Treatment Of Pancreatic Cancer Cells Research Articles

Dimethylaminoparthenolide and gemcitabine: a survival study using a genetically engineered mouse model of pancreatic cancer

BackgroundPancreatic cancer remains one of the deadliest cancers due to lack of early detection and absence of effective treatments. Gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer, has limited clinical benefit. Treatment of pancreatic cancer cells with gemcitabine has been shown to induce the activity of the transcription factor nuclear factor-kappaB (NF-κB) which regulates the expression of genes involved in the inflammatory response and tumorigenesis. It has therefore been proposed that gemcitabine-induced NF-κB activation may result in chemoresistance. We hypothesize that NF-κB suppression by the novel inhibitor dimethylaminoparthenolide (DMAPT) may enhance the effect of gemcitabine in pancreatic cancer.MethodsThe efficacy of DMAPT and gemcitabine was evaluated in a chemoprevention trial using the mutant Kras and p53-expressing LSL-KrasG12D/+; LSL-Trp53R172H; Pdx-1-Cre mouse model of pancreatic cancer. Mice were randomized to treatment groups (placebo, DMAPT [40 mg/kg/day], gemcitabine [50 mg/kg twice weekly], and the combination DMAPT/gemcitabine). Treatment was continued until mice showed signs of ill health at which time they were sacrificed. Plasma cytokine levels were determined using a Bio-Plex immunoassay. Statistical tests used included log-rank test, ANOVA with Dunnett’s post-test, Student’s t-test, and Fisher exact test.ResultsGemcitabine or the combination DMAPT/gemcitabine significantly increased median survival and decreased the incidence and multiplicity of pancreatic adenocarcinomas. The DMAPT/gemcitabine combination also significantly decreased tumor size and the incidence of metastasis to the liver. No significant differences in the percentages of normal pancreatic ducts or premalignant pancreatic lesions were observed between the treatment groups. Pancreata in which no tumors formed were analyzed to determine the extent of pre-neoplasia; mostly normal ducts or low grade pancreatic lesions were observed, suggesting prevention of higher grade lesions in these animals. While gemcitabine treatment increased the levels of the inflammatory cytokines interleukin 1α (IL-1α), IL-1β, and IL-17 in mouse plasma, DMAPT and DMAPT/gemcitabine reduced the levels of the inflammatory cytokines IL-12p40, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 beta (MIP-1β), eotaxin, and tumor necrosis factor-alpha (TNF-α), all of which are NF-κB target genes.ConclusionIn summary, these findings provide preclinical evidence supporting further evaluation of agents such as DMAPT and gemcitabine for the prevention and treatment of pancreatic cancer.

Abstract 1660: Therapeutic targeting cross talk between ROS/autophagy and apoptosis for pancreatic cancer by a herbal extract.

Abstract 5-year survival for pancreatic cancer (PanCA) continuous to be lower than any other solid tumor due to late stage diagnosis combined with lack of effective therapeutic options. Therefore developing novel strategies possibly based on molecular targets is critical for successful management of PanCA. Recent studies from our laboratory show potential anti-pancreatic cancer activity for NexrutineR (Nx), a bark extract isolated from traditional Chinese medicine Phellodendron amurense. These studies also show that Nx reduces pancreatic fibrosis in preclinical animal model through targeting two critical oncogenic transcription factors including NF-κB and Stat3. Nonetheless, the precise molecular mechanism associated with the observed biological activity is unclear. We investigated whether Nx inhibits pancreatic cell proliferation and pancreatic fibrosis via reactive oxygen species (ROS)-mediated activation of autophagy. Our data show that Nx can induce autophagy at earlier time points (4h) but inhibit autophagy at later time points (24h) in human pancreatic cancer cell lines Capan-2 and BxPC-3 as evidenced by autophagosome formation, alteration in the expression and levels of various autophagy related genes. Interestingly Nx also induced apoptosis at 24h time point but not at 4h suggesting that autophagy precedes apoptosis. Further under these experimental conditions, treatment of pancreatic cancer cells with Nx enhanced intracellular production of ROS implicating a cross talk between Nx-induced ROS, autophagy and apoptosis. Overall, our findings divulge an important role for ROS/autophagy/apoptosis in Nx-mediated anti-pancreatic cancer effects. Supported by NCCAM (APK). Citation Format: Gong Jingjing, Addanki P. Kumar. Therapeutic targeting cross talk between ROS/autophagy and apoptosis for pancreatic cancer by a herbal extract. [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 1660. doi:10.1158/1538-7445.AM2013-1660

Genistein Inhibits Cell Growth and Induces Apoptosis Through Up-regulation of miR-34a in Pancreatic Cancer Cells

Pancreatic cancer (PC) is the fourth most common cause of cancer-related deaths in the United States, suggesting that designing novel therapeutic strategy is required to improve the survival outcome of patients diagnosed with PC. Recently, microRNAs (miRNA) have been found to be involved in the regulation of multiple aspects of tumor development and progression including PC. In this study, we investigate whether miR-34a plays a critical role in the control of cell growth and apoptosis in PC cells. We found that Re-expression (forced expression) of miR-34a inhibits cell growth and induces apoptosis, with concomitant down-regulation of Notch-1 signaling pathway, one of the target of miR-34a. Moreover, treatment of PC cells with a natural compound genistein led to the up-regulation of miR-34a, resulting in the down-regulation of Notch-1, which was correlated with inhibition of cell growth, and induction of apoptosis. Our findings suggest that genistein could function as a non-toxic activator of a miRNA that can suppress the proliferation of PC cells.

Hypoxia-Induced Aggressiveness of Pancreatic Cancer Cells Is Due to Increased Expression of VEGF, IL-6 and miR-21, Which Can Be Attenuated by CDF Treatment

Hypoxia is known to play critical roles in cell survival, angiogenesis, tumor invasion, and metastasis. Hypoxia mediated over-expression of hypoxia-inducible factor (HIF) has been shown to be associated with therapeutic resistance, and contributes to poor prognosis of cancer patients. Emerging evidence suggest that hypoxia and HIF pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation-all which lead to therapeutic resistance. However, the precise molecular mechanism(s) by which hypoxia/HIF drives these events are not fully understood. Here, we show, for the first time, that hypoxia leads to increased expression of VEGF, IL-6, and CSC signature genes Nanog, Oct4 and EZH2 consistent with increased cell migration/invasion and angiogenesis, and the formation of pancreatospheres, concomitant with increased expression of miR-21 and miR-210 in human pancreatic cancer (PC) cells. The treatment of PC cells with CDF, a novel synthetic compound inhibited the production of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 and miR-210 under hypoxia. CDF also led to decreased cell migration/invasion, angiogenesis, and formation of pancreatospheres under hypoxia. Moreover, CDF decreased gene expression of miR-21, miR-210, IL-6, HIF-1α, VEGF, and CSC signatures in vivo in a mouse orthotopic model of human PC. Collectively, these results suggest that the anti-tumor activity of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become a novel, and effective anti-tumor agent for PC therapy.

Pristimerin Causes G1 Arrest, Induces Apoptosis, and Enhances the Chemosensitivity to Gemcitabine in Pancreatic Cancer Cells

Despite rapid advances in chemotherapy and surgical resection strategies, pancreatic cancer remains the fourth leading cause of cancer related deaths in the United States with a 5-year survival rate of less than 5%. Therefore, novel therapeutic agents for the prevention and treatment of pancreatic cancer are urgently needed. The aim of this study was to investigate the effect of pristimerin, a quinonemethide triterpenoid compound isolated from Celastraceae and Hippocrateaceae, on inhibition of cell proliferation and induction of apoptosis in three pancreatic cancer cells, BxPC-3, PANC-1 and AsPC-1, in both monotherapy and in combination with gemcitabine. Treatment with pristimerin decreased the cell proliferation of all three pancreatic cancer cells in a dose- and time-dependent manner. Treatment of pancreatic cancer cells with pristimerin also resulted in G1-phase arrest which was strongly associated with a marked decrease in the level of cyclins (D1 and E) and cyclin-dependent kinases (cdk2, cdk4 and cdk6 ) with concomitant induction of WAF1/p21 and KIP1/p27. Pristimerin treatment also resulted in apoptotic cell death, cleavage of caspase-3, modulation in the expressions of Bcl-2 family proteins, inhibition of the translocation and DNA-binding activity of NF-κB. In addition, pristimerin potentiated the growth inhibition and apoptosis inducing effects of gemcitabine in all three pancreatic cancer cells, at least in part, by inhibiting constitutive as well as gemcitabine-induced activation of NF-κB in both its DNA-binding activity and transcriptional activity. Taken together, these data provide the first evidence that pristimerin has strong potential for development as a novel agent against pancreatic cancer.

Targeting the MAGE A3 antigen in pancreatic cancer

Pancreatic cancer is the fourth-leading cause of death in the United States and one of the most aggressive known malignancies. New and innovative advances in treatment are desperately needed. One promising area of investigational treatment for pancreatic cancer involves the use of immunotherapy. The development of immunotherapy for pancreatic cancer has been hampered by difficulty in generating tumor-reactive lymphocytes from resected specimens and by a lack of appropriate target antigens expressed on tumor cells. Innovative strategies have been developed with the use of peripheral blood lymphocytes that are genetically engineered to express T-cell receptors targeting common tumor antigens, including cancer-testis antigens, such as the MAGE-A3 antigen. Cancer-testis antigens pose excellent targets for immunotherapy because they are expressed in cancer and in the testis, an immune-privileged site, but have limited expression in normal tissue. An additional advantage in targeting cancer-testis antigens for immunotherapy is that their expression can be selectively up-regulated in tumor cells via epigenetic regulation with chromatin remodeling agents. Current interest in targeting cancer-testis antigens in pancreatic cancer is well-founded because cancer-testis antigens have been shown to be expressed in pancreatic cancer as potential targets for therapy. In our studies, we validated the expression pattern of cancer-testis antigens in resected specimens of pancreatic cancer and tested the hypothesis that treatment of pancreatic cancer cells with chromatin remodeling agents would render them more sensitive to antigen-specific T lymphocytes. We focused predominately on the MAGE-A3 antigen because it is highly expressed in pancreatic cancer, and several immunotherapeutic strategies are in clinical trials targeting this specific antigen. The results of these studies have important translational implications and provide the rationale for combined treatment with chromatin remodeling agents and immunotherapeutic approaches for pancreatic cancer.

SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of β-catenin

Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of β-catenin, we postulated that β-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target β-catenin in a colon cancer model, suppresses β-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of β-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced β-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of β-catenin. Treatment with MG132, a proteasomal inhibitor, restored β-catenin protein levels, suggesting that SIRT1-mediated degradation of β-catenin requires proteasomal activity. It was reported that inhibition of GSK-3β or Siah-1 stabilizes β-catenin in colon cancer cells, but suppression of GSK-3β or Siah-1 using siRNA in the presence of resveratrol instead diminished β-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3β and Siah-1 are not involved in SIRT1-mediated degradation of β-catenin in the cells. Finally, activation of SIRT1 inhibited the proliferation of Panc-PAUF cells by down-regulation of cyclin-D1, a target molecule of β-catenin. These results suggest that SIRT1 activation may be a therapeutic strategy for treatment of pancreatic cancer cells that express PAUF via the down-regulation of β-catenin.

BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1

We report that Binder of Arl Two (BART) plays a role in inhibiting cell invasion by regulating the activity of the Rho small guanosine triphosphatase protein Rac1 in pancreatic cancer cells. BART was originally identified as a binding partner of ADP-ribosylation factor-like 2, a small G protein implicated as a regulator of microtubule dynamics and folding. BART interacts with active forms of Rac1, and the BART-Rac1 complex localizes at the leading edges of migrating cancer cells. Suppression of BART increases active Rac1, thereby increasing cell invasion. Treatment of pancreatic cancer cells in which BART is stably knocked down with a Rac1 inhibitor decreases invasiveness. Thus, BART-dependent inhibition of cell invasion is likely associated with decreased active Rac1. Suppression of BART induces membrane ruffling and lamellipodial protrusion and increases peripheral actin structures in membrane ruffles at the edges of lamellipodia. The Rac1 inhibitor inhibits the lamellipodia formation that is stimulated by suppression of BART. Our results imply that BART regulates actin-cytoskeleton rearrangements at membrane ruffles through modulation of the activity of Rac1, which, in turn, inhibits pancreatic cancer cell invasion.

Abstract 1614: Grape seed proanthocyanidins inhibit pancreatic cancer cell growth in vitro and in vivo through induction of apoptosis and inhibition of PI3K/Akt pathway

Abstract The American Cancer Society reported that 35,420 Americans died in 2009 because of pancreatic cancer. It is an aggressive malignancy that is frequently diagnosed at an advanced stage with poor prognosis. Phosphoinositide 3-kinase (PI3K)/Akt is a potent survival pathway that may mediate resistance to chemotherapeutic drugs and conventional therapies in pancreatic cancer patients. Thus, PI3K/Akt is considered as a promising therapeutic target for this cancer. Dietary phytochemicals offer promising options for the development of effective strategies for the prevention of cancer, and thus can be utilized as complementary and alternative medicine. Therefore, to develop newer and more effective chemotherapeutic agent for pancreatic cancer, we have examined the effect of grape seed proanthocyanidins (GSPs) on the growth of pancreatic cancer cell lines and the molecular mechanisms underlying this effect using PANC-1, AsPC-1 and Miapaca-2 cell lines as a model. The effect of GSPs on pancreatic cancer cell lines was studied on cell proliferation, cell cycle regulation, apoptosis and PI3K/Akt pathway using MTT assay, western blotting and FACS analysis. In vitro treatment of pancreatic cancer cells (Miapaca-2, PANC-1, and AsPC-1) with GSPs resulted in: (i) growth inhibition, which was associated with G2/M-phase arrest, and includes the inhibition of the expressions of cyclin B1, Cdc25B and Cdc25c proteins. (ii) Induction of apoptosis of pancreatic cancer cells by GSPs was associated with the enhanced expression of Bax, and caspase-3 activation while reduced levels of anti-apoptotic proteins (Bcl2, Bcl-xl). (iii) Blocking of PI3K/Akt pathway. Exposure of Miapaca-2 and PANC-1 cancer cells to PI3K inhibitor (wortmannin) also resulted in a dose-dependent induction of apoptosis in these cells. The in vitro data were further verified using in vivo tumor xenograft model. Administration of GSPs (0.2% and 0.5%, w/w) as a supplement of an AIN76A control diet resulted in a dose-dependent inhibition of the growth of Miapaca-2 tumor xenografts in athymic nude mice (35-65%; P<0.05-0.01) compared to non-GSPs-treated controls. The growth inhibitory effect of dietary GSPs on the pancreatic xenograft tumors was associated with the induction of apoptotic cell death of tumor cells, and induction of Bax expression and activation of caspase-3 while decreased the expression of anti-apoptotic proteins. Further, the levels of phospho-Akt and the proteins of PI3K family (PI3K 110 and PI3K 85) were markedly decreased in the xenograft tumors treated with GSPs compared with controls. Together, this preclinical study provides evidence that the chemotherapeutic effect of GSPs on the growth of pancreatic cancer cells in vitro and in vivo is mediated, at least in part, through the induction of apoptosis, G2/M phase arrest and blocking of PI3K/Akt cell survival pathway. 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 1614. doi:1538-7445.AM2012-1614

Abstract 1195: Erk activity promotes Notch-dependent HES1 expression in pancreatic cancer cells

Abstract The oncogene Ras is well-recognized for its involvement in tumorigenesis in part through activation of downstream pathways including the PI3K/Akt and Mek/Erk pathways. Recent studies have suggested that the ability of Ras to transform cells also depends on cooperation with the Notch signalling pathway. However, the molecular and cellular mechanisms involved in this cooperation remain largely unknown. The aim of this study was to evaluate whether specific activation of the Ras/Raf/Mek/Erk pathway impacts Notch-dependent signalling. METHODS. Experiments were done using the pancreatic cancer cells MIA PaCa-2 and BxPC-3. Both cell lines express endogenous levels of the activated Notch1 (NIC). To inhibit Mek/Erk activity, cells were treated with the Mek inhibitor U0126 (10uM). Activation of the Mek/Erk pathway was achieved by treatment with phorbol 12-myristate 13-acetate (PMA, 100nM). The hes1-luciferase reporter gene was used to evaluate Notch-dependent transcriptional activity. The gamma-secretase inhibitor DAPT (25µM) was used to inhibit Notch activation. RESULTS. 1- Treatment of pancreatic cancer cells with U0126 reduced the activity of the hes1-luciferase reporter gene to the same extent than treatment with the gamma-secretase inhibitor DAPT. 2- Combined treatment (U0126 + DAPT) did not result in an additive inhibition of hes1-luciferase activity suggesting a Notch-dependent impact of the Mek inhibitor on hes1 transcription. 3- Increased HES1 mRNA expression levels were observed following treatment with PMA, an effect prevented by addition of U0126. 4- Massive activation of the Notch receptors by treatment with EGTA promoted HES1 protein expression. 5- Interestingly, strong stimulation of Erk activity by PMA promoted the EGTA-induced HES1 protein expression. 6- Conversely, inhibition of Erk activity completely prevented both the EGTA-induced and EGTA+PMA-induced HES1 protein expression. CONCLUSION. Taken together, our results suggest that Erk activity promotes Notch-dependent transcriptional activity and consequently promotes the expression of Notch target genes such as HES1. 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 1195. doi:1538-7445.AM2012-1195

Immunotherapy Synergizes with Chemotherapy Targeting Pancreatic Cancer

Pancreatic adenocarcinoma is a devastating disease and has an extremely poor prognosis. Despite aggressive treatment approaches, including surgery, chemotherapy and radiation, the overall 5‐year survival rate is less than 5%. Therefore, the development of therapeutic strategies for advanced pancreatic cancer has traditionally been considered particularly challenging to improve clinical outcomes. Although immunotherapy that is designed to target tumor-associated antigens (TAAs) is a promising treatment approach, immunotherapy alone is limited by the number of cytotoxic T lymphocytes (CTLs) able to penetrate the large established pancreatic tumor. Even if large numbers of antigen-specific polyclonal CTLs were generated in vitro and injected into the patients, CTLs cannot penetrate into tumor sites because of stroma cells such as cancer-associated fibroblasts, tolerogenic dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), immunosuppressive tumor-associated macrophages and Tregs. Moreover, these cells produce immunosuppressive cytokines such as IL-10 and TGF-β; thus, clinical responses by immunotherapy alone cannot induce efficient antitumor immunity in patients with advanced pancreatic cancer. On the other hand, cytotoxic chemotherapy is well known to blunt immune responses, because of its toxicity for dividing cells including peripheral lymphoid tissue as well as the bone marrow. However, increasing evidence has been mounting to suggest that immunotherapy has the possibility of achieving better success when used in combination with chemotherapy [1]. For example, necrotic or apoptotic tumor cells induced by chemotherapy can be phagocytosed by DCs that are potent antigen-presenting cells, processed and presented to immune lymphocytes, followed by induction of antitumor immune responses. Different chemotherapeutic agents may kill tumor cells through an apparently homogeneous apoptotic pathway. Of note, treatment of pancreatic cancer cells with a standard cytotoxic agent for pancreatic cancer, gemcitabine, results in enhanced cross-presentation of TAAs by DCs and CTL induction [2]. Moreover, gemcitabine can also inhibit Tregs, B cells and MDSCs [2,3], but induce the proliferation of DCs [4]. These phenomena suggest that gemcitabine induces efficient CTL responses, improves the penetration of CTLs into the tumor parenchyma, and enhances tumor cell sensitivity to lyze antigen-specific CTLs. Thus, immunotherapy may mediate a potent antitumor effect when c ombined with chemotherapy.

Abstract 4100: HuR regulates death receptor-5 (DR5, TRAIL-R2)-targeted treatment of pancreatic cancer cells

Abstract Apoptosis has been identified as a core signaling pathway disrupted in pancreatic ductal adenocarcinoma (PDA) tumorigenesis. Death Receptor 5 (DR5, TRAIL-R2) is a membrane bound protein that initiates the extrinsic apoptotic pathway upon ligand exposure and is currently being explored as a ‘druggable’ target in multiple cancers including PDA. Identifying a mechanism that regulates DR5 in the tumor microenvironment (e.g. hypoxia, chemotherapeutic exposure) is critical for optimizing DR5 based-therapies. Human antigen R (HuR), an RNA binding protein, controls post-transcriptional gene expression by binding to specific regions of 3’and 5’ UTRs of mRNA target genes. Previously, HuR, a pro-survival molecule, has been shown to play an important role in the intrinsic apoptotic pathway. We identified DR5 mRNA as a HuR target in PDA cells and explored the significance of HuR's role in functionally regulating the extrinsic apoptotic pathway in PDA cells. We also explored HuR as a modulator of DR5-targeted therapy for the treatment of PDA. Ribonucleoprotein immunoprecipitation (RNP-IP) assays were performed on PDA cells using HuR antibody (Ab) compared to a control (IgG Ab) under stress conditions, 3 hours with 1μM of the standard of care drug for PDA, gemcitabine; and 75 μM of a PARP inhibitor (PARPi). mRNA was converted to cDNA using RT-PCR, and then analyzed by qPCR. DR5 mRNA was validated as a HuR target with a 6-fold greater binding to HuR compared to the control. Strikingly, this binding increases 12- and 24-fold upon treatment with gemcitabine and the PARPi respectively. Silencing HuR expression, through siRNA transfections, leads to an increase of DR5 protein expression at 24 and 48 hours in multiple PDA cell lines. Additionally, silencing of HuR significantly enhances the action of a DR5-specific monoclonal Ab (0.8 mg/mL) against PDA cells within 36 hours (a 20% detected increase in cell death compared to control cells), most likely due to an enhanced availability of the DR5 receptor. Finally, in a training set of PDA clinical specimens, we found a significant inverse correlation between high/low HuR cytoplasmic expression and low/high DR5 levels (p value=0.03). In over 80% (26 of 31) of the specimens HuR cytoplasmic levels inversely correlated with DR5 expression levels, providing further evidence that elevated cytoplasmic HuR is repressing DR5 protein levels in patient tumor cells. In sum, we have shown that ‘activated HuR’ represses DR5 protein expression in PDA cells. Therefore, we conclude that low cytoplasmic HuR levels allow for greater availability of the target DR5, and will thus accordingly enhance the efficacy of DR5-targeted therapy. Thus, manipulating and/or utilizing HuR expression levels may serve as a clinically informative tool for optimizing DR5-targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4100. doi:10.1158/1538-7445.AM2011-4100

Role of endogenous cholecystokinin on growth of human pancreatic cancer

Cholecystokinin (CCK) and gastrin stimulate growth of pancreatic cancer. Although down-regulation of gastrin inhibits growth of pancreatic cancer, the contribution of endogenous CCK to tumor growth is unknown. The purpose of this study was to evaluate the role of endogenous CCK on autocrine growth of pancreatic cancer. Pancreatic cancer cell lines were analyzed for CCK mRNA and peptide expression by real-time RT-PCR and radioimmunoassay, respectively. The effect of endogenous CCK on growth was evaluated by treating cancer cells with CCK neutralizing antibodies and by down-regulating CCK mRNA by RNAi. Wild-type pancreatic cancer cells expressed significantly lower CCK mRNA and peptide levels than gastrin. Neither treatment of pancreatic cancer cells with CCK antibodies nor the down-regulation of CCK mRNA and peptide by shRNAs altered growth in vitro or in vivo. Conversely, when gastrin mRNA expression was down-regulated, the same cells failed to produce tumors in spite of having sustained levels of endogenous CCK. Pancreatic cancer cells produce CCK and gastrin; however, the autocrine production of gastrin is more important for stimulating tumor growth.

Loss of tyrosine phosphatase‐dependent inhibition promotes activation of tyrosine kinase c‐Src in detached pancreatic cells

Despite an intense focus on novel therapeutic strategies, pancreatic adenocarcinoma remains one of the deadliest human malignancies. The frequent and rapid mortality associated with pancreatic cancer may be attributed to several factors, including late diagnosis, rapid tumor invasion into surrounding tissues, and formation of distant metastases. Both local invasion and metastasis require disruption of tumor cell contacts with the extracellular matrix. Detachment of normal cells from the extracellular matrix leads to a form of programmed cell death termed anoikis. Pancreatic cancer cells avert anoikis by activation of signaling pathways that allow for adhesion-independent survival. In the present studies, cellular signaling pathways activated in detached pancreatic cancer cells were examined. We demonstrate a rapid and robust activation of Src kinase in detached pancreatic cancer cells, relative to adherent. Src autophosphorylation rapidly returned to baseline levels upon reattachment to tissue culture plastic, in the presence or absence of specific extracellular matrix proteins. Treatment of pancreatic cancer cells with tyrosine phosphatase inhibitors increased steady-state Src autophosphorylation in adherent cells and abrogated the detachment-induced increase in Src autophosphorylation. Src was found to co-immunoprecipitate with the Src homology 2 (SH2) domain containing protein tyrosine phosphatase (SHP-2) in pancreatic cancer cells, suggesting that SHP-2 may participate in regulation of Src autophosphorylation in adherent cells. Src family kinase (SFK) dependent increases in Akt and Jun N-terminal kinase (JNK) phosphorylation were observed in detached cells, indicating the potential for Src-dependent activation of survival and stress pathways in pancreatic cancer cells that have detached from the extracellular matrix.

The role of autophagy in the treatment of pancreatic cancer with gemcitabine and ionizing radiation

Autophagy has recently emerged as a significant mechanism in cancer treatment. Although gemcitabine and/or ionizing radiation are important modalities in the treatment of pancreatic cancer, the contribution of autophagy in such treatment has not been fully elucidated. This study investigated the role of autophagy in the treatment of pancreatic cancer with gemcitabine and ionizing radiation. To evaluate the effect of gemcitabine and/or ionizing radiation on autophagy, several human pancreatic cancer cell lines were used. The treatment of pancreatic cancer cell cultures in vitro and in vivo with gemcitabine and ionizing radiation resulted in synergistic cytotoxicity. After treatment with gemcitabine, the autophagy-related protein light chain 3-II (LC3-II) was upregulated. When gemcitabine was combined with ionizing radiation treatment, LC3-II upregulation was enhanced. In addition, electron microscopy of pancreatic cancer cells treated with gemcitabine and/or ionizing radiation detected the induction of autophagy. The blockage of autophagy by 3-methyladenine indicated that autophagy contributed to cell death after gemcitabine treatment and enhanced its cytotoxicity. The inhibitory effect and immune reactivity of the autophagy-related proteins LC3 and beclin-1 were the strongest after the combination treatment. In conclusion, these results suggest that autophagy can be activated by gemcitabine and/or ionizing radiation in the treatment of pancreatic cancer cells and that activated autophagy plays a role in cancer suppression. These findings may have important implications for future therapeutic strategies using gemcitabine and ionizing radiation against pancreatic cancer.

MiR-146a Suppresses Invasion of Pancreatic Cancer Cells

The aggressive course of pancreatic cancer is believed to reflect its unusually invasive and metastatic nature, which is associated with epidermal growth factor receptor (EGFR) overexpression and NF-kappaB activation. MicroRNAs (miRNA) have been implicated in the regulation of various pathobiological processes in cancer, including metastasis in pancreatic cancer and in other human malignancies. In this study, we report lower expression of miR-146a in pancreatic cancer cells compared with normal human pancreatic duct epithelial cells. Reexpression of miR-146a inhibited the invasive capacity of pancreatic cancer cells with concomitant downregulation of EGFR and the NF-kappaB regulatory kinase interleukin 1 receptor-associated kinase 1 (IRAK-1). Cellular mechanism studies revealed crosstalk between EGFR, IRAK-1, IkappaBalpha, NF-kappaB, and MTA-2, a transcription factor that regulates metastasis. Treatment of pancreatic cancer cells with the natural products 3,3'-diinodolylmethane (DIM) or isoflavone, which increased miR-146a expression, caused a downregulation of EGFR, MTA-2, IRAK-1, and NF-kappaB, resulting in an inhibition of pancreatic cancer cell invasion. Our findings reveal DIM and isoflavone as nontoxic activators of a miRNA that can block pancreatic cancer cell invasion and metastasis, offering starting points to design novel anticancer agents.

Curcumin Inhibits Constitutive STAT3 Phosphorylation in Human Pancreatic Cancer Cell lines and Downregulation of Survivin/BIRC5 Gene Expression

ABSTRACTThe purpose of this study was to determine the effect of curcumin on Survivin/BIRC5 and on the role of signal transducer and activator of transcription 3 (STAT3) activation in Survivin/ BIRC5. We incubated two pancreatic cancer cell lines with different amounts of curcumin. This resulted in a downregulation of proliferation in all cell lines tested. The expression of Survivin/BIRC5 on mRNA and protein level was significantly downregulated and the phosphorylation of STAT3 was blocked. Treatment of pancreatic cancer cells with curcumin resulted in an induction of apoptosis. The results indicate that curcumin inhibits several key factors in cancer cellular pathways and may be of interest in pancreatic cancer.

Endocrine glands-derived vascular endothelial growth factor protects pancreatic cancer cells from apoptosis via upregulation of the myeloid cell leukemia-1 protein

Endocrine glands-derived vascular endothelial growth factor (EG-VEGF, also termed as Prok1)—a novel cytokine that selectively acts on the endothelial cells of endocrine glands—was recently reported to be involved in the regulation of tumor cell growth and survival. However, its roles in the regulation of pancreatic cancer progression remain unclear. In this report, we investigated the suppressive effects of EG-VEGF on pancreatic cancer cell apoptosis and the relevant mechanisms. By using reverse-transcriptase polymerase chain reaction (RT-PCR) we found that the Mia PaCa II cells of the pancreatic cancer cell line express the mRNAs of both EG-VEGF (Prok1) and its receptors. EG-VEGF protects pancreatic cancer cells from apoptosis through upregulation of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein of the bcl-2 family. Treatment of pancreatic cancer cells with EG-VEGF results in the rapid phosphorylation of mitogen-activated protein kinase (MAPK), STAT3, and AKT, which are involved in the upregulation of Mcl-1 expression. EG-VEGF (Prok1) protects Mia PaCa II cells from apoptosis through G protein-coupled receptor (GPR)-induced activation of multiple signal pathways, and hence can be a novel target for pancreatic cancer therapy.

From a Natural Product Lead to the Identification of Potent and Selective Benzofuran-3-yl-(indol-3-yl)maleimides as Glycogen Synthase Kinase 3β Inhibitors That Suppress Proliferation and Survival of Pancreatic Cancer Cells

Recent studies have demonstrated that glycogen synthase kinase 3beta (GSK-3beta) is overexpressed in human colon and pancreatic carcinomas, contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3beta inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3beta and an enhanced selectivity against cyclin-dependent kinase 2 (CDK-2). Selected GSK-3beta inhibitors were tested in the pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds, namely compounds 5, 6, 11, 20, and 26, demonstrate antiproliferative activity against some or all of the pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of pancreatic cancer cells with GSK-3beta inhibitors 5 and 26 resulted in suppression of GSK-3beta activity and a distinct decrease of the X-linked inhibitor of apoptosis (XIAP) expression, leading to significant apoptosis. The present data suggest a possible role for GSK-3beta inhibitors in cancer therapy, in addition to their more prominent applications in CNS disorders.

Wilms’ tumour gene 1 (WT1) as a target in curcumin treatment of pancreatic cancer cells

The transcription factor WT1 plays an important role in cellular proliferation and survival of various cancer cells, and is frequently expressed in pancreatic cancer. Curcumin has been shown to be a potentially effective agent in pancreatic cancer. In this context, the purpose of this study was to determine the role of WT1 in a curcumin-treated pancreatic cancer cell line. To study the effect of curcumin on the expression of WT1, we incubated the pancreatic cancer cell line PANC-1 with different amounts of curcumin. The expression of WT1 on mRNA and protein level was measured with real-time RT-PCR and Western blot analysis. The incubation of the pancreatic cancer cell line PANC-1 with curcumin resulted in an inhibition of cellular proliferation as measured with MTT assay. The expression of WT1 on mRNA and protein level was significantly down-regulated in a concentration-dependent manner after treatment with curcumin. The WT1 mRNA levels were decreased by 20%, 25%, 40%, 78% and 88% in response to 10, 20, 30, 40 and 50 μM curcumin. The use of small inhibitory RNA (siRNA) targeting WT1 down-regulated the expression of WT1 about 90%. Combined treatment with curcumin and siRNA targeting WT1 resulted in a significant inhibition of cell proliferation compared to curcumin-treated cells alone. In conclusion, WT1 is involved in cellular proliferation of PANC-1 cells. Targeting WT1 gene expression with siRNA may enhance the efficacy of curcumin to inhibit cell proliferation.