HPAF-II Cells Research Articles

Abstract 5534: Novel pluronic F127-coated paclitaxel nanoparticles formulation for pancreatic cancer

Abstract Pancreatic cancer (PanCa) is one of the deadliest cancers which accounts for 46,420 new cancer cases and almost 39,590 deaths in the United States in 2014. There is no effective treatment modality for PanCa that can significantly improve the overall survival rate of patients. Paclitaxel is a mitotic inhibitor widely used in chemotherapy. Recent FDA approved treatment regimen of Abraxane® (paclitaxel nanoformulation) with gemcitabine has been increased overall survival of PanCa patients from 6.7 months (Gemcitabine) to 8.5 months (Abraxane® + Gemcitabine). Targeted delivery of paclitaxel may lead to significant improvement in treating pancreatic cancer. For this purpose, we have engineered a unique, pluronic F127-coated paclitaxel loaded nanoparticles (PPNPs) formulation. This formulation is composed of a PLGA core that is subsequently coated with poly(vinyl alcohol) (PVA), poly(l-lysine) (PLL), and pluronic polymer (F127) for efficient drug delivery and active tumors targeting. This formulation has several unique properties: (a) the PLGA core is capable of loading paclitaxel and its sustained release (b) pluronic-polymer layer coating provides stability and also reverses multi-drug resistance in cancer cells (c) the polyethylene glycol chains of pluronic F127 polymer act as a stealth polymer which diminishes the nonspecific uptake of formulation, and (d) amine functional groups on NPs (PLL) are useful for antibody conjugation through a PEG-linker for specific targeting of tumor/cancer cells. The PPNPs formulation was characterized for particle size, chemical composition, and thermal characteristics. The PPNPs exhibited superior dose-dependent inhibition effects on the proliferation of Colo-357, HPAF-II, BxPC-3, MiaPaca-2, Panca-1, and AsPC-1 pancreatic cancer cell lines, over free PTX and other conventional PTX nanoformulation (Abraxane®). Similar inhibition effects were also observed in colony formation assay in BxPC-3 cells. The improved anti-cancer effects of this formulation was achieved due to its accumulation in the cytosolic compartment and less presence in early endosome/late endosome/lysosome indicates their escape from lysosomal degradation. In addition, our data suggest that F127-stabilized PPNPs exhibit superior anti-cancer potential and is capable of regulating proteins associated with sonic hedgehog, multi drug resistance and miR-21 signaling pathways and inducing apoptosis which, eventually lead to the gemcitabine chemo-sensitization. Interestingly, MUC13-conjugated nanoformulation exhibited efficient targeting in HPAF-II cells. In conclusion, our data suggest that the PPNPs nanoformulation can be used for the efficient, targeted delivery of paclitaxel to tumors and may useful for treatment of pancreatic cancer alone and/or in combination with gemcitabine. Citation Format: Murali M. Yallapu, Neeraj Chauhan, Sheema Khan, Meena Jaggi, Aditya Ganju, Diane M. Maher, Mara C. Ebeling, Subhash C. Chauhan. Novel pluronic F127-coated paclitaxel nanoparticles formulation for pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5534. doi:10.1158/1538-7445.AM2015-5534

Abstract 4468: Anticancer activity of novel cucurbitacin analogue in pancreatic cancer

Abstract Background: Human pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States. Accumulating studies have witnessed the malfunction of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), enhances patient survival by only few months. Cucurbitacins, naturally occurring dietary tetracyclic triterpenoid compounds, have shown promising anti-cancer activities. Herein, we investigated the potency and anti-cancer efficacy of a novel analogue of cucurbitacin (Cuc D) in pancreatic cancer cell lines and in a xenograft mouse model. Additionally, we determined efficacy of this analogue on MicroRNAs (miRNAs) which are important regulators of genes that have crucial roles in pancreatic tumorigenesis. Methods: The effect of Cuc D on the growth of pancreatic cancer cells was determined by cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96 h. The clonogenic potential of cancer cells was also studied using the colony formation assay. Tumor suppressor miR-145, which is downregulated in pancreatic cancer, directly target MUC13. Thus the effect of Cuc D was also investigated on the expression of miR-145 through qPCR analysis. Immunoblotting techniques were performed to study the known direct targets of miRNA-145 and its associated proteins. The anti-cancer potential of Cuc D in pancreatic cancer was also evaluated in vivo using a xenograft mouse model. Results: Our results demonstrate potent anticancer effects of Cuc D on pancreatic cancer cells. Cuc D induces dose and time dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell line models at nanomolar concentrations (100-500 nM). It also inhibits colony formation and invasiveness of pancreatic cancer cells. Furthermore, Cuc D blocks the cell cycle progression in G2/M phase and decreases the mitochondrial membrane potential in pancreatic cancer cells. Notably, Cuc D significantly increases the expression of tumor suppressor miR-145 in HPAF-II cells as observed by qPCR. Furthermore, Cuc D decreases the expression of MUC13 and its associated proteins including pAKT and HER2. In addition, it restores the expression of p53 level as studied by immunofluorescence technique. The expression of key oncogenic proteins including NF-κB, STAT3 (Tyr-705) and Mcl-1 were also downregulated. The levels of PTEN and p27 (Kip1) tumor suppressor genes were increased after Cuc D treatment. Additionally, in vivo administration of Cuc D effectively inhibited pancreatic tumor growth in xenograft mouse model. Conclusion: Overall, this study suggests that Cuc D modulates the expression of key oncogenes and tumor suppressors, thus it can be a promising therapeutic modality for pancreatic cancer prevention and treatment. Citation Format: Mohammed Sikander, Sheema Khan, Neeraj Chauhan, Mohd Saif Zaman, Murali Mohan Yallapu, Fathi T. Halaweish, Bhavin Chauhan, Shabnam Malik, Meena Jaggi, Subhash C. Chauhan. Anticancer activity of novel cucurbitacin analogue in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2015-4468

Abstract B53: Effects of metformin and ATP-competitive inhibitor of mTOR on targeted-metabolomic profile in HPAF-II pancreatic cancer cell lines

Abstract Introduction: Pancreatic cancer (PC) is a devastating disease attributable to late diagnosis and is resistant to conventional treatment. Thus, there is an urgent need to develop new strategies for intervention of this lethal disease. Pancreatic ductal adenocarcinoma (PDAC) is the most common and most aggressive type of PC. Recent reports indicate the 50% of PDAC patients are diabetic at the time of diagnosis. To that end, tumor-related diabetes is now considered Type 3cDM. The mammalian Target of Rapamycin mTOR (also known as mechanistic TOR) is a conserved serine/threonine kinase and master regulator of metabolism, cell growth and proliferation. mTOR is dysregulated in a variety of tumors including pancreatic cancer. Furthermore, the anti-diabetic drug, metformin, which indirectly inhibits mTOR by activation of AMPK, has emerged as a potential therapeutic target in the treatment of PC, particularly when associated with type 3c Diabetes Mellitus. Mounting evidence links glycolytic metabolic disturbances to the adverse pancreatic cancer prognosis. The objective of this study is to determine the targeted-metabolomic profile in human PDAC cell line (HPAF-II) and the possible synergism between pan mTOR inhibition by ATP competitors (which inhibits both mTORC1 and mTORC2) and metformin administration as potential targets for therapeutic intervention in pancreatic cancer. Methods: Well differentiated pancreatic cancer cell line HPAF-II (CRL# 1997) were cultured in DMEM media supplemented with 10% fetal bovine serum in the presence of either Torin 2 (ATP-competitive mTOR inhibitor), metformin, both, or vehicle control for 1 hour and 24 hours in five replicate samples per group. We utilized targeted LC/MS/MS to characterize the alterations in the glycolytic and tricarboxylic acid (TCA) cycle metabolomics, and employed Western Blot analysis for cell signaling activation by phosphorylation. Comparisons between groups were analyzed using one-way Analysis of Variance (ANOVA) for detection of the overall statistical significance followed by secondary post-hoc analysis to determine differences between groups. The significance level was assessed at p < 0.05. Results: After 1 hour incubation with Torin 2, AMP concentration was reduced compared to the control and other treatment groups (p < 0.03). After 24 hours incubation, Torin-2 significantly decreased the glycolysis intermediates (fructose 1, 6 bisphosphate (FBP), and 2 phosphoglycerate/3 phosphoglycerate (2PG/3PG), TCA intermediate metabolites (acetyl CoA, citrate/isocitrate, and malate), as well as the electron acceptors (NAD+, and FAD). Metformin treatment alone reduced FBP and citrate/ citrate/isocitrate ratio. However, when HPAF-II cells were incubated with both Torin-2 and metformin, there was significant reduction in nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), suggesting decreased levels of energy equivalents available to the electron transport chain. To confirm that Torin 2 blocked both mTORC1 and mTORC2, whole cell lysates from all treatment and control groups where subjected to Western Blot analysis. Torin 2 treatment decreased the autophosphoyration of mTOR (S2481), and phosphorylation of its downstream target phospho S6 (pS6) confirming the inhibition of mTORC1, and decreased Akt phosphorylation at S473 (phospho-Akt-S473) indicating the inhibition of mTORC2, while rapamycin had no effect on Akt phosphorylation. Conclusion: The targeted metabolomics data indicate that mTORC1 and mTORC2 inhibition by Torin 2 reduced glycolytic intermediates and TCA metabolites in HPAF- II and may synergize with metformin to decrease the electron donors NAD+ and FAD by the TCA cycle which may lead to reduced energy production. The work on this abstract utilized Metabolomics Core Services supported by grant U24 DK097153 of NIH to the University of Michigan. Citation Format: Ghada A. Soliman, Asserewou Etekpo. Effects of metformin and ATP-competitive inhibitor of mTOR on targeted-metabolomic profile in HPAF-II pancreatic cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B53.

Aurora kinase A inhibition in combination with proapoptotic BH3-mimetic for treatment of pancreatic cancer.

316 Background: BH3-mimetics were designed to inhibit the anti-apoptotic members of the Bcl-2 family of proteins. BH3-mimetics in clinical development do not inhibit the Bcl-2 protein Mcl1, which is often active in pancreatic cancer. However, Mcl1 level can be decreased by mitotic arrest. Therefore, drugs that cause mitotic arrest may augment apoptosis induced by BH3-mimetics. Aurora Kinase A (AKA), also commonly overexpressed in pancreatic cancer, regulates mitotic spindle assembly and centrosome maturation and its inhibition leads to M-phase arrest. We hypothesized that inhibiting AKA would increase sensitivity of pancreatic cancer cells to BH3-mimetic-induced apoptosis. Methods: Pancreatic cancer cell lines (AsPC-1, PANC-1, MIA PaCa-2, HPAF-II) were evaluated for AKA protein expression by Western blot (WB). Baseline cell cycle distribution was evaluated by flow cytometry. All four cell lines were treated with a BH3-mimetic (ABT-263) alone, an AKA inhibitor (MLN8237) alone, or the combination in comparison to untreated controls. Cell viability was measured using the Celltiter-Fluor assay. Apoptosis was evaluated by WB for cleaved PARP and caspase 3. Percentage of cells undergoing apoptosis was quantitated using flow cytometry of annexin V and propidium iodide stained cells. Results: AKA expression varied between cell lines but correlated with percentage of cells in G2/M. Level of AKA expression did not appear to correlate with response to MLN8237. However, MLN8237 did induce mitotic arrest resulting in an increase in the G2/M fraction. Cell viability assays revealed limited sensitivity to MLN8237 or ABT-263 alone. MIA PaCa-2 cells were the most sensitive to MLN8237 with IC50 ~0.09µM and HPAF-II cells were the most sensitive to ABT-263 with IC50 0.6µM. MLN8237 lowered the IC50of ABT-263 in all 4 cell lines. Chou-Talalay plots exhibited a combination index < 1 in each cell line, confirming synergy. WB for cleaved PARP and caspase 3 showed increased apoptosis with the combination. The percentage of cells undergoing apoptosis also increased with the combination in each cell line. Conclusions: Inhibition of AKA synergistically augments BH3-mimetic-induced apoptosis in pancreatic cancer cells.

ING3 as a novel regulator of pancreatic cancer growth and aggressiveness.

304 Background: Recent studies demonstrate that chromatin regulation by posttranslational means, such as histone methylation or acetylation, is an essential control mechanism in tumorigenesis and tumor progression. Accumulative findings support the notion that pancreatic cancer tumorigenesis is not only governed by genetic alterations but also aberrant epigenetic regulation. The aim of the present study was to identify and assess the role of chromatin regulators in pancreatic cancer. Methods: The gene expression profile of chromatin regulators was assessed using gene expression microarray analysis in pancreatic cancer and uninvolved human tissues. Validation of microarray findings was performed by qRT-PCR in two extended cohorts of patients and by immunohistochemistry in pancreas tumor tissue microarrays. PANC-1, MIA Paca-2, Capan-2, HPAF-II and AsPC-1 cell lines were used for in vitro assays. Efficiency of knockdown experiments, performed by RNAi interference assays and shRNA-expressing lentiviral vectors, was evidenced by qRT-PCR and Western Blot Analysis. Cell proliferation, invasion and colony formation assays were conducted in genetically modified cells. The significance of variability between the results from each group and corresponding control was determined by unpaired t-test. Results: Differential expression analysis of chromatin regulators in pancreatic cancer versus uninvolved tissues demonstrates that 27 epigenetic molecules are significantly de-regulated (>1.5 fold, P<0.05). By hierarchical clustering, the samples are classified into two major groups that reflect the normal and cancer state. The decreased expression of inhibitor of growth family member 3 (ING3), a component of the NuA4 histone acetyl-transferase complex, was further validated by qRT-PCR and immunohistochemistry analysis in tumor tissues. Transient or stable silencing of ING3 caused significant increase of viability, proliferation, colony formation and invasion in several pancreatic cell lines. Conclusions: Our data show that human pancreatic cancer is characterized by loss of ING3 expression and we defined a potential role of the latter in pancreatic cancer cell growth and invasion.

Abstract 4214: Antitumoral effects of orexins and their receptors OX1R in pancreatic ductal adenocarcinomas (PDAC)

Abstract Orexins A and B were identified as hypothalamic ligands for two serpentine G protein-coupled receptors, OX1R and OX2R. Aberrant expression of OX1R was detected in colonic cancer cells and hepatic metastasis. Orexins induce apoptosis in human colon cancer cell lines and in mice models, resulting in massive reduction of tumor cell growth. Pancreatic ductal adenocarcinomas (PDAC) are highly malignant neoplasms with poor prognosis. Chemotherapy treatment shows a poor response rate. Our previous data suggested the expression of OX1R in 98% of tested PDAC. The aims of this study were 1) to investigate the presence of OX1R in human PDAC cell lines and to analyse orexin-A (OX-A) effects in relation to apoptosis and 2) to develop an in vivo heterotopic xenografted model from the cell lines expressing OX1R, for the study of tumor growth in response to OX-A. The expression of OX1R was studied at mRNA (RT-PCR), proteins (immunocytochemistry) and functional levels (number of viable cells after OX-A treatment), in 3 PDAC cell lines (AsPC-1, HPAF-II and SW 1990). The development of an animal model (heterotopic xenograft) from the cell line expressing OX1R, has allowed studying the effect of OX-A in tumor growth. Resected tumors were analyzed by immunohistochemistry. Only AsPC-1 cell line expresses OX1R. The treatment with OX-A promoted a 32% cell growth inhibition. OX-A injection in nude mice xenografted with AsPC-1 cells, has declined 49% of tumor progression in treated cases. This effect was dose-dependent and more important in weekly or twice-weekly treatments. All the tumors corresponded to poorly differentiated adenocarcinomas expressing OX1R. Induction of apoptosis was observed in OX-A treated tumors (activated caspase-3). To generalize further the role of OX1R in inhibition of cell growth and induction of apoptosis, we considered HPAF-II cells expressing the recombinant hOX1R. Orexin-A strongly inhibited HPAF-II/OX1R cell growth in vitro and tumor growth in vivo, whereas no effect of orexins could be detected in the parental HPAF-II cell line. The role of OX1R in controlling apoptosis is directly demonstrated by the expression of recombinant OX1R in HPAF-II cells that confers the ability of orexins to promote apoptosis in this cell line. This work has demonstrated the antitumor and proapoptotic effect of orexins in PDAC, using in vitro and in vivo models. In this context, orexines receptors may establish a new therapeutic target in pancreatic antineoplastic therapies. Citation Format: Thierry Voisin, Daniela Speisky, Anne Couvelard, Alain Couvineau. Antitumoral effects of orexins and their receptors OX1R in pancreatic ductal adenocarcinomas (PDAC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4214. doi:10.1158/1538-7445.AM2014-4214

Abstract 5437: Dual targeting of ErbB-2 and ErbB-3: A new potential strategy for the treatment of pancreatic cancer

Abstract Background: Pancreatic cancer (PC) is one of the most lethal cancers and there is an urgent need to find out new therapeutic approaches. Deregulated signaling through ErbB-1 and ErbB-2, members of the epidermal growth factor receptor family, frequently occurs in PC. However, attempts aiming at inhibiting ErbB-1 and ErbB-2 have revealed only a modest impact on disease outcome. A growing body of evidence suggests that ErbB-3 and its ligand NRG-1β are key players in driving oncogenic signaling and resistance to targeted therapy in PC. We have recently developed a novel humanized ErbB-3 antibody, named EV20, which displayed a potent antitumor activity by promoting receptor downregulation in vitro and in vivo. Here we hypothesized that targeting of ErbB-3 with EV20 would enhance the therapeutic effect of ErbB-2 inhibitors in PC. Materials and Methods: Three different PC cell lines (BxPC-3, HPAF II and SW1990) were chosen based on their metastatic origin, genetic background (KRAS status) and responsiveness to erlotinib. The expression of ErbB-1, ErbB-2 and ErbB-3 was analyzed by FACS and WB. The effects of trastuzumab, cetuximab, EV20 and their combinations on NRG-1β-induced activation of the ErbB3/PI3K/Akt axis, cell proliferation, and in vivo tumor growth were evaluated by standard procedures. Results: ErbB-1 was overexpressed by all the cell lines, whereas the expression levels of ErbB-2 and ErbB-3 were moderate in HPAFII cells and low in BxPC-3 and SW1990 cells. NRG-1β was more effective than EGF (an ErbB-1 ligand) in activating ErbB-3 downstream signaling, which was not inhibited by cetuximab, despite a high ErbB-1 expression in the cells. Conversely, inhibition of ErbB-2 and/or ErbB-3 resulted in a marked suppression of NRG-1β-driven Akt activation. Finally, EV20 in combination with trastuzumab exerted a superior antitumor activity compared to single agent alone in terms of cell proliferation and growth of PC xenografts in nude mice. Conclusions: Dual targeting of ErbB-2 and ErbB-3 could represent a newer therapeutic approach in PC. Note: This abstract was not presented at the meeting. Citation Format: Gianluca Sala, Reza Ghasemi, Emily Capone, Ilario Giovanni Rapposelli, Sara Traini, Cosmo Rossi, Pier Giorgio Natali, Stefano Iacobelli. Dual targeting of ErbB-2 and ErbB-3: A new potential strategy for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5437. doi:10.1158/1538-7445.AM2014-5437

Abstract 2714: A multi-targeted approach for pancreatic cancer treatment by a novel cucurbitacin analogue

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in United States. Tremendous efforts have witnessed the failure of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), extends patient survival by only a few weeks. Numerous studies implicate the need for the drugs that can target multiple signaling events in pancreatic cancer. Recently, cucurbitacins, tetracyclic triterpenoid compund/molecules, belonging to a family of Cucurbitaceae have shown promising anti-cancer activities such as antiproliferation, cell cycle arrest, and apoptosis induction. Here, we report the anticancer activity of a novel analogue of cucurbitacin (Cuc D) that is able to target important signaling proteins involved in pancreatic cancer cell proliferation, invasion and metastasis. Methods: Herein, we investigated the use of Cuc D for the treatment of pancreatic cancer using a panel of pancreatic cancer cells. The effect of Cuc D on the growth of pancreatic cancer cells was determined by CellTiter 96® AQueous One Solution cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96h. The clonogenic potential of cancer cells was also studied using the colony formation assay. microRNA-21 expression levels were investigated through Real-time PCR. Immunoblotting techniques were performed to determine the effects of Cuc D at the molecular level. Results: Our results indicate the anticancer effects of Cuc D in pancreatic cancer cells. Cuc D induces concentration dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell lines at nano molar concentrations. It also inhibits colony formation and metastasis potential of pancreatic cancer cells in dose and time dependent manner. Importantly, Cuc D targets and inhibits proteins, MUC13, Bcl2 and survivin (inhibitor of apoptosis protein family member: IAP) that are involved in proliferation, metastasis and tumor progression as seen through Western blotting. Additionally, the treatment of Cuc D induces the phosphorylation of p53 (p-p53; ser-15) in pancreatic cancer cells. We also observed that Cuc D effectively inhibits miR-21 levels in pancreatic cancer, HPAF-II cells as seen through Real time PCR analysis. miR-21 is an oncogenic miRNA that is overexpressed in pancreatic cancer. Conclusion: Our findings demonstrate that Cuc D exerts multi-focal action in pancreatic cancer cells targeting multiple signaling events. Overall, this study suggests that Cuc D can be a potential and promising therapeutic modality for pancreatic cancer treatment. Citation Format: Mohammed Sikander, Mohd Saif Zaman, Neeraj Chauhan, Murali M. Yallapu, Sheema Khan, Fathi T. Halaweish, Bhavin Chauhan, Meena Jaggi, Subhash C. Chauhan. A multi-targeted approach for pancreatic cancer treatment by a novel cucurbitacin analogue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2714. doi:10.1158/1538-7445.AM2014-2714

Dual targeting of ErbB-2/ErbB-3 results in enhanced antitumor activity in preclinical models of pancreatic cancer.

ErbB-3 and its ligand NRG-1β are key players in driving oncogenic signaling and resistance to therapy through the activation of the PI3K/Akt pathway. We have recently reported that EV20, a humanized anti-ErbB3 antibody, possesses a marked antitumor activity in a variety of human tumor models, including pancreatic cancer (PC). Here, we report that despite epidermal growth factor receptor overexpression, PC cells are more sensitive to NRG-1β than EGF in terms of Akt activation and cell proliferation. Using stable ErbB-3-knocked down cells and EV20 in combination with trastuzumab, we showed that dual targeting of ErbB-2 and ErbB-3 was necessary to completely abrogate ErbB-3 signaling and to impair cell proliferation. Similarly, enhanced therapeutic efficacy of the antibody combination was seen in xenografts originating from K-Ras-mutated HPAF-II and SW1990 cells, without increasing the toxicity. These results indicate that dual targeting of ErbB-2 and ErbB-3 could represent a new therapeutic approach in PC.

Novel synthetic oleanane triterpenoid AMR-MeOAc inhibits K-Ras through ERK, Akt and survivin in pancreatic cancer cells

K-Ras activating mutations are a major problem that drives aggressive tumor growth and metastasis in pancreatic cancer. Currently, there are no effective targeted therapies for this genetically defined subset of cancers harboring oncogenic K-Ras mutations that confer drug resistance, aggressive tumor growth, metastasis and poor clinical outcome. We identified a novel synthetic oleanane triterpenoid compound designated AMR-MeOAc that effectively kills K-Ras mutant pancreatic cancer HPAF-II cells. The cytotoxic effects correlated with apoptosis induction, as was evidenced by increase of apoptosis cells upon the treatment of AMR-MeOAc in HPAF-II cells. Our studies revealed that AMR-MeOAc treatment inhibits cancer associated survival gene survivin. Moreover, AMR-MeOAc also led to down regulation of Akt, ERK1/2 and survivin protein levels. Our results indicate that AMR-MeOAc or its active analogs could be a novel class of anticancer agents against K-Ras driven human pancreatic cancer.

Ellagic Acid and Embelin Affect Key Cellular Components of Pancreatic Adenocarcinoma, Cancer, and Stellate Cells

Ellagic acid is a polyphenolic phytochemical present in many fruits and nuts with anticancer properties demonstrated in experimental tumor studies. Embelin is a benzoquinone phytochemical isolated from the Japanese herb Ardisiae Japonicae and has been shown to induce apoptosis in cancer cells. We found that ellagic acid and embelin each dose-dependently increased apoptosis and inhibited proliferation in human pancreatic cancer cells, MIA PaCa-2 and HPAF-II cells, and in pancreatic stellate cells, which are progenitors of pancreatic cancer desmoplasia. In each of these cell types, combinations of ellagic acid and embelin at low micromolar concentrations (0.5–3 μM) induced synergistic increases in apoptosis and decreases in proliferation. Ellagic acid decreased NF-κB transcriptional activity, whereas embelin decreased STAT-3 phosphorylation and protein expression of its downstream target survivin in cancer cells. In vivo dietary ellagic acid alone or in combination with embelin decreased tumor size and tumor cellularity in a subcutaneous xenograft mouse model of pancreatic cancer. These results show that ellagic acid and embelin interact with divergent intracellular signaling pathways resulting in augmentation of apoptosis and inhibition of proliferation at low micromolar concentrations for the key cellular components of pancreatic adenocarcinoma.

Novel Curcumin-Loaded Magnetic Nanoparticles for Pancreatic Cancer Treatment

Curcumin (CUR), a naturally occurring polyphenol derived from the root of Curcuma longa, has showed potent anticancer and cancer prevention activity in a variety of cancers. However, the clinical translation of CUR has been significantly hampered due to its extensive degradation, suboptimal pharmacokinetics, and poor bioavailability. To address these clinically relevant issues, we have developed a novel CUR-loaded magnetic nanoparticle (MNP-CUR) formulation. Herein, we have evaluated the in vitro and in vivo therapeutic efficacy of this novel MNP-CUR formulation in pancreatic cancer. Human pancreatic cancer cells (HPAF-II and Panc-1) exhibited efficient internalization of the MNP-CUR formulation in a dose-dependent manner. As a result, the MNP-CUR formulation effectively inhibited growth of HPAF-II and Panc-1 cells in cell proliferation and colony formation assays. The MNP-CUR formulation suppressed pancreatic tumor growth in an HPAF-II xenograft mouse model and improved the survival of mice by delaying tumor growth. The growth-inhibitory effect of MNP-CUR formulation correlated with the suppression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-extra large (Bcl-xL), induced myeloid leukemia cell differentiation protein (Mcl-1), cell surface-associated Mucin 1 (MUC1), collagen I, and enhanced membrane β-catenin expression. MNP-CUR formulation did not show any sign of hemotoxicity and was stable after incubation with human serum proteins. In addition, the MNP-CUR formulation improved serum bioavailability of CUR in mice up to 2.5-fold as compared with free CUR. Biodistribution studies show that a significant amount of MNP-CUR formulation was able to reach the pancreatic xenograft tumor(s), which suggests its clinical translational potential. In conclusion, this study suggests that our novel MNP-CUR formulation can be valuable for the treatment of pancreatic cancer.

Novel mechanism of cytokine-induced disruption of epithelial barriers

The ductal epithelium plays a key role in physiological secretion of pancreatic enzymes into the digestive system. Loss of barrier properties of the pancreatic duct may contribute to the development of pancreatitis and metastatic dissemination of pancreatic tumors. Proinflammatory cytokines are essential mediators of pancreatic inflammation and tumor progression; however, their effects on the integrity and barrier properties of the ductal epithelium have not been previously addressed. In the present study, we investigate mechanisms of cytokine-induced disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium. Exposure of HPAF-II human pancreatic epithelial cell monolayers to interferon (IFN)γ disrupted integrity and function of apical junctions as manifested by increased epithelial permeability and cytosolic translocation of AJ and TJ proteins. Tumor necrosis factor (TNF)α potentiated the effects of IFNγ on pancreatic epithelial junctions. The cytokine-induced increase in epithelial permeability and AJ/TJ disassembly was attenuated by pharmacological inhibition of Janus kinase (JAK) and protein kinase D (PKD). Loss of apical junctions in IFNγ/TNFα-treated HPAF-II cells was accompanied by JAK and PKD dependent decrease in expression of AJ (E-cadherin, p120 catenin) and TJ (occludin, ZO-1) proteins. Depletion of E-cadherin or p120 catenin recapitulated the effects of cytokines on HPAF-II cell permeability and junctions. Our data suggests that proinflammatory cytokines disrupt pancreatic epithelial barrier via expressional downregulation of key structural components of AJs and TJs. This mechanism is likely to be important for pancreatic inflammatory injury and tumorigenesis.

Abstract 4503: Novel curcumin loaded magnetic nanoparticles for pancreatic cancer treatment.

Abstract Curcumin (CUR), a naturally occurring polyphenol derived from the root of Curcuma longa, has demonstrated potent anti-cancer and cancer prevention activity in a variety of cancers. However, the clinical translation of curcumin has been significantly hampered due its extensive degradation, suboptimal pharmacokinetics and poor bioavailability. To address these clinically relevant issues, we have developed a novel curcumin loaded magnetic nanoparticle (MNP-CUR) formulation. Herein, we have evaluated the in vitro and in vivo therapeutic efficacy of this novel MNP-CUR formulation in pancreatic cancer. Human pancreatic cancer cells (HPAFII and Panc-1) exhibited efficient internalization of the MNP-CUR formulation in a dose dependent manner. As a result, MNP-CUR formulation effectively inhibited growth of HPAFII and Panc-1 cells in cell proliferation and colony formation assays. Similarly, the MNP-CUR formulation suppressed pancreatic tumor growth in an HPAFII xenograft mice model and improved mice survival by delaying tumor growth. The growth inhibitory effect of MNP-CUR formulation was correlated with the suppression of PCNA, Bcl-xL, Mcl-1, MUC1, collagen I and enhanced membrane β-catenin expression. Interestingly, our MNP-CUR formulation did not show any sign of hemotoxicity and stable after incubation with human serum proteins. Additionally, our MNP-CUR formulation improved serum bioavailability of curcumin in mice up to 2.5 fold as compared to curcumin in Tween-20. Furthermore, biodistribution studies of our MNP-CUR formulation demonstrate a significant amount of formulation was able to reach the pancreatic xenograft tumor(s) which suggests its clinical translational potential. In conclusion, our study suggests that our novel MNP-CUR formulation can be valuable for the treatment of pancreatic cancer. Citation Format: Murali M. Yallapu, Mara C. Ebleling, Sheema Khan, Neeraj Chauhan, Brij K. Gupta, Vasudha Sundram, Meena Jaggi, Subhash C. Chauhan. Novel curcumin loaded magnetic nanoparticles for pancreatic cancer treatment. [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 4503. doi:10.1158/1538-7445.AM2013-4503

Lipocalin2 Promotes Invasion, Tumorigenicity and Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma

Lipocalin 2 (LCN2) is a small secreted protein and its elevated expression has been observed in pancreatic as well as other cancer types. LCN2 has been reported to promote resistance to drug-induced apoptosis, enhance invasion through its physical association with matrix metalloproteinase-9, and promote in vivo tumor growth. LCN2 was found to be commonly expressed in patient PDAC samples and its pattern of immunohistochemical staining intensified with increasing severity in high-grade precursor lesions. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high LCN2 expression significantly reduced attachment, invasion, and tumour growth in vivo, but not proliferation or motility. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high expression significantly reduced attachment, invasion, and tumour growth in vivo. In contrast, LCN2 overexpression in PANC1, with low endogenous expression, significantly increased invasion, attachment, and enhanced tumor growth. Suppression of LCN2 in BxPC3 and HPAF-II cells increased their sensitivity to gemcitabine in vitro, and in vivo when BxPC3 was tested. Furthermore, LCN2 promotes expression of VEGF and HIF1A which contribute to enhanced vascularity. These overall results demonstrate that LCN2 plays an important role in the malignant progression of pancreatic ductal carcinoma and is a potential therapeutic target for this disease.

Pancreatic cancer cells retain the epithelial-related phenotype and modify mitotic spindle microtubules after the administration of ukrain in vitro

The aim of this study is to characterize the phenotype of pancreatic ductal adenocarcinoma (PDAC) cells in relation to the expression of epithelial-to-mesenchymal transition (EMT) markers and determine whether ukrain, an anticancer drug based on the alkaloids extracted from greater celandine, modulates in vitro the malignant behavior of PDAC cells in order to extend our understanding of its therapeutic potential. Three cell lines (HPAF-II, HPAC, and PL45) were treated with ukrain (5, 10, and 20 μmol/l) for 48 h or left untreated (control). Cell proliferation was assessed by growth curves. Apoptosis was determined by Hoechst nuclear staining and by cytochrome c and caspase-8 expressions. The EMT markers E-cadherin, β-catenin, and vimentin, as well as actin and tubulin cytoskeletons, were analyzed by immunofluorescence. Interphase and mitotic microtubules as well as abnormal mitotic figures were studied by fluorescence microscopy after tubulin immunolabeling. Ukrain strongly suppressed cell proliferation and induced apoptosis possibly through an extrinsic pathway as cytochrome c immunoreactivity suggested that the integrity of the mitochondria was not affected. Tubulin expression indicated an antiproliferative effect of ukrain on the basis of alterations in mitotic spindle microtubule dynamics, leading to abnormal mitosis. Membranous E-cadherin/β-catenin immunoreactivity was similarly expressed in control-treated and ukrain-treated cells, although the drug upregulated E-cadherin in cell lysates. Our results suggest that ukrain exerts its chemotherapeutic action on PDAC cells targeting mitotic spindle microtubules, leading to abnormal mitosis and apoptosis, and favoring cell cohesiveness. The differentiated epithelial phenotype of HPAF-II, HPAC, and PL45 cell lines concomitant with a highly invasive potential suggests that further experiments will be necessary to definitively clarify the role of EMT in PDAC progression.

Abstract 62: Vitamin D3 induces 15-Hydroxyprostaglandin Dehydrogenase to suppress pancreatic cancer cell proliferation

Abstract BACKGROUND: Excess prostaglandin E2 (PGE2) mediates growth and survival of several cancers, including prostate and pancreatic cancers (PaCa). 15-Hydroxyprostaglandin dehydrogenase (PGDH) catalyzes PGE2 to an inactive 15-keto metabolite. We have previously shown that PGDH is down-regulated in human PaCa, contributing to accumulated levels of PGE2 that can subsequently feed back to suppress further PGDH expression. In contrast, PGDH expression has been reportedly up-regulated by vitamin D3 (VD3) in prostate cancer cells but has not been confirmed in pancreatic cancer cells. AIM: The study focuses on whether VD3 up-regulates PGDH expression in pancreatic cancer cells. METHODS: Human PaCa cell lines BxPC-3, HPAF-II and MIA PaCa-2 were treated with VD3 were analyzed for vitamin D receptor (VDR), cyclooxygenase-2 (COX-2) and PGDH expression by RT-PCR. Protein extracts were analyzed by Western blotting for VDR, COX-2, PGDH, SNAI1, SNAI2 and GAPDH expression. Cellular proliferation was analyzed by MTT assay. PGE2 was measured by ELISA. RESULTS: Dose-dependent treatment with VD3 at 1000 nM resulted in a maximal decrease in proliferation by 1.3 ± 0.1-fold in BxPC-3, 1.7 ± 0.3-fold in HPAF-II expressing elevated VDR and 1.1 ± 0.3-fold in MIA PaCa-2. VD3 at 10 nM resulted a time-dependent increase in PGDH mRNA expression by 3.8 ± 0.3-fold in BxPC-3 and 7.7 ± 0.6-fold in HPAF-II, whereas expression remained unchanged in MIA PaCa-2. Conversely, COX-2 mRNA expression decreased by 0.5 ± 0.1-fold in BxPC-3 and 0.6 ± 0.1-fold in HPAF-II, but not in MIA PaCa-2. PGDH protein expression increased at 96 and 120 hrs following treatment with VD3 in BxPC-3 and HPAF-II whereas COX-2 protein expression decreased in HPAF-II cells but was abolished by 96 and 120 hrs in BxPC-3 cells. PGDH protein expression was increased at 120 hrs in MIA PaCa-2 following addition of VD3 but COX-2 protein expression in this cell line was not detected. VD3 decreased the PGDH transcriptional regulators SNAI1 protein expression by 120 hrs in BxPC-3 and HPAF-II cells but not MIA PaCa-2 whereas SNAI2 decreased in all cell types by 96 hrs. Knockdown of SNAI by siRNA at 100 nM in HPAF-II resulted in a 1.3 ± 0.3-fold decrease proliferation and 1.4 ± 0.1-fold decrease in PGE2 production. Combined treatment with 100 nM of siSNAI and 10 nM of VD3 did not result in synergistic decrease in proliferation or PGE2 production. CONCLUSION: PaCa cells expressing vitamin D receptors responded to VD3 treatment with a corresponding increase in PGDH coupled with a decrease in COX-2 expression. VD3 or siSNAI resulted in a decrease in cellular proliferation and PGE2 production but the combined treatment did not augment further decreases, suggesting that the decrease by VD3 could be mediated through SNAI regulation of PGDH. 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 62. doi:1538-7445.AM2012-62

Pro-tumour activity of interleukin-22 in HPAFII human pancreatic cancer cells

Interleukin (IL)-22 is a cytokine involved in inflammatory and wound healing processes that is secreted primarily by T helper type 17 (Th17) cells. IL-22 receptor (IL-22R) expression is limited to epithelial cells of the digestive organs, respiratory tract and skin. Most tumours originating in these sites over-express IL-22R. Interestingly, there is an increase in Th17 frequency within the peripheral blood and tumour microenvironment of advanced cancer patients. Subsequently, IL-17 has been shown to display both pro-tumour and anti-tumour functions. Because many tumours lack expression of the IL-17 receptor, the effects of IL-17 on tumour growth are generated by cells that surround the tumour cells. Like IL-17, high levels of IL-22 have been detected in tumour tissues and the peripheral blood of cancer patients; however, the direct effect of IL-22 on tumour cells has remained largely unknown. In this report, we show that IL-22 stimulated production of vascular endothelial growth factor (VEGF) and the anti-apoptotic factor Bcl-X(L) in IL-22R-positive HPAFII human pancreatic cancer cells. Additionally, IL-22 augmented HPAFII cell production of immunosuppressive cytokines. We show further that IL-22 activation of HPAFII cells diminished T cell production of interferon (IFN)-γ through the action of IL-10. Strikingly, we show for the first time that IL-22 can fully protect cancer cells from natural killer (NK) cell-mediated cytotoxicity by stimulating tumour production of IL-10 and transforming growth factor (TGF)-β1. Our data support the idea that IL-22 may act to promote the pathogenesis of cancers rather than function in anti-tumour immunity.

Determination of Rottlerin, a Natural Protein Kinases C Inhibitor, in Pancreatic Cancer Cells and Mouse Xenografts by RP-HPLC Method

Rottlerin is a natural polyphenolic ketone isolated from the pericarps of Mallotus phillippinensis. In previous studies we showed that parenteral administration of rottlerin reduced tumor growth in murine xenograft models of pancreatic cancer. The aim of this study was to develop a simple and validated method for the quantitative determination of rottlerin in plasma and tumor tissues of mice fed a rottlerin diet. A xenograft model of pancreatic cancer was prepared by injection of 2×106 HPAF-II cells subcutaneously into nude mice. One week before tumor implantation, mice were randomly allocated to standard diet (AIN76A) and standard diet supplement with 0.012% rottlerin (n=6 per group). Mice were sacrificed after 6 weeks on diets. Rottlerin was extracted from the plasma and tissues using protein precipitation-extraction and analyzed by reverse-phase HPLC-DAD method. The same HPLC method was also applied to determine rottlerin levels in conditioned culture media and in cell lysates from HPAF-II cells exposed to 25 µM concentration of rottlerin. A substantial amount of rottlerin was detected in tumor (2.11 ± 0.25 nmol/g tissue) and plasma (2.88 ± 0.41 µM) in mice fed rottlerin diet. In addition, significant levels of rottlerin (57.4 ± 5.4 nmol/mg protein) were detected in cell lysates from rottlerin-treated HPAF-II cells. These data indicate that rottlerin is efficiently absorbed in cells and tissues both in vivo and in vitro and suggest a strong potential for rottlerin as a preventive or adjuvant supplement for pancreatic cancer.

Concomitant inhibition of HSP90, its mitochondrial localized homologue TRAP1 and HSP27 by green tea in pancreatic cancer HPAF‐II cells

Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer.