Cancer Cell MIA PaCa-2 Research Articles

Abstract 5553: Development of novel selenium-NSAIDs as potential cancer therapeutics.

Abstract Several non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to be effective chemopreventive agents in both pre-clinical and clinical studies on various cancer types. Here in, we developed several selenium (Se)-NSAID hybrid molecules and evaluated their efficacy as cancer therapeutics. The rationale for introducing Se into the NSAIDs is that this redox-active trace element is an essential micronutrient and a normal component of diets with chemopreventive and anticancer properties. Our recent studies have also shown that appropriately designed organoselenium compounds exhibited promising anti-tumor properties in various preclinical cancer models. We hypothesized that the rational incorporation of Se into NSAIDs should enhance their anti-cancer properties and therefore, we synthesized 16 novel Se-NSAID hybrid agents designed by incorporating Se moieties into the structures of aspirin, ibuprofen, and naproxen. In the first phase, a selenocyanate moiety was incorporated to these NSAIDs and screened for their cytotoxicity in various cancer cell lines. The cell viability results at 24 and 48 h showed that the introduction of this Se functionality on aspirin scaffold achieved a dramatic increase in the activity as compared to aspirin, as well as other NSAIDs or their Se hybrids. Thus, we continued optimization of aspirin with a variety of Se moieties, such as selenides and selenomethyl functionalities. Most of these compounds reduced cell viability in a dose dependent manner, while aspirin had no significant effect. Based on these studies we identified two Se-aspirin hybrid compounds; ASD-43, having a selenide functionality and ASD-49, with a selenocyanate functionality, as the most potent compounds for further development. The IC50 values after 48 h treatment in HCT116 cells were 2.5μM and 1.0 μM and in MiaPaCa-2 cells were 5.0 μM and 2.5 μM for ASD-43 and ASD-49, respectively. To further establish the cytotoxic potential of these compounds, an esterase staining (live and dead) assay was performed in colon (HCT116) and pancreatic (MiaPaCa-2) cancer cells. Treatment of ASD-43 and ASD-49 reduced cell viabilities dose dependently in both HCT116 and MiaPaCa-2 cells, while aspirin had no significant effects. In addition, both these compounds induced caspase-mediated PARP cleavage as early as 24 h in HCT116 cells. In conclusion, new Se-NSAID hybrid agents have been developed which showed a solid potential to be effective cancer therapeutics in our initial studies. Detailed results of these investigations will be presented. Citation Format: Daniel Plano, Deepkamal Karelia, Manoj Pandey, Shantu Amin, Arun K. Sharma. Development of novel selenium-NSAIDs as potential cancer therapeutics. [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 5553. doi:10.1158/1538-7445.AM2013-5553

Abstract 1705: Bioenergetic changes in glycolysis, Kreb's cycle, and glutaminolysis in pancreatic cells treated with mitochondria-targeted cationic drugs.

Abstract Human pancreatic ductal adenocarcinoma (PDAC) is an incurable and highly aggressive human cancer. Current chemotherapy provides a slight increase in survival. New therapeutic strategies to treat PDAC are desperately needed. In this study we used a relatively non-toxic mitochondria-targeted cationic nitroxide (Mito-CP) in combination with antiglycolytic agents (2-deoxyglucose, 2-DG) to selectively inhibit pancreatic cancer cell proliferation. We used high-throughput XF24 extracellular flux analyzer and mass spectrometry-based metabolomics approach to investigate cellular bioenergetics changes in pancreatic cancer cells (MiaPaCa-2 and Panc-1 cells). Results indicate that at nanomolar concentrations, Mito-CP alone potently inhibited proliferation of MiaPaCa-2 cells and synergized with 2-DG in inhibiting intracellular ATP levels. In the presence of glycolytic inhibitors, i.e. 2-DG or 3-bromopyruvate, Mito-CP exhibited enhanced toxicity in both pancreatic cell lines. LC-MS-based metabolomics studies indicate that Mito-CP inhibits Kreb's cycle metabolites (citric acid, cis-aconitic and isocitric acids) but significantly enhances intracellular glutamine, alpha-ketoglutarate, succinate, fumarate, and malate. This enhancement is abolished in glutamine-free media. We conclude that strategies to modulate bioenergetic metabolism with relatively non-toxic mitochondria-targeted nitroxides in combination with glycolysis or glutaminolysis inhibitors may provide a novel route for treating patients with PDAC with minimal toxic side effects. Citation Format: Gang Cheng, Jacek Zielonka, Joy Joseph, Balaraman Kalyanaraman. Bioenergetic changes in glycolysis, Kreb's cycle, and glutaminolysis in pancreatic cells treated with mitochondria-targeted cationic drugs. [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 1705. doi:10.1158/1538-7445.AM2013-1705

Induction of Apoptosis and Anoikis by Bit1 in Pancreatic Cancer Cells

Pancreatic cancer is a highly aggressive disease with a very high mortality rate among all human cancers. The poor prognosis is in part due to intrinsic resistance to the apoptosis-inducing effects of radio- and chemotherapy. To find alternative cell death pathways that can bypass the apoptotic resistance of pancreatic cancer cells, we examined the role of the novel anoikis effector Bit1 (Bcl-2 inhibitor of transcription) in the survival and apoptotic resistance of pancreatic cancer cells. Bit1 is a mitochondrial protein that induces a caspase-independent apoptosis upon its release into the cytosol following loss of integrin-mediated attachment to extracellular matrix (anoikis). In this report, we observed that ectopic expression of Bit1 in the cytosol reduced viability and induced caspase-independent apoptosis in human pancreatic cancer cell lines, Miapaca-2 and PANC-1. While increased expression of mitochondrial Bit1 in these cells did not induce apoptosis under attached conditions, detachment significantly induced higher level of apoptosis in mitochondrial Bit1-transfected cells than in control transfected cells. Conversely, downregulation of endogenous Bit1 in PANC-1 cells further enhanced their anoikis resistance. Furthermore, exogenous expression of mitochondrial Bit1 in Miapaca-2 cells inhibited their anchorage-independent growth and enhanced their sensitivity to etoposide-mediated apoptosis. Mechanistically, we found that the Bit1 apoptosis function is in part dependent on the groucho related Amino-terminal Enhancer of Split (AES) expression and is abrogated by the transcriptional corepressor TLE1 protein. Consistent with our in vitro findings that Bit1 is an effector of apoptosis in pancreatic tumor cells, we find that Bit1 is significantly downregulated in a fraction of advanced stages of human pancreatic carcinoma tissues. Taken together, these findings indicate that the Bit1-apoptotic pathway can be targeted to trigger cell death in pancreatic cancer cells and implicate Bit1 as a novel therapeutic agent in attenuating pancreatic chemoresistance.

Abstract A65: Activation of ERK1/2 using combination of aspirin, curcumin and sulforaphane regimen leads to NF-κB inhibition and apoptosis in pancreatic cancer cells

Abstract Pancreatic cancer ranks as the fourth most deadly form of cancer in the United States with approximately 37,000 deaths each year. Due to poor prognosis, attention has recently focused on the chemoprevention of this disease rather than on diagnosis and treatment. The present study aims to evaluate the combinatorial chemopreventive effects of low doses of aspirin (ASP), curcumin (CUR), sulforaphane (SFN) alone and in combination ACS (ASP+CUR+SFN) on two pancreatic cancer cell lines, MIA PaCa-2 and Panc-1. Results demonstrated that low doses of ASP (1mM), CUR (10μM) and SFN (5μM) alone did not reduce cell viability, whereas combination ACS reduced cell viability by ∼70% (P<0.001). ACS treatment also induced cell apoptosis by ∼60% accompanied by activation of caspase- 3 and Poly (ADP-ribose) polymerase (PARP) proteins. The NF-κB DNA binding activity was inhibited by ∼45% (P<0.01) and ∼75% (P<0.001) in MIA PaCa-2 and Panc-1 cells respectively. The reduction in NF-κB activity was accompanied by down-regulation of phospho IκBα and phospho Akt proteins. Mechanistic studies revealed that ACS promoted reduction in cell viability, associated with increased expression of phospho extracellular signal-regulated kinase 1/2 (ERK1/2), phospho c-Jun, phospho p38 MAPK and phospho p53 proteins. Furthermore, to evaluate the significance of activation of ERK1/2 pathway on ACS induced reduction in cell viability and NF-κB, the cells were pretreated with U0126 (ERK1/2 inhibitor). The inhibition of ERK1/2 by U0126 pretreatment abolished the effect of ACS on cell viability and NF-κB DNA binding activity. Overall, our results for the first time, illustrate that low dose ACS combinations impart synergistic suppressive activity than chemopreventive agents alone thus confirming the importance of novel combination chemoprevention strategies against pancreatic cancer. Citation Format: Arvind Thakkar, Dhruvitkumar Sutaria, Karthik Grandhi, Ying Huang, Jeffrey Wang, Sunil Prabhu. Activation of ERK1/2 using combination of aspirin, curcumin, and sulforaphane regimen leads to NF-κB inhibition and apoptosis in pancreatic cancer cells. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A65.

MiRNA profiling in pancreatic cancer and restoration of chemosensitivity

Pancreatic cancers relapse due to small but distinct population of cancer stem cells (CSCs) which are in turn regulated by miRNAs. The present study identifies a series of miRNAs which were either upregulated (e.g. miR-146) or downregulated (e.g. miRNA-205, miRNA-7) in gemcitabine resistant MIA PaCa-2 cancer cells and clinical metastatic pancreatic cancer tissues. Gemcitabine resistant MIA PaCa-2 cells possessed distinct ALDH-positive CSC fraction expressing stem cell markers OCT3/4 and CD44 and chemoresistance marker class IIIβ-tubulin (TUBB3) which decreases on transfection with miR-205 resulting in the restoration of chemosensitivity to gemcitabine.

Efficacy of gemcetabine and LY294002 on the growth of human pancreatic cancer cell lines BxPc-3 and MiaPaCa-2

Objective To investigate the effects of gemcetabine and LY294002 monotherapy or combination on the proliferation and poptosis of pancreatic cancer cell lines BxPc-3 and MiaPaCa-2.Methods Cell proliferation and poptosis were detected by MTT and Annexin V-FTTC,respectively.Results Both gemcetabine and LY294002 could inhabit the proliferation of the two cell lines.Their inhibitory effects were increased accompanied with increased drug concentrations and the cell survival rates was negatively correlated with logarithmic of the drug concentrations (r＜-0.95,P＜0.01).The inhibitory effects of gemcetabine and LY294002 to the BxPc-3 proliferation were significantly stronger than to the MiaPaCa-2(P＜0.05).For BxPc-3 and MiaPaCa-2,the IC50 of gemcetabine were(10.07±1.83),(36.45±2.71)μmol/L(P＜0.05),and the IC50 of LY294002 were(7.84±1.48),(17.89±1.98)μmol/L(P＜0.05),respectively.Gemcetabine and LY294002 could induce cell apoptosis(P＜0.01).Though both the concurrent or consecutive use of these two drugs could promote cell apoptosis,the effect of the concurrent group was significantly stronger(P＜0.05).The order of these two drugs in the concurrent group had no significant influence on their effects(P＞0.05).Conclusion Both gemcetabine and LY294002 could inhibit the proliferation of pancreatic cancer cell lines.Their concurrent application shows a significant inhibitory effect on the cell apoptosis. Key words: Pancreatic cancer; Gemcetabine; LY294002; Apoptosis

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 1612: Bitter melon juice induces apoptotic death of human pancreatic carcinoma cells

Abstract Pancreatic cancer is one of the deadliest cancers. The prognosis of this malignancy is extremely poor in general and especially at advanced stages of the disease, suggesting that newer preventive/intervention strategies are necessary to improve the outcome of this malignancy. Plant based therapies are often used as alternative and complementary treatments for various diseases including cancer. Bitter melon (Momordica Charantia) is a fruit from the perennially growing climber of Cucurbitacae family. It is a commonly consumed vegetable and is also used as a traditional medicine for treating disease conditions such as diabetes, ulcers, microbial and viral infections, etc. In recent years, the interest in its anti-cancer potential has grown considerably in the scientific community; however, literature still lacks detailed studies on this aspect. In the present study, we assessed the efficacy and associated mechanisms of bitter melon juice (BMJ) against pancreatic cancer. For this, we selected two human pancreatic cancer cell lines, BxPC-3 and MiaPaCa-2, which differ in K-ras status; a major genetic alteration in most pancreatic cancer cases. We observed that treatment of these cells with BMJ (2-4% v/v) significantly decreases the viability by 59-94% and 40-91% after 48h in case of BxPC-3 and MiaPaCa-2 cells, respectively. Decrease in cell viability was largely due to apoptosis induction as evidenced by cell death ELISA and western blot analysis for cleaved caspases-9 and -3. A significant increase in the release of cytochrome-c in the cytoplasmic fraction was also observed, thereby indicating that BMJ activates the intrinsic pathway of apoptosis in these cell lines. Additionally, BMJ significantly increased the levels of pro-apoptotic protein Bak and decreased the levels of anti-apoptotic protein Bcl-XL in both the cell lines. In other studies, BMJ treatment down-regulated XIAP and survivin, but increased p21 and Chop, and caused an activation of ERK and p38 in both cell lines without any effect on JNK. Furthermore, BMJ treatment resulted in significant up regulation in AMPK activation thereby suggesting it might be impinging on additional pathways. In tumor xenograft study, administration of lyophilized BMJ powder in diet (1.5%, w/w) to athymic nude mice for 42 days caused a significant inhibition in MiaPaca-2 xenograft growth in terms of a decrease in tumor volume by 43% (P<0.05) and tumor weight by 52% (P<0.05) without affecting body weight and food intake. Taken together, our findings demonstrate the anti-cancer efficacy of BMJ towards the human pancreatic carcinoma cells irrespective of their genetic background, suggesting that more studies are needed to establish the translational potential of BMJ in human pancreatic cancer control. 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 1612. doi:1538-7445.AM2012-1612

Abstract 1136: Synergistic inhibition of bioenergetic pathways in pancreatic cancer cells by mitochondria-targeted nitroxide and glycolytic inhibitors

Abstract Pancreatic adenocarcinoma is one of the most aggressive cancers with a high fatality rate. Currently available chemotherapy for treating pancreatic cancers is ineffective. Emerging research is focused on drugs targeting bioenergetic pathways in tumor cells. Despite decades of evidence that cancer cells have increased aerobic glycolysis, glycolytic inhibition has failed as a chemotherapeutic approach, due in part to enhanced systemic toxicity of antiglycolytic agents. We hypothesized that a combined inhibition of glycolysis and mitochondrial function may represent an alternate therapeutic approach that can potentially overcome the insufficiency of glycolytic inhibition alone. In this study we investigated the chemotherapeutic efficacy of combining mitochondria-targeted nitroxide, Mito-CP (a nitroxide, carboxy proxyl conjugated to a triphenylphosphonium (TPP+) moiety), and inhibitors of glycolysis, 2-deoxy-D-glucose (2-DG) or 3-bromopyruvate (3-BP) in pancreatic cancer cell lines, PANC-1 and MiaPaCa-2. The relative cytotoxicity and mitochondrial bioenergetic changes in pancreatic cancer cells were assessed. We observed that Mito-CP synergized with glycolytic inhibitors (2-DG or 3-BP) in inducing cell death. Using a Seahorse XF24 extracellular flux analyzer, we determined the combined effects of Mito-CP and 2-DG or 3-BP on cellular mitochondrial function and glycolytic function. The combined treatments induced fast and irreversible inhibition of mitochondrial functions and intracellular ATP depletion. Collectively, these results suggest that targeting mitochondrial bioenergetic metabolism with Mito-CP and glycolytic inhibitors such as 2-DG and 3-BP may be a promising chemotherapeutic strategy in pancreatic cancer. 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 1136. doi:1538-7445.AM2012-1136

Abstract 2765A: Co-treatment of human pancreatic cancer MiaPaCa-2 Cells with PNC-27 and Gemzar

Abstract Novel anti-cancer PNC-27 peptide, derived from the p53 binding domain of MDM2, induces necrosis of human pancreatic MiaPaCa-2 cancer cells but not normal cells in vitro, in a dose dependent fashion. Recent studies support a membranolytic mechanism of action for the peptide, initiated by binding of peptide monomers to the cells’ plasma membrane that subsequently aggregate to form transmembrane pores. Gemzar, on the other hand, is an established, non-selective chemotherapeutic used to treat pancreatic cancer. As a nucleoside analog, Gemzar inhibits DNA replication, resulting in cell cycle arrest, and ultimately apoptosis. Speculation regarding whether the two drugs, with different mechanisms, could produce a combined killing effect if administered simultaneously is investigated. Cell proliferation assays (MTT), cell cytotoxicity assays (LDH) and confocal microscopy were used to study the susceptibility of the MiaPaCa-2 cells to the co-treatment of PNC-27 and Gemzar. To determine the working concentration of PNC-27 that would result in pore formation but would not be cytotoxic to the cells, a dose response assay with serial dilutions was conducted. Results revealed that while MiaPaCa-2 cells treated with PNC-27 in DMEM have an LD50 = 150 ug/mL, the working concentration that would enable minimal pore formation without cell death was determined to be ∼10 ug/mL. Through confocal microscopy, it was then determined that propidium iodide (PI) (668.4 Da), a marker for dead cells and that is impermeable for intact cells, would nevertheless be able to enter the cell cytoplasm via PNC-27 pores. The results found PI staining in the cytoplasm but complete absence of nuclear staining. This observation not only indicates the sustained viability of the cells treated with 10 ug/mL PNC-27, but also supports the hypothesis that PI entered the cells through the PNC-27 induced pores. To explore a combined cytotoxic effect of Gemzar and PNC-27, MTT assays were carried out with Gemzar concentrations between 0 uM-100 uM for either 24 or 48 hours, in presence or absence of PNC-27. Our results demonstrate that co-treatment of MiaPaCa-2 cells with PNC-27 and Gemzar leads to a greater cytotoxic effect than with either Gemzar or PNC-27 alone. Additionally, the 48hr treatment resulted in a greater cytotoxic effect than did the 24hr condition. Our results indicate that the increased permeability of the cancer cells to Gemzar via the PNC-27-induced pores may allow for a more rapid mechanism of entry for Gemzar. Due to the selectivity of PNC-27, more Gemzar is able to target the cancer cells which have compromised membrane integrity caused by PNC-27. Therefore, co-treatment with Gemzar may allow for a dose reduction of Gemzar due to increased accessibility of the chemotherapeutic to its site of action. By reducing the dose of Gemzar required to achieve a significant cytotoxic effect, the severity of the side-effects of the drug experienced by patients may also be reduced. 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 2765A. doi:1538-7445.AM2012-2765A

Inhibition of Glycogen Phosphorylation Induces Changes in Cellular Proteome and Signaling Pathways in MIA Pancreatic Cancer Cells

Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells. We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (5-chloro-1H-indole-2-carboxylic acid [1-(4-fuorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2 oxoethyl] amide) (25, 50, and 100 μM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC (stable isotope labeling with amino acids in cell culture) method. A total of 22 protein spots and 4 phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of mitogen-activated protein kinase/extracellular signal-regulated kinase and tumor necrosis factor α/nuclear factor κB. Signaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through 1 pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown, and posttranslational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways.

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

Abstract 3545: Autophagy inhibition as a therapeutic strategy in cancer

Abstract Autophagy is an evolutionarily conserved process involving degradation of cellular components in lysosomes to maintain protein and organelle quality control, and temporarily sustain cell viability in response to stressful stimuli. Several studies have demonstrated the dual role of autophagy in tumorigenesis as both a tumor suppressor and a protector of cancer cell survival under stress. It is well recognized that anticancer therapies, such as chemotherapy and irradiation (RT), induce autophagy as a pro-survival response, likely resulting in therapeutic resistance. Therefore, concurrent autophagy inhibition as a chemo- and RT-sensitizer has generated great interest in cancer therapy. We generated Beclin1+/+ and Beclin1+/- immortalized mouse mammary epithelial cells (iMMECs) stably expressing the apoptosis inhibitor BCL-2 and the autophagy marker EGFP-LC3 to investigate how acute autophagy inhibition and chronic autophagy deregulation impact tumor sensitivity to anticancer drugs commonly used against breast cancer. We also used human cancer cell lines, including MCF-7, PANC-1, MIA PaCa-2 and HCT-116 cells, to explore whether pharmacologic autophagy inhibition or genetic knockdown of autophagy regulators exhibits a synergistic anticancer effect with newer antineoplastic drugs, such as mTOR inhibitors (CCI-779 and RAD001) and novel antimicrotubule agents (ixabepilone). Treatment efficacy was evaluated by clonogenic survival and cytotoxicity assays in 2D- and 3D-epithelial cell culture in vitro and by tumor responsiveness assays in mice in vivo. Autophagy inhibition sensitized Beclin1+/+ apoptosis-defective iMMECs to doxorubicin, but not to paclitaxel, and had an even greater impact on autophagy-deficient cells. Moreover, autophagy inhibition via choloroquine or Beclin1 knockdown enhanced the treatment efficacy of mTOR inhibitors on MCF-7, PANC-1, MIA PaCa-2 and HCT116 cells in vitro and in xenograft tumors in nude mice in vivo. These studies provide insight into how autophagy functional status in tumors impacts treatment responsiveness and into how to best incorporate pharmacologic autophagy modulation in the decision tree for cancer treatment. 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 3545. doi:10.1158/1538-7445.AM2011-3545

Abstract 859: EGCG inhibits pancreatic cancer growth and stem cell characteristics in xenografts and KrasG12D transgenic mice models

Abstract FoxO (forkhead box O) transcription factors are crucial regulators of cell fate. This function of FoxO proteins relies on their ability to control diverse functions such as proliferation, differentiation, DNA repair and apoptosis. Constitutively active AKT and ERK in cancer cells have been shown to phosphorylate FoxO transcription factors (FKHR, FKHRL1 and AFX), which bind to protein 14-3-3 and sequester in the cytoplasm. In the absence of survival signals, FoxO proteins dephosphorylate, translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest and apoptosis. EGCG, a component of green tea, exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines through diverse mechanisms. However, the mechanisms by which EGCG inhibits cell proliferation and induces apoptosis are poorly understood. EGCG inhibited viability and induced apoptosis in human pancreatic cancer cell lines. EGCG induces PTEN expression and inhibits Akt phosphorylation in pancreatic cancer Hs766T, AsPC-1, MIA PaCa-2 and PANC-1 cells. Treatment of PANC-1 cells with EGCG caused a decrease in ERK phosphorylation, and an increase in JNK phosphorylation. Overexpression of FOXO1, FOXO3a, and FOXO4 inhibited cell viability in PANC-1 cells. The inhibitory effects of EGCG on cell viability were further enhanced when cells were transfected with FOXO1, FOXO3a, and FOXO4. EGCG enhanced FOXO-DNA binding and transcriptional activities. Interestingly, EGCG inhibited pancreatic cancer growth and/or development in xenograft, orthotopic and KrasG12D transgenic mice models. EGCG also inhibited pancreatic cancer stem cell characteristics. In conclusion, our data demonstrate that EGCG can inhibit pancreatic cancer growth and/or development through multiple mechanisms and thus can be used for the management of pancreatic cancer. 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 859. doi:10.1158/1538-7445.AM2011-859

Abstract 2192: KL1 internal repeat mediates klotho tumor suppressor activities and inhibits bFGF and IGF-1 signaling in pancreatic cancer

Abstract Klotho is a transmembranal protein, which can be cleaved, shed and act as a circulating hormone. Klotho deficient mice present a syndrome of early aging, while overexpression of klotho extends lifespan. Klotho is a potent inhibitor of the insulin growth factor (IGF)-1 and the basic fibroblast growth factor (FGF) pathways. Klotho is also an essential co-factor for the activity of FGF23 and thus serves as a major regulator of phosphate homeostasis. We have recently identified klotho as a tumor suppressor in breast cancer. As klotho is expressed in the normal pancreas and as the IGF-1 and FGF pathways are involved in pancreatic cancer development, we undertook to study the expression and activity of klotho in pancreatic cancer. Immunohistochemistry analysis revealed significantly reduced expression of klotho in pancreatic cancer compared to normal pancreas, and low levels of klotho mRNA were noted in the pancreatic cancer cells Panc1, Colo357 and MiaPaCa2 compared to normal pancreas cells. Further studies revealed that klotho silencing is mediated by epigenetic mechanisms. Overexpression of klotho or treatment with soluble klotho (8pM) reduced clonal growth of pancreatic cancer cell lines, and a synergistic effect was observed in combination with chemotherapeutic drugs. Daily injections of klotho (10μg/kg) inhibited tumor formation of Panc1 cells in athymic mice. Klotho inhibited activation of the IGF-1 pathway in pancreatic cancer cells. As klotho further enhanced growth inhibition by the IGF-1R inhibitor AG1024, modulation of additional signaling pathways by klotho was suspected. Indeed, klotho overexpression inhibited the bFGF pathway in these cells. The extracellular region of klotho is composed of two internal repeats, KL1 and KL2, and each of them can be cleaved and act separately. While both domains share homology to glycosidase, a structural model suggested that only KL1 retains enzymatic activity. While the KL2 domain did not show growth inhibitory activities, KL1 effectively slowed growth of pancreatic cancer cells, both in vitro and in vivo and was found to be a potent inhibitor of the IGF-1 and bFGF pathways. Importantly, KL1 did not promote FGF23 signaling and therefore, in contrary to the full-length protein, its administration to mice did not affect phosphate homeostasis. In conclusion, these studies indicate klotho as a potent tumor suppressor in pancreatic cancer and suggest, for the first time, that klotho growth inhibitory activities are mediated by the KL1 domain, possibly due to its putative enzymatic activities. As the KL1 domain is not involved in regulation of phosphate homeostasis, its administration may serve as a potential safe and effective novel strategy for the treatment of pancreatic cancer. 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 2192. doi:10.1158/1538-7445.AM2011-2192

Abstract 704: Upregulation of the human equilibrative nucleoside transporter 1 (hENT1) expression by indole-3-carbinol (I3C) in pancreatic cancer cell lines

Abstract Pancreatic adenocarcinoma is the fourth most common cause of death in cancer in the Western world. In the past years, gemcitabine (GEM) has become the standard chemotherapeutic agent for patients with pancreatic cancer. However, a substantial number of patients fail to respond to therapy due to inherent or developed GEM chemoresistance. GEM is transported into cells mainly by the human equilibrative nucleoside transporter 1 (hENT1). Overexpression of hENT1 mRNA and protein in pancreatic carcinoma is known to correlate with better outcomes. In recent years, indole-3-carbinol (I3C), a dietary compound in cruciferous vegetables, has been shown to alter cancer cell survival by inducing apoptosis in vivo. I3C has also been shown to reverse multiple drug resistance by inhibiting efflux transporter, P-glycoprotein (P-gp) expression. In this study, the potential of I3C to modulate hENT1 expression and activity was evaluated in a panel of human pancreatic cancer cell lines, AsPC-1, BxPC-3, MiaPaca2, PANC-1 and PL-45. Our data showed that I3C at 50 and 100 uM significantly increased hENT1 mRNA and protein expression in BxPC-3 cell, MiaPaca2 cells and PANC-1 cells compared with the vehicle controls. In contrast, no such effect was observed in AsPC-1 and PL-45 cells with same treatment. The potential synergistic cytotoxic effect of I3C and GEM was also examined in all five pancreatic cancer cell lines. Pancreatic cancer cells were incubated with or without I3C at 50 uM for 24 h and then treated with GEM at different concentrations with or without I3C at 50 uM and a specific hENT1 inhibitor, NBMPR, at 50nM for 48 hours. MTS assay was used for cell viability measurement. Our data showed that I3C decreased IC50 by 6.8, 8.1 and 8.4 folds in PANC-1, MiaPaca2 and BxPC-3 cells respectively, and only 2.7 and 2.0 folds in AsPC-1 and PL-45 cells respectively, compared with GEM alone treated cells. Investigation also found that NBMPR, a specific inhibitor of hENT1, only abrogated a part of the augmented effect of I3C in response to GEM in all five pancreatic cell lines. The present study provides evidence that increasing hENT1 expression is a novel mechanism of the antitumor effect of I3C. The combination of GEM and I3C may be an effective strategy to overcome the GEM chemoresistance in pancreatic cancer patients. This affirms our hypothesis that I3C could be an adjuvant therapy improving the response to GEM treatment in patients with pancreatic cancer. 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 704. doi:10.1158/1538-7445.AM2011-704

Abstract 2657: Radiosensitization of pancreatic cancer cells by combined inhibition of Chk1 and PARP1 using AZD7762 and olaparib

Abstract Small molecule inhibitors targeted to key DNA damage response/repair proteins, such as Chk1 (checkpoint kinase 1) and PARP1 (poly (ADP-ribose) polymerase-1) are being developed to sensitize tumor cells to chemo- and radio- therapy. We have recently shown that inhibition of HRR (homologous recombination repair) by the Chk1/2 inhibitor, AZD7762 (AstraZeneca) is a mechanism of radiosensitization in pancreatic cancer cells. Others have demonstrated that AZD7762 selectively sensitizes p53 mutant tumor cells. PARP inhibitors have demonstrated preferential efficacy as radiation sensitizers in cells with other DNA damage repair defects, including HRR. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 (via olaparib, AZD2281) would selectively sensitize p53 mutant pancreatic cancer cells to radiation. To test this hypothesis we assessed radiosensitization by clonogenic survival in two p53 mutant pancreatic cancer cell lines, MiaPaCa-2 and MPanc96. Cells were treated with AZD7762 (100nM) and/or olaparib (1uM) for 1 hour before radiation (0-8Gy), and for 23 hours after radiation, followed by processing for clonogenic survival. We verified that treatment with olaparib inhibited poly (ADP-ribose). We found that olaparib or AZD7762 alone produced similar magnitudes of radiosensitization in MiaPaCa-2 cells (1.5 ± 0.1 and 1.5 ± 0.01, respectively). However, the combination of olaparib and AZD7762 produced significantly greater radiosensitization than either agent alone (2.4 ± 0.1, P<0.05), which appeared to be greater than additive. Similarly, in a second cell line, MPanc96, the combination of olaparib and AZD7762 produced significantly greater radiosensitization (3.0 ± 0.4, P<0.05) than either olaparib (1.5 ± 0.1) or AZD7782 alone (2.0 ± 0.1). The combination of olaparib with AZD7762 produced minimal toxicity with clonogenic surviving fractions in MiaPaCa-2 cells of 1.1 ± 0.1 (olaparib), 0.8 ± 0.1 (AZD7762), and 0.7 ± 0.1 (olaparib + AZD7762) and in MPanc96 cells of >1.0 ± 0.2 (olaparib), 0.5 ± 0.1 (AZD7762), and 0.6 ± 0.2 (olaparib + AZD7762). Radiosensitization by olaparib and AZD7762 was accompanied by an increase in γH2AX and S345 Chk1 suggesting that DNA damage was increased under these conditions. Taken together, these findings demonstrate that the combination of olaparib and AZD7762 induces profound radiosensitization of p53 mutant pancreatic cancer cell lines. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRAC1/2 deficient-like phenotype in p53 mutant tumor cells, thus resulting in synergistic radiosensitization when used in combination with PARP1 inhibition. Supported by R01CA78554. 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 2657. doi:10.1158/1538-7445.AM2011-2657

Effects and mechanisms of pancreatic stellate cells on proliferation of pacnreatic cancer cells

Objective To assess the effects of pancreatic stellate cells (PSC) on proliferation of pancreatic cancer cells and to reveal its possible mechanism. Methods Expression and secretion of fibroblast growth factor (FGF) ligands in both pancreatic cancer cells and stellate cells were determined by immunoblotting analysis. Mmethyl thiazol tetrazolium (MTT) assay was used to examine the effects of conditioned medium (CDM) of PSC on the proliferation of pancreatic cancer cells. An in vivo tumorigenicity assay was used to evaluate the effect of PSC on xenograft formation in cancer cells. Results FGF2 was predominantly expressed and secreted by PSC. Yet MIA PaCa-2 and PANC-1 pancreatic cancer cells mainly expressed FGF5. CDM of PSC enhanced the proliferation of MIA PaCa-2 and PANC-1 cells for ( 1. 2993 ±0. 1170 ,P ＜0. 05 ) times and ( 1. 2447 ±0. 0123 ,P ＜0. 05 ) times, respectively. Neutralizing the effects of FGFs in the CDM by heparin-sepharose precipitation abolished this effect. The volume and weight of subcutaneous tumors in nude mice by injection of combination of pancreatic cancer cells with PSC were significantly greater than those by injection of PANC-1 cells alone (380. 13 mm3 and 170 g versus 601.31 mm3and 349 g,P ＜0. 05, respectively). The CDM of PSC reduced the antiproliferative effect by cyclopamine on pancreatic cancer cells ( P ＜ 0. 01, respectively). Conclusion We identified in this study a mechanism based on stroma-tumor interactions involving PSC that can contribute to enhance the proliferation of human pancreatic cancer cells. Key words: Pancreatic stellate cell; Pancreatic cancer; FGF

Cytotoxicity Effects of Amoora rohituka and chittagonga on Breast and Pancreatic Cancer Cells

Chemotherapeutic agents for cancer are highly toxic to healthy tissues and hence alternative medicine avenues are widely researched. Majority of the recent studies on alternative medicine suggested that Amoora rohituka possesses considerable antitumor and antibacterial properties. In this work, rohituka and chittagonga, fractionated with petroleum ether, dichloromethane, and ethanol, were explored for their anticancer potential against two breast cancer (MCF-7 and HTB-126) and three pancreatic cancer (Panc-1, Mia-Paca2, and Capan1). The human foreskin fibroblast, Hs68, was also included. Cytotoxicity of each extract was analyzed using the MTT assay and label-free photonic crystal biosensor assay. A concentration series of each extract was performed on the six cell lines. For MCF-7 cancer cells, the chittagonga (Pet-Ether and CH2Cl2) and rohituka (Pet-Ether) extracts induced cytotoxicity; the chittagonga (EtoAC) and rohituka (MeOH) extracts did not induce cytotoxicity. For HTB126, Panc-1, Mia-Paca2, and Capan-1 cancer cells, only the chittagonga CH2Cl2 extract showed a significant cytotoxic effect. The extracts were not cytotoxic to normal fibroblast Hs68 cells, which may be correlated to the specificity of Amoora extracts in targeting cancerous cells. Based on these results, further examination of the potential anticancer properties Amoora species and the identification of the active ingredients of these extracts is warranted.

Hedgehog signaling and desmoplasia are regulated by hypoxia in pancreatic cancer

Abstract Pancreatic cancer is one of the most aggressive solid tumors, with less than 5% of patients surviving 5 years. Chemotherapy treatments have modest improvement in survival rates and surgical resection in most cases is not an option. New strategies for therapy are needed to improve overall survival in this deadly disease. Pancreatic tumors are notoriously hypoxic and characterized by a dense fibroinflammatory stromal reaction (desmoplasia). We hypothesized that signals emanating from tumor cells exposed to hypoxia enable pancreatic tumors to thrive. To explore this we conducted a transcriptome microarray study comparing genes transcribed in normoxia and hypoxia (1% O2 for 24 hrs). We have found that hypoxia significantly increases the levels of the hedgehog (Hh) pathway ligand, sonic hedgehog (sHh), and its receptor, patched (PTCH), in two pancreatic cancer cell lines, MiaPaca 2 and Panc-1. In addition, siRNA to HIF-1α showed that this increase is HIF-1α dependent. Western blotting confirmed a hypoxia/HIF-1α-dependent increase in sHh protein levels in both MiaPaca-2 and Panc-1 cells. To further explore the hypoxia-induced increase in expression of hedgehog pathway components we used a specific pathway array profiling the expression of 84 key genes involved in the hedgehog signaling. REN, (retinoic acid, EGF and NGF induced gene), was the only other hedgehog pathway gene to increase in hypoxia. Panc-1 cells transfected with a GLI-luciferase reporter of hedgehog activation, showed no increase in GLI activity in either normoxia, hypoxia or upon addition of conditioned media containing sHh, suggesting there is not an autocrine stimulation of the cancer cells themselves by the secreted sHh. This may be explained by the increase in PTCH and REN, both inhibitors of the hedgehog pathway. sHh is a known key factor promoting stromal desmoplasia in pancreatic cancer and therefore we tested whether sHh produced by cancer cells can activate fibroblasts. Indeed, NIH-3T3 fibroblasts stably transfected with GLI-luciferase (NIH-3T3/GLI-luc) showed an increase in GLI activity when co-cultured with MiaPaca-2 cells in hypoxia, but not normoxia, or when treated with conditioned media from Panc-1 or MiaPaca-2 cells grown in hypoxia. A 3-D system simulating conditions of tumor growth and demonstrating a hypoxic core within the cancer cells mass showed GLI transactivation in NIH-3T3/GLI-luc cells only when cells were co-cultured with pancreatic cancer cells (Panc-1, MiaPaca-2, Capan-2 or Su-86), but not when cells were cultured alone. Using siRNA this GLI activation was shown to be dependent on both HIF-1α and sHh. Finally, an immunohistochemical study in pancreatic cancer tumor samples showed a significant positive correlation between tumor and stromal HIF-1α staining and stromal sHh. Thus, we propose a new mechanism of the tumor's response to hypoxia in which pancreatic tumor cells secrete sHh that acts on stroma to increase fibrosis which in turn leads to decreased blood flow, and thus a continuous cycle of more hypoxia and Hh signaling. [Supported by CA109552-05]