Cancer Cell Line MIA PaCa-2 Research Articles

Abstract 4738: The early response protein EGR-1 mediates vitamin E γ-tocotrienol-induced apoptosis in pancreatic cancer cells

Abstract Background: Vitamin E tocotrienols are bioactive components of cereal foods such as oats, barley, rice bran, and palm, that have demonstrated anticancer and chemopreventive activities in various types of cancer. Recently we have shown that γ-tocotrienol is the most bioactive tocotrienol against pancreatic cancer both in vitro as well as in vivo. However, the molecular mechanism of action of tocotrienols in pancreatic cancer has not been fully elucidated. In this work, we investigated the early genes associated with Vitamin E γ-tocotrienol induced apoptosis, and therefore identify novel pathways related with its anticancer effects. Methods: Human pancreatic cancer MiaPaca-2 cells were treated with γ-tocotrienol (50 µM) and vehicle (ethanol) for 3 h and were subjected to microarray analysis using costume Affymetrix for the identification of early regulatory genes. Early growth response protein-1 (EGR-1) was one of those early response genes induced by γ-tocotrienol. Then we evaluated the protein expression of EGR-1 by real-time PCR and western-blot in pancreatic cancer cell lines (MiaPaca-2 and Panc-1) after treatment with γ-tocotrienol. Mechanistic studies were conducted trough siRNA knockdown of EGR-1 and the rescue of Vitamin of γ-tocotrienol-induced apoptosis, anti-proliferative effects and signaling pathways. Results: We identified 8 genes that were induced and 8 genes that were suppressed early after treatment with γ-tocotrienol in MiaPaca-2 cells. Then we selected and focused on the EGR-1 for signaling pathway analysis. Induction of protein expression was noticed in both MiaPac-2 cells and Panc-1 (6 fold and 10 fold respectively;p<0.05) after 24 h γ-tocotrienol treatment. γ-tocotrienol-induced apoptosis in MiaPac-2 cells and Panc-1 cells (PARP1 cleavage and Annexin V) was associated with induction of p-JNK, p-C-JUN, and EGR-1. Knockdown of EGR-1 in this two cell lines partially rescued γ-tocotrienol-induced apoptosis. Conclusion: Our data identifies EGR-1 as an early gene which is induced by Vitamin E γ-tocotrienol in pancreatic cancer cells. Activation of the EGR-1 pathway, may constitute a novel potential strategy for inhibiting pancreatic tumor growth. 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 4738. doi:1538-7445.AM2012-4738

Abstract 1402: A xenograft pancreatic cancer mouse model to study the function of extracellular DNA in metastasis

Abstract Pancreatic cancer is one of the most lethal cancers with the lowest survival rate of all solid cancers. This is largely due to its highly aggressive metastasis, which almost always leads to death. Understanding the mechanisms and the process of pancreatic cancer metastasis is critical towards its prevention and treatments. Although the etiology of pancreatic cancer is not well understood, the relationship between inflammation and the development of this cancer has been well recognized. Extracellular DNA (exDNA) is a newly discovered component associated with inflammation. exDNA production can be induced by interleukin 8 (IL-8), one of the major inflammatory and angiogenic cytokine. IL-8 exhibits a significantly elevated level in pancreatic cancer cell lines MiaPaCa-2 and BxPC3 and has been shown to play a role in stimulating pancreatic tumorigenesis and metastasis. With this background, we initiated a study to examine the presence of exDNA associated with pancreatic cancer cells. We have discovered a large amount of extracellular DNA associated with cultured pancreatic cancer cells but not with the normal pancreatic cells. Here, we tested the hypothesis that pancreatic cancer cells produce extracellular DNA to promote their metastasis. Our in vitro study showed that removal of the exDNA by DNase I inhibited metastasis-associated characteristics of cancer cells including cell migration, invasion, and adhesion. To test our hypothesis in vivo, we generated the orthotopic xenograft pancreatic cancer mouse model. We used the pancreatic cancer cell line MiaPaCa-2/LucE expressing luciferase to induce tumors. We assessed the effect of DNase I treatment on pancreatic cancer metastasis from pancreases, the primary tumor site, to different organs. Tumors were examined directly in size and weight, and indirectly by the bioluminescent signal intensity that corresponds to the tumor size. Our preliminary data showed that continuous applications of a low dosage of DNase I reduced the tumor incidences and the tumor size measured by the chemiluminescence in all the organs examined. When mouse tumor tissues were stained with DAPI, a DNA-specific fluorescent dye and examined through immunohistochemistry (IHC) with DNA-specific antibody, high level of exDNA was found in the tumor tissues. More exDNA was found to be associated with the inflammatory area in the tumor tissues. We conclude that extracellular DNA play a novel role in metastatic behaviors of pancreatic cancer cells in vitro and facilitate metastasis in vivo. To our knowledge, this is the first report on the presence of exDNA associated with pancreatic cancer tissues and the first report on the inhibition of DNase I treatment on pancreatic cancer metastasis. Our results open new avenues for understanding the metastasis of pancreatic cancer, and facilitate the identification of new drug targets and development of new treatments. 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 1402. doi:1538-7445.AM2012-1402

Abietane diterpenes induce cytotoxic effects in human pancreatic cancer cell line MIA PaCa-2 through different modes of action

Abietane diterpenes, especially those containing quinone moieties, are often reported to have cytotoxic effects on cancer cell lines. They deserve greater attention because several cancer chemotherapeutic agents also possess the quinone structural feature. To date, very little is known about their cytotoxic molecular modes of action. In the present study, five diterpenes, 7 alpha-acetoxyroyleanone, horminone, royleanone, 7-ketoroyleanone and sugiol which have been previously isolated from the medicinal plant Peltodon longipes were shown to possess cytotoxic activity against the human pancreatic cancer cell line MIA PaCa-2. 7 alpha-Acetoxyroyleanone, horminone and royleanone were demonstrated to possess alkylating properties using the nucleophile 4-(4-nitrobenzyl)pyridine. However, no clear correlation between the alkylating properties and cytotoxicity of these diterpenes was observed. Furthermore, the relaxation activity of human DNA topoisomerases I and II was found to be influenced by these compounds, with 7-ketoroyleanone and sugiol being the most active. These two diterpenes preferentially inhibited topoisomerase I and exhibited lower IC50 values than the classical topoisomerase I inhibitor camptothecin. Molecular docking studies revealed possible interactions of diterpenes with topoisomerase I, indicating that these compounds do not form the drug–enzyme–DNA covalent ternary complex as observed with camptothecin. A binding pocket located at the surface of the DNA-interaction site was proposed. Moreover, the ability of the five diterpenes to generate DNA-strand breaks in single cells was confirmed using the alkaline comet assay. As expected, these diterpenes also influenced cell cycle progression and arrested cells in different phases of the cell cycle, primarily the G1/G0 and S-phases. Interestingly, the diterpenes only exhibited a slight ability to induce apoptotic cell death and failed to generate intracellular reactive oxygen species. These results provide additional understanding of the cytotoxic effects of abietane diterpenes. Depending on their functional groups, we propose that abietane diterpenes utilise different mechanisms to induce cell death.

Paradoxical cross-talk between the Stat3 and MAPK pathways in CCL25-CCR9 mediated pancreatic cancer growth and proliferation.

231 Background: Chemokine receptors have been shown to regulate the progression of several malignancies. Signal transducer and activator of transcription 3 (Stat3) may contribute to the invasive phenotype of several human cancers, but its upstream signals have not been well characterized. Our objective was to investigate the relationship between the CCL25-CCR9 axis and Stat3 signaling in pancreatic cancer cells. Methods: We exposed two established human pancreatic cancer cell lines PANC-1 and MIAPaCa-2 to the cytokine CCL25 (800 ug/uL for 20 min) and measured the activation of Stat3 (phosho-Stat3) by Western blot assay. Stattic, a small molecule Stat3 inhibitor, was used (20uM) to antagonize Stat3 signaling. We also measured activation level of extracellular signal-regulated kinase (phospho-ERK) following CCL25 exposure and used UO126 (10uM), a small molecule MEK inhibitor, to antagonize the MAPK pathway. Changes in cell proliferation were measured by CellTiter Glo Fluorescence assay. Results: Constitutive phosphorylation of Stat3 was observed in both pancreatic cancer cell lines. Exposure of MIAPaCa-2 to CCL25 further increased phospho-Stat3 levels on Western blot assay. We also observed a concomitant increase in phospho-ERK levels with exposure to CCL25 in both cell lines. Exposure of pancreatic cancer cells to CCL25 significantly increased cell proliferation. To determine the mechanism of CCR9-mediated cell proliferation, we used stattic and UO126 to specifically inhibit Stat3 and MEK activation, respectively. Interestingly, pre-treatment with stattic prior to CCL25 exposure resulted in a paradoxical enhancement of phospho-ERK levels. Conversely, inhibition of the MAPK pathway with UO126 led to a paradoxical enhancement of phospho-Stat3 levels. Conclusions: Our results demonstrate that CCL25 activates Stat3 and MAPK pathways to contribute to pancreatic cancer proliferation. We also show potential cross-talk between Stat3 and MAPK pathways, wherein antagonism of one pathway resulted in paradoxical activation of the other pathway. Our findings suggest that therapeutic targeting of downstream pathways may require a multi-drug approach.

Anti Reverse Transcriptase and Anticancer activity of stem ethanol extracts of <i>Excoecaria agallocha</i> (Euphorbiaceae)

Excoecaria agallocha L. (Euphorbiaceae) is a mangrove plant widely used in folklore medicine for the treatment of several diseases. In the present study anti HIV and anticancer properties of active fraction of stem ethanol extracts of the plant were investigated. The fraction showed significant anti-reverse transcriptase activity in the enzyme-based direct binding assay which was as good as that of the standard synthetic inhibitor. Anticancer activity of the same fraction was determined using MTS in vitro assay. It showed potent cytotoxicity against pancreatic cancer cell lines Capan-1 and Miapaca-2 with IC50 values of 4 μg/ml and 7 μg/ml respectively. These results clearly indicate strong anti HIV and anticancer properties in stem extracts of E. agallocha. Key words: mangrove; pancreatic cancer; activity guided fractionation; anti HIV properties DOI: http://dx.doi.org/10.4038/cjsbs.v40i2.3931 CJSBS 2011; 40(2): 147-155

Abstract A31: Differential processing of let-7a precursors influences gemcitabine chemosensitivity in pancreatic cancer: Role of ribonucleotide reductase subunit M2

Abstract Ribonucleotide reductase subunit M2 (RRM2) involved in deoxyribonucleotide synthesis acts as a key determinant of chemoresistance to nucleoside drugs (e.g., gemcitabine) used in treating pancreatic cancer. While silencing RRM2 expression by synthetic means (e.g., siRNA) improves the chemosensitivity of pancreatic cancer cells to gemcitabine, targeting endogenous molecules to advance gemcitabine chemotherapeutic responses has never been explored. Based on existing evidence and computational predictions, we hypothesized that the let-7 tumor suppressor microRNAs will inhibit RRM2-mediated gemcitabine chemoresistance in pancreatic cancer. Reduced expression of the majority of let-7 miRNAs with an inverse relationship to RRM2 expression was identified in inherently gemcitabine-resistant pancreatic cancer cells. Direct binding of let-7 miRNA to the 3′-UTR of RRM2 transcripts identified post-transcriptional repression of RRM2 as a novel phenomenon influencing gemcitabine chemosensitivity. Intriguingly, overexpression of human precursor-let-7 miRNAs led to differential RRM2 expression and gemcitabine chemosensitization in a poorly differentiated gemcitabine-resistant pancreatic cancer cell line MIA PaCa-2. Defective processing of the let-7a precursors to mature forms explained, in part, the discrepancies observed with let-7a expressional outcomes. Screening putative regulators of let-7a biogenesis in MIA PaCa-2 cells identified LIN-28 and SET oncoprotein to differentially modulate let-7 biogenesis and chemosensitivity in gemcitabine-sensitive versus-resistant pancreatic cancer cells. Additionally, acquired gemcitabine chemoresistance was found to be independent of let-7 alterations. These data demonstrate an intricate post-transcriptional regulation of RRM2 expression by let-7 and that the manipulation of let-7 processing machinery may provide a mechanism for improving gemcitabine chemosensitivity in innately-resistant pancreatic cancer cells. Citation Format: Rajgopal Govindarajan. Differential processing of let-7a precursors influences gemcitabine chemosensitivity in pancreatic cancer: Role of ribonucleotide reductase subunit M2 [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A31.

Targeting X-linked inhibitor of apoptosis protein inhibits pancreatic cancer cell growth through p-Akt depletion

To determine whether lentivirus-mediated shRNA targeting the X-linked inhibitor of apoptosis protein (XIAP) gene could be exploited in the treatment of pancreatic cancer. Human pancreatic cancer cells Panc-1, Mia-paca2, Bxpc-3 and SW1990, infected with lentivirus, were analyzed by real-time polymerase chain reaction (PCR). Western blotting was used to examine XIAP protein levels, survivin and p-Akt to confirm the result of real-time PCR and determine the possible mechanism. The 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure IC₅₀ to determine chemosensitivity to the chemotherapeutic drugs 5-fluorouracil (5-FU) and gemcitabine. A colony assay, MTT assay and a tumorigenicity experiment were used to study cell proliferation in vitro and in vivo. Caspase-3/7 activity, 4',6-diamidino-2-phenylindole-staining and flow cytometric measurements were used to study apoptosis in SW1990 cells. XIAP proteins were found to be differentially expressed among pancreatic cancer cell lines Panc-1, Mia-paca2, Bxpc-3 and SW1990. Data of real-time PCR and Western blotting showed that XIAP was reduced persistently and markedly by lentivirus-mediated shRNA. Downregulation of XIAP by transfection with XIAP shRNA resulted in decreased p-Akt expression. XIAP shRNA also inhibited the growth of pancreatic cancer cells in vitro and in vivo, enhanced drug-induced apoptosis and increased chemosensitivity to 5-FU and gemcitabine. Results also suggest that inhibition of XIAP and subsequent p-Akt depletion may have an anti-tumor effect through attenuating the ability of cancer cells to survive. Lentivirus-mediated gene therapy is an attractive strategy in the treatment of pancreatic cancer and justifies the use of lentivirus in pancreatic cancer gene therapy studies.

Preparation and evaluation of a set of bis(methoxycarbonylmethylthio) heteroquinones as CDC25B phosphatase inhibitors

A set of new heteroquinone derivatives bearing two methoxycarbonylmethylthio groups on the benzoquinone ring were synthesized and evaluated for CDC25B phosphatase inhibitory activity. All compounds inhibited the enzyme with IC 50 values in the micromolar range regardless of the size and heteroatoms constituting the heterocycle fused to the quinone ring. Moreover, these quinonoid-based compounds showed moderate antiproliferative activity toward two cancer cell lines (HeLa and MiaPaca-2). These results provide additional data for CDC25 inhibition by quinone-type derivatives and highlight the importance of substituents on the quinonic moiety.

Gene Expression Analysis In Gemcitabine Resistant Cells Derived From Human Pancreatic Cancer Cell

To analyze the expression of genes involved in gemcitabine resistance formation in gemcitabineresistant cells derived from human pancreatic cancer cell, we established several cell lines with different acquired resistance to gemcitabine from pancreatic cancer cell line MIA-PaCa2, and analyzed the expression of the genes that are considered to be associated with the resistance to gemcitabine using PCR and real-time PCR. We found that in these gemcitabine-resistant cell lines, the expression of human equilibrative nucleoside transporter 2 (hENT2) and deoxycytidylate deaminase (DCTD) was lower than that of the parental cell line MIA-PaCa2 in the earlier stage of the cells incubated with 300nM gemcitabine; and, in the later stage, the expression of hENT2 recovered, but the expression levels of ribonucletide reductase subunit M1 (RRM1) and M2 (RRM2) were increased. These findings suggest that the decreased accumulation of gemcitabine and the increased expression of RRM1 and RRM2 are involved in gemcitabine-resistance formation in MIA-PaCa2.

KAI1-induced autophagy inhibiting apoptosis through the downregulation of Caspase-3 activation

Objective To explore the pathway of KAI1 induced autophagy regulating apoptosis in human pancreatic cancer cell line MiaPaCa-2.Methods There were three groups in the experiment,which were extracellular regulated protein kinases (ERK) phosphorylation inhibitor PD98059 pretreated group,Caspase-3 activation inhibitor VAD-FMK pretreated group and no PD98059 or VAD-FMK pretreated groups.And each group was divided into three sub groups with different treatment,which were adenovirus AD5-null vector infected control group,the human KAI1 gene recombinant adenovirus vector AD5 KAI1 infected group and autophagy inhibitor 3-MA pretreated and AD5-KAI1 infected group.The cell apoptosis was observed by AnnexinV-FITC/PI double staining.Caspase-3 activation level was evaluated by flow cytometry.ERK phosphorylation and poly(ADPribose) polymerase (PARP) cleavage were determined by Western blot.Results After the cancer cells infected with AD5 KAI1,KAI1 protein was expressed and GFP-LC3 green particles increased.Caspase-3 activation,PARP cleavage,ERK phosphorylation and apoptosis increased obviously.After autophagy inhibitor 3-MA pretreated,the percentage of apoptosis increased from (63.0 ± 7.9)％ to (88.0±4.5) ％ and Caspase-3 activation increased from (34.0±2.8) ％ to (44.2±4.0) ％ and PARP cleavage more.The apoptosis induced by 3-MA could be totally inhibited by Caspase-3 activation inhibitor VAD-FMK pretreated but could not be inhibited by ERK phosphorylation inhibitor PD98059.Conclusion KAI1- induced autophagy inhibits apoptosis through the downregulation of Caspase-3activation and PARP cleavage instead of ERK phosphorylation. Key words: Genes, Tumor Suppressor; Autophagy; Apoptosis; Caspase 3; Phosphorylation

Antitumor Indolequinones Induced Apoptosis in Human Pancreatic Cancer Cells via Inhibition of Thioredoxin Reductase and Activation of Redox Signaling

Indolequinones (IQs) were developed as potential antitumor agents against human pancreatic cancer. IQs exhibited potent antitumor activity against the human pancreatic cancer cell line MIA PaCa-2 with growth inhibitory IC(50) values in the low nanomolar range. IQs were found to induce time- and concentration-dependent apoptosis and to be potent inhibitors of thioredoxin reductase 1 (TR1) in MIA PaCa-2 cells at concentrations equivalent to those inducing growth-inhibitory effects. The mechanism of inhibition of TR1 by the IQs was studied in detail in cell-free systems using purified enzyme. The C-terminal selenocysteine of TR1 was characterized as the primary adduction site of the IQ-derived reactive iminium using liquid chromatography-tandem mass spectrometry analysis. Inhibition of TR1 by IQs in MIA PaCa-2 cells resulted in a shift of thioredoxin-1 redox state to the oxidized form and activation of the p38/c-Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathway. Oxidized thioredoxin is known to activate apoptosis signal-regulating kinase 1, an upstream activator of p38/JNK in the MAPK signaling cascade and this was confirmed in our study providing a potential mechanism for IQ-induced apoptosis. These data describe the redox and signaling events involved in the mechanism of growth inhibition induced by novel inhibitors of TR1 in human pancreatic cancer cells.

Antibacterial Activity and Cytotoxicity of Selected Egyptian Medicinal Plants

Medicinal plants have been used as a source of remedies since ancient times in Egypt. The present study was designed to investigate the antibacterial activity and the cytotoxicity of the organic extracts from 16 selected medicinal plants of Egypt. The study was also extended to the isolation of the antiproliferative compound jaeschkeanadiol p-hydroxybenzoate (FH-25) from Ferula hermonis. The microbroth dilution was used to determine the minimal inhibitory concentration (MIC) of the samples against twelve bacterial strains belonging to four species, Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli, while a resazurin assay was used to assess the cytotoxicity of the extracts on the human pancreatic cancer cell line MiaPaCa-2, breast cancer cell line MCF-7, CCRF-CEM leukemia cells, and their multidrug resistant subline, CEM/ADR5000. The results of the MIC determination indicated that all the studied crude extracts were able to inhibit the growth of at least one of the tested bacterial species, the best activity being recorded with the crude extracts from F. hermonis and Vitis vinifera, whichwere active against 91.7% and 83.3% of the studied bacteria, respectively. The lowest MIC value of 128 μg/mL was recorded against P. stuartii ATCC 29916 and E. coli ATCC 10536 with the extract from V. vinifera and Commiphora molmol, respectively. In the cytotoxicity study, IC50 values below 20 μg/mL were recorded for the crude extract of F. hermonis on all four studied cancer cell lines. FH-25 also showed good cytotoxicity against MCF-7 cells (IC50: 2.47 μg/mL). Finally, the results of the present investigation provided supportive data for the possible use of the plant extracts investigated herein, mostly F. hermonis and V. vinifera in the treatment of bacterial infections and jaeschkeanadiol p-hydroxybenzoate in the control of cancer diseases.

Abstract B63: Synergistic combination between resveratrol and tanshinone I for effective prevention of pancreatic cancer

Abstract Pancreatic cancer is the fourth most common cause of cancer-related mortality in the United States. Pancreatic cancer is often dubbed the silent killer because it seldom causes symptoms until advanced. As a result, few pancreatic cancers are operable at diagnosis. Furthermore, pancreatic cancer is often resistant to conventional radiotherapy or chemotherapy, and the morbidity rate almost equals the mortality rate. Therefore, search for effective regimens with moderate side effects for pancreatic cancer therapy and prevention is an urgent necessity. Chinese herbal medicines have been used for centuries with demonstrated safety profiles, thus their bioactive ingredients may be effective candidates for the prevention and/or treatment of pancreatic cancer. Danshen (Salvia miltiorrhiza) has been widely adopted in the traditional Chinese medicinal preparations for treatment of cardiovascular disease with minimal side effects. In the present study, we evaluated the effect of several major Danshen components, tanshinone IIA (T2A), tanshinone I (T1) and cryptotanshinone (CT) on pancreatic cancer cells. We found that CT, T2A and T1 all showed inhibitory effects on the growth of human pancreatic cancer cell lines MIA PaCa-2 and ASPC-1 with IC50 around 30μM, 3–8μM and 3–5μM, respectively. The drug-resistant human pancreatic cancer cell line PANC-1 showed less sensitivity to CT and T2A treatment but was very sensitive to T1 treatment with IC50 around 3.5μM. The potent T1 activity against pancreatic cancer cells was associated with downregulation of several protein markers related to cell cycle (cyclin D and cyclin B) and anti-apoptosis (Bcl2 and survivin), c-myc and Aurora A and B kinases in both MIA PaCa-2 and PANC-1 cells. To further identify more potent synergistic combination effect of natural bioactive compounds against pancreatic cancer, we determined the anti-growth activity of the combination between T1 and resveratrol, a natural compound found in the skin of red grape and other fruits. T1 and resveratrol combinations consistently showed synergistic effect on inhibiting the growth of MIA PaCa-2, ASPC-1 and PANC-1 cells. Protein assays showed that the synergistic combination effect of T1 and resveratrol was associated with combined downregulation of c-myc and Aurora A protein levels, suggesting that T1 and resveratrol may inhibit the growth of pancreatic cancer cells in part via combined inhibition on the functions of c-myc and Aurora A. Aurora A is the kinase that plays a critical role in controlling mitosis and cell growth. We further confirmed if Aurora A was a functional target for the synergistic combination effect of T1 and resveratrol. Knockdown of Aurora A dramatically inhibited PANC-1 cell growth and decreased the T1 and resveratrol combination activity, suggesting that Aurora A could be one of the targets by the T1 and resveratrol combination effect. Aurora A gene promoter contains the c-myc binding site, suggesting that Aurora A may be regulated by c-myc and the inhibition of c-myc expression may be one of the causes of reduced Aurora A expression following T1 treatment. To confirm this, we treated PANC-1 cells with a c-myc inhibitor and found decreased Aurora A gene expression in a time-dependent manner, indicating the regulation of c-myc on Aurora A gene expression. Our results suggest that the T1 and resveratrol combination may serve as a novel chemopreventive regimen for effective prevention of pancreatic cancer. Supported in part by DOD (PC073988) and NCI/NIH (R21CA127794) Citation Information: Cancer Prev Res 2011;4(10 Suppl):B63.

Radiosensitization Effect of PARP Inhibitor in Cells Exposed to Low and High LET Radiation

To improve radiotherapy of cancer patients, development of efficient radiosensitizer is one of the most important issues in clinical cancer treatment. Poly(ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme that promotes base excision repair and DNA strand break repair. Inhibitors of PARP have been shown to enhance the cytotoxic effects of ionizing radiation and DNA damaging agents. We investigated the impact of inhibition of PARP on responses to γ-irradiation (low LET (liner energy transfer) radiation) and carbon-ion irradiation (high LET radiation) in human pancreatic cancer cell line MiaPaCa2.

Triphendiol (NV-196), development of a novel therapy for pancreatic cancer

Despite incremental progress in the treatment of pancreatic adenocarcinoma, the prognosis of patients remains poor. Here, we report the preclinical studies in pancreatic cancer cells that demonstrate the efficacy of triphendiol (NV-196, a synthetic isoflavene) both as a monotherapy and as a gemcitabine sensitizer. The in-vitro effects of triphendiol on the pancreatic cancer cell lines HPAC and MIAPaCa-2 were determined using cell proliferation, flow cytometry, and western blot analysis. The antiproliferative activity of triphendiol was also investigated in two xenograft models of pancreatic cancer (HPAC and MIAPaCa-2). As a monotherapy, triphendiol-inhibited cell proliferation-induced p53-independent G2/M cell cycle arrest and activation of the intrinsic (mitochondrial) apoptosis pathway. Triphendiol-induced apoptosis was caspase independent and death receptor independent, whereas cell necrosis was caspase mediated. Using combination index analysis, we have shown that pretreatment of pancreatic cancer cells with triphendiol enhanced the cytotoxic effect of gemcitabine, the standard of care used to treat advanced pancreatic cancer. In xenograft models of pancreatic cancer, the rate of tumor proliferation on mice coadministered with triphendiol and gemcitabine was significantly reduced when compared with the corresponding tumor proliferation rates from the respective monotherapy-control and vehicle-control groups. Triphendiol was recently granted Investigational New Drug status by the US Food and Drug Administration. These data justify the commencement of clinical studies investigating the utility of combining triphendiol and gemcitabine in patients with early-stage and late-stage pancreatic cancer.

Effects of MEK signaling inhibitor on the growth of human pacreatic cancer cell lines and the expression of cell cyde associated genes

Objective To examine the effects of the MEK inhibitor on human pancreatic cancer cells, and to explore the molecular mechanisms. Methods Human pancreatic cancer cell lines CFPANC1, PANC1 and MiaPaCa2 were treated with MEK inhibitor PD98059 or DMSO, the sensitivity was analyzed by an MTT assay, and cell cycle distribution was evaluated by flow cytometry( FCM), The transcriptional level and protein expression of tumor suppressor genes were detected by real-time RT-PCR and western blot respectively. DNA methyltransferase (Dnmt)1, 3a and 3b were also assayed by western blot, The methylation status of the promoter of the p16INK4A gene was assayed by methylation-specific PCR (MSP). Results PD98059 inhibited to various degrees the growth of three pancreatic cancer cell lines, accompanied by G0-G1 cell cycle arrest. PD98059 up-regulated the expression of p16INK4a, p21WAF1, p27KIP1 mRNA, demethylated the hypermethylation status of p16INK4a gene promoter, and decreased Dnmtl and Dnmt3b in CFPANC1 and PANC1 cell lines. PD98059 only increased the expression of p27KIP1, while the changes of p16INK4a, p21WAF1 and Dnmt were less marked in MiaPaCa2 cell line. Conclusions MEK inhibitor PD98059 down-regulate the activation of Dnmt and up-regulate tumor supress genes, along with the inhibition of cell proliferation and cell cycle progression. Key words: Pancreas; Tunor cell line; MEK inhibitor; Cell cycle; DNA methyltransferase; Tumor suppressor genes

Omeprazole Inhibits Proliferation and Modulates Autophagy in Pancreatic Cancer Cells

BackgroundOmeprazole has recently been described as a modulator of tumour chemoresistance, although its underlying molecular mechanisms remain controversial. Since pancreatic tumours are highly chemoresistant, a logical step would be to investigate the pharmacodynamic, morphological and biochemical effects of omeprazole on pancreatic cancer cell lines.Methodology/Principal FindingsDose-effect curves of omeprazole, pantoprazole, gemcitabine, 5-fluorouracil and the combinations of omeprazole and 5-fluorouracil or gemcitabine were generated for the pancreatic cancer cell lines MiaPaCa-2, ASPC-1, Colo357, PancTu-1, Panc1 and Panc89. They revealed that omeprazole inhibited proliferation at probably non-toxic concentrations and reversed the hormesis phenomena of 5-fluorouracil. Electron microscopy showed that omeprazole led to accumulation of phagophores and early autophagosomes in ASPC-1 and MiaPaCa-2 cells. Signal changes indicating inhibited proliferation and programmed cell death were found by proton NMR spectroscopy of both cell lines when treated with omeprazole which was identified intracellularly. Omeprazole modulates the lysosomal transport pathway as shown by Western blot analysis of the expression of LAMP-1, Cathepsin-D and β-COP in lysosome- and Golgi complex containing cell fractions. Acridine orange staining revealed that the pump function of the vATPase was not specifically inhibited by omeprazole. Gene expression of the autophagy-related LC3 gene as well as of Bad, Mdr-1, Atg12 and the vATPase was analysed after treatment of cells with 5-fluorouracil and omeprazole and confirmed the above mentioned results.ConclusionsWe hypothesise that omeprazole interacts with the regulatory functions of the vATPase without inhibiting its pump function. A modulation of the lysosomal transport pathway and autophagy is caused in pancreatic cancer cells leading to programmed cell death. This may circumvent common resistance mechanisms of pancreatic cancer. Since omeprazole use has already been established in clinical practice these results could lead to new clinical applications.

The effect of dual PI3K/mTOR inhibitor NVP-BGT226 on cell cycle and survivin and STAT3 gene expression in human pancreatic cancer cell lines.

e14523 Background: The phosphatidylinositol-3-kinase (PI3K) pathway is one of the most commonly activated signaling pathways in pancreatic cancer. The inhibition of this transducing pathway may open new options for the treatment of pancreatic cancer. NVP-BGT226 is a novel dual class PI3K/mammalian target of rapamycin (mTOR) inhibitor that has entered Phase I/II clinical trials. Methods: We have tested the effects of NVP-BGT226 on cell viability (M'TT assay), apoptosis (AnnexinV/PI-FACS and PARP1 cleavage), cell cycle (FACS PI/RNAse-staining) and expression of Survivin, STAT3 and BCL-xL with Real-time RT-PCR and Western blot in the human pancreatic cancer cell lines Panc-1, BxPc-3, AsPC-1 and MiaPaCa-2. Results: The cell viability decreased within 24-72 hours exposure to about 50% compared to untreated control cells in a concentration and but not time dependent manner. Apoptosis was not detected, but cell cycle analysis revealed that NVP-BGT226 induced predominantly a G0/G1-arrest to about 90-95%. Analysis with Real-time RT-PCR and Western blot showed a remarkable reduction of Survivin and STAT3 expression but an increase of the anti-apoptotic BCL-xL gene expression. Conclusions: The PI3K/mTOR inhibitor NVP-BGT226 induces G0/G1-arrest and and acts via downregulation of Survivin and STAT3.

Abstract 3782: Chloroquine induces defective autophagic flux and enhances pancreatic cancer cell death induced by pegylated-arginine deiminase

Abstract We have previously shown that a majority of human pancreatic adenocarcinomas are deficient in argininosuccinate synthetase (ASS), a key enzyme in arginine synthesis and therefore sensitive to arginine deprivation by arginine deiminase (ADI). ASS deficient cell lines undergo cell death when deprived of arginine, but also undergo autophagy. The role of autophagy and its contribution to cell death is controversial. We hypothesized that autophagy is protective in the setting of arginine deprivation and that inhibition of autophagy by hydroxychloroquine (ChQ) would increase cell death. Methods: The human pancreatic cancer cell line MIA-PaCa2 was treated in vitro and in vivo with pegylated arginine deiminase (PEG-ADI) alone or in the presence of ChQ. Cell death was measured by propidium iodide-FACS and apoptosis was evaluated by Annexin V-PI flow cytometry. Caspase 3 cleavage was evaluated by western blotting and ELISA as was expression of LC3. In vivo experiments were conducted by creation of subcutaneous xenografts in athymic mice followed by treatment with intraperitoneal injections of PBS, PEG-ADI, ChQ and combination of PEG-ADI and ChQ. At sacrifice, tumors were removed for analysis. Tumor lysates were analyzed by western blot for caspase 3 and p62. Immunohistochemistry for activated caspase 3 and DNA fragmentation (TUNEL) were also performed. Results: In vitro treatment of MIA-PaCa2 with PEG-ADI induced caspase dependent cell death as well as autophagy at 72 hours. ChQ at low doses inhibited autophagy induced by PEG-ADI, but increased cell death. In vivo, addition of ChQ to a sub-therapeutic dose of PEG-ADI increased tumor suppression decreased autophagy and increased caspase 3 activation compared to either treatment alone. Conclusion: Arginine deprivation induces autophagy and cell death in cell lines deficient in ASS. Autophagy appears to play a protective role, and its inactivation by hydroxychloroquine leads to enhanced tumor suppression. 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 3782. doi:10.1158/1538-7445.AM2011-3782

Abstract 4776: Calmodulin modulates Fas-induced survival signals in pancreatic cancer cells

Abstract Pancreatic cancer remains a devastating malignancy with poor prognosis, which is largely resistant to current therapies. Death receptor-initiated apoptotic pathways have been implicated both in pancreatic cancer pathogenesis and therapy. To understand the resistance of pancreatic tumor to Fas death receptor-induced apoptosis, we investigated the molecular mechanisms of Fas-activated survival signaling in pancreatic cancer cells. Using lentivirus-mediated RNA interfering strategy, we generated pancreatic cancer cell lines MiaPaCa-2 and BxPc-3 with stable knockdown of the Fas associated protein with death domain (FADD), the adaptor that mediates downstream signaling upon Fas activation. Knockdown of FADD rendered these Fas-sensitive pancreatic cells resistant to apoptosis induced by the Fas agonist antibody, CH-11. Consistently, CH-11 induced recruitment of caspase-8 into the Fas-activated death inducing signaling complex was blocked in FADD knockdown cells, which was associated with no activation of caspase-8 and caspase-3. By contrast, CH-11 promoted the survival of the FADD knockdown MiaPaCa2 and BxPc3 cells in a concentration-dependant manner, as measured by MTS assay. Increased phosphorylation of ERK was demonstrated in the FADD knockdown cells in response to CH-11 treatment. The pharmacological inhibitor of ERK, PD98059, abrogated CH-11-promoted cell survival in FADD knockdown MiaPaCa-2 and BxPC-3 cells. Furthermore, increased phosphorylation of Src was demonstrated in FADD knockdown cells in response to CH-11. The Fas-induced activation of Src was blocked by the specific Src inhibitor, PP2, which further inhibited the Fas-induced activation of ERK and cell survival of the FADD knockdown cells. To elucidate the mechanisms underlying Fas-induced Src activation, we analyzed Fas-associated proteins by immunoprecipitaion of Fas in the FADD knockdown cells. Increased Src and phosphorylated Src was indentified in the Fas-associated protein complex activation by CH-11. Concomitantly, increased recruitment of calmodulin (CaM) into the Fas-associated protein complex was demonstrated as well. Trifluoperazine, a CaM antagonist, inhibited the Fas-induced recruitment of CaM, Src and phosphorylated Src. Consistently, trifluoperazine inhibited Fas-induced activation of Src and blocked Fas-promoted cell survival in the FADD knockdown cells. These results demonstrate that Fas induces FADD-independent survival signaling in pancreatic cancer cells via CaM-mediated activation of Src-ERK signaling pathways. Understanding the molecular mechanisms responsible for the resistance of pancreatic cells to apoptosis induced by Fas-death receptor signaling may provide molecular insights into designing novel therapies to treat pancreatic tumors. 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 4776. doi:10.1158/1538-7445.AM2011-4776