic fluid obtained after pancreatic cancer resection, however, invasiveness of the cancer cells in such conditions was practically abolished by NK4. Consistently, the ascitic fluid in patients who had undergone pancreatic cancer surgery contained high levels of HGF. These findings mean that HGF is probably involved in invasion, dissemination, and metastasis of pancreatic cancer, particularly through tumour-stromal interaction and after resection of the pancreatic cancer. NK4, an effective antagonist of HGF, may prove to have the potential for anti-invasion/metastasis. © 2001 Cancer Research Campaign http://www.bjcancer.com