Cancer Cell Line MIA PaCa-2 Research Articles

271 - Catalase as a Potential Biomarker of Pharmacological Ascorbate Cancer Therapy

Pharmacological ascorbate (P-AscH–) has been shown to induce toxicity in a various cancer cell lines both in vitro and in vivo. P-AscH– can readily oxidize and deliver a high flux of H2O2, which can easily cross cell membrane and convert to more reactive species inside the cell. We hypothesize differential sensitivity of cells to P-AscH– is due to their ability to remove H2O2. The principal enzymes responsible for the elimination of H2O2 are catalase, glutathione peroxidase (GPx) and peroxiredoxins (Prx). Among these, catalase is the principal enzyme that involved in the detoxification of H2O2 at high concentration. In this study, we compared catalase levels and peroxide removal capacity in several pancreatic cancer cell lines MIA PaCa-2, AsPC-1, 403, 339 and Panc-1. Preliminary results indicated that MIA PaCa-2 cells had the lowest level of catalase and were most sensitive to P-AscH–, while Panc-1 had the highest level of catalase and were the least sensitive. In mice with pre-established xenograft pancreatic tumor, P-AscH– (4g/kg, i.p. twice daily) showed a greater inhibition of tumor growth for MIA PaCa-2 xenografts in comparison to Panc-1 xenografts. Furthermore, immunofluorescent staining of catalase in xenograft tumors also indicated that MIA PaCa-2 had lower level of catalase than Panc-1 in vivo. Taken together, catalase activity in tumors may predict which cancers will respond to P-AscH– therapy. Supported by VA Merit grant and NIH grants CA184051, CA137230, and CA169046.

RETRACTED ARTICLE: Linc-ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR-124/PTBP1/PKM2 axis

In this study, we investigated the regulation of linc-ROR on autophagy and gemcitabine resistance of pancreatic cancer cells and further studied the underlying involvement of the miR-124/PTBP1/PKM2 axis in this regulation. Pancreatic cancer cell lines PANC-1 and MIAPaCa-2 cells were used as in vitro model. Autophagy was assessed by western blot of LC3 I/II and observation GFP-LC3 puncta. Cell viability was examined using CCK-8 assay. Cell apoptosis was examined by flow cytometric analysis of Annexin V/PI staining. QRT-PCR, RNA fluorescence in situ hybridization and dual luciferase assay were used to study the expression and the binding between linc-ROR and miR-124. Linc-ROR siRNA significantly sensitized PANC-1 and MIAPaCa-2 cells to gemcitabine, while linc-ROR overexpression significantly reduced the sensitivity. Linc-ROR knockdown reduced basal autophagy, while linc-ROR overexpression markedly increased basal autophagy in the cells. Linc-ROR siRNA showed similar effect as 3-MA on enhancing gemcitabine-induced cell apoptosis and also reduced PKM2 expression. MiR-124 overexpression restored PKM1 and reduced PKM2 levels in the cells. In addition, miR-124 mimics also alleviated autophagy in pancreatic cancer cells. Both miR-124 mimics and PKM2 siRNA enhanced gemcitabine-induced cell apoptosis. In both pancreatic cell lines and PADC tissues, linc-ROR is negatively correlated with miR-124 expression. In addition, dual luciferase assay verified two 8mer binding sites between miR-124 and linc-ROR. Linc-ROR confers gemcitabine resistance to pancreatic cancer cells at least partly via inducing autophagy. There is a linc-ROR/miR-124/PTBP1/PKM2 axis involved in regulation of gemcitabine resistance in pancreatic cancer cells.

128P - Modulation of specificity protein 1 (SP1) is a novel therapeutic strategy for pancreatic cancer

Pancreatic cancer is one of the most aggressive and lethal malignancies. Specificity Protein 1 (SP1) is a transcription factor regulates and promotes tumor progression. Some studies have reported SP1 promotes epithelial-mesenchymal transition (EMT) and is associated with aggressive and poor patient prognosis. However, there is a paucity of clinical evidence regarding the role of SP1 in pancreatic cancer. In this study, we aimed to confirm the function of SP1 in invasiveness of pancreatic cancer cells and to evaluate the clinical impact in patients with pancreatic cancer. Between June 2002 and December 2012, 81 patients underwent radical curative resection for pancreatic cancer at Gangnam Severance Hospital, Seoul, Korea. Pancreatic cancer cell lines MIA PaCa-2, PANC-1, AsPC-1, and BxPC-3 were used for in vitro study. To evaluate the endogenous expression level of SP1, we purified the whole RNA and protein to perform the qPCR, RT-PCR and Western blot. si-SP1 was used for specifically inhibit the function of SP1. The invasive potential of pancreatic cancer cells were assessed in matrigel coated chambers. Among the 81 patients, 32 (39.5%) were positive for SP1. On univariate and multivariate analyses, poor differentiation tumor and SP1-positive status were identified as independent prognostic factors for DFS. High expression of SP1 was observed and correlated with the expression of mesenchymal markers (Snail, L1CAM, Vimentin) unlike epithelial markers (CDH1). Importantly, silencing of SP1 showed markedly decrease in motility and the invasiveness of cancer cells (p < 0.001) as determined from transwell invasion and transendothelial migration assays, respectively. Our results demonstrated that SP1 is an independent marker for metastatic disease and death in patients with pancreatic cancer. Additionally, our in vitro study demonstrated that SP1 expression promotes EMT and the invasiveness of pancreatic cancer cell lines, a finding compatible with the results of previous in vitro studies. These findings suggest that SP1 can potentially be a valuable target for the improvement of survival rates in patients with pancreatic cancer.

MicroRNA miR-374, a potential radiosensitizer for carbon ion beam radiotherapy.

In this study, we compared the microRNA(miRNA) profiles of a control and X-ray- and carbon ion beam-resistant cells to identify miRNAs that can be used as radiosensitizers and biomarkers. Mouse squamous cell carcinoma line NR-S1, its X-ray-resistant derivative X60, and its carbon ion beam‑resistant derivative C30 were subjected to miRNA microarray analysis. Expression of miRNAs shown to be upregulated or downregulated in the microarray analysis was confirmed by qRT-PCR. Downregulated miRNAs were overexpressed in human pancreatic cancer cell lines PANC1 and MIA PaCa-2, and the resulting cells were tested for radiosensitivity using colony-forming and sphere-forming assays. Of 1,265miRNAs analyzed, 4were downregulated and11 were upregulated in X-ray-resistant and carbon ion beam-resistant cells. Two of the downregulated miRNAs, miR-196 and miR-374, were selected for overexpression in PANC1 and MIA PaCa-2 cells. Overexpression of miR-374 sensitized PANC-1 and MIA PaCa-2 cells toward carbon ion beam radiation. miRNA miR-374 has the potential to be a new radiosensitizer for carbon ion beam radiotherapy and a new biomarker to determine the optimal treatment for cancer.

Antitumor effects of the hyaluronan inhibitor 4-methylumbelliferone on pancreatic cancer.

Hyaluronan (HA) is a major component of the extracellular matrix (ECM), and influences tumor invasion and metastasis. In a previous study, the present authors reported for the first time that 4-methylumbelliferone (MU) inhibited HA synthesis and suppressed tumor growth. However, the localization of HA and the changes in ECM morphology caused by MU in pancreatic cancer remain to be examined in detail. In the present study, the cytotoxicity of MU and its effect on cellular proliferation was evaluated in the human pancreatic cancer cell line MIA PaCa-2. The amount of HA synthesized and the retention of HA around the cells were quantitatively and immunohistochemically analyzed in vitro and in vivo. Structural changes in the ECM in the tumor tissue were investigated using an electron microscope. MU treatment led to a decrease in extracellular HA retention, as evidenced by a particle exclusion assay and immunohistochemical staining. Cell proliferation was suppressed by MU in a dose-dependent manner. The release of lactate dehydrogenase into the culture medium due to damage to the cellular membrane did not increase following MU administration. In tumor-inoculated mice, MU suppressed any increase in tumor volume and decreased the quantity of HA. Electron microscopy revealed that MU attenuated the intercellular space and caused it to be less cohesive. These data indicate that MU inhibits HA synthesis and reduces the amount of HA in the ECM while exhibiting no obvious cytotoxic effect. These findings suggest that MU has potential as a novel therapeutic agent for pancreatic cancer.

Novel REIC/Dkk-3-encoding adenoviral vector as a promising therapeutic agent for pancreatic cancer.

Reduced expression in immortalized cells (REIC)/dickkopf-3 (Dkk-3), a tumor suppressor gene, is downregulated in various cancers. We previously reported the tumor-inhibitory effects of the REIC/Dkk-3 gene, delivered by a conventional adenoviral vector (Ad-CAG-REIC) in pancreatic cancer. Here, we developed an Ad-REIC vector with a novel gene expression system, termed the super gene expression (SGE) system, and assessed its therapeutic effects relative to those of Ad-CAG-REIC in pancreatic cancer cells. Human pancreatic cancer cell lines ASPC1 and MIAPaCa2 were used. REIC/Dkk-3 expression was assessed by western blot analysis. Relative cell viability and apoptotic effects were examined in vitro. The anti-tumor effects of Ad-REIC treatment were assessed in the mouse xenograft model. Compared with Ad-CAG-REIC, Ad-SGE-REIC elicited a significant increase in REIC protein expression in the cells studied. Relative to Ad-CAG-REIC, Ad-SGE-REIC reduced cell viability and induced apoptosis in the ASPC1 and MIAPaCa2 cell lines in vitro, and achieved superior tumor growth inhibition in the mouse xenograft model. Compared with conventional Ad-REIC agents, Ad-SGE-REIC provided enhanced inhibitory effects against tumor growth. Our results indicate that Ad-SGE-REIC is an innovative therapeutic tool for pancreatic cancer.

A novel colchicine-based microtubule inhibitor exhibits potent antitumor activity by inducing mitochondrial mediated apoptosis in MIA PaCa-2 pancreatic cancer cells.

Colchicine, an antimitotic alkaloid isolated from Colchicum autumnale, is a classical drug for treatment of gout and familial Mediterranean fever. It causes antiproliferative effects through the inhibition of microtubule formation, which leads to mitotic arrest and cell death by apoptosis. Here, we report that a novel colchicine analog, 4o (N-[(7S)-1,2,3-trimethoxy-9-oxo-10-[3-(trifluoromethyl)-4-chlorophenylamino]-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide), which exhibited potent anticancer activities both in vitro and in vivo. In this study, 4o with excellent pharmacokinetic profile and no P-gp induction liability displayed strong inhibition of proliferation against various human cancer cell lines. However, pancreatic cancer cell line MIA PaCa-2 was found to be more sensitive towards 4o and showed strong inhibition in concentration and time-dependent manner. By increasing intracellular reactive oxygen species (ROS) levels, 4o induced endoplasmic reticular stress and mitochondrial dysfunction in MIA PaCa-2 cells. Blockage of ROS production reversed 4o-induced endoplasmic reticulum (ER) stress, calcium release, and cell death. More importantly, it revealed that increased ROS generation might be an effective strategy in treating human pancreatic cancer. Further 4o treatment induced mitotic arrest, altered the expression of cell cycle-associated proteins, and disrupted the microtubules in MIA PaCa-2 cells. 4o treatment caused loss of mitochondrial membrane potential, cytochrome c release, upregulation of Bax, downregulation of Bcl-2, and cleavage of caspase-3, thereby showing activation of mitochondrial mediated apoptosis. The in vivo anticancer activity of the compound was studied using sarcoma-180 (ascitic) and leukemia (P388 lymphocytic and L1210 lymphoid) models in mice and showed promising antitumor activity with the least toxicity unlike colchicine. Such studies have hitherto not been reported. Taken together, these findings highlighted that 4o, a potent derivative of colchicine, causes tumor regression with reduced toxicity and provides a novel anticancer candidate for the therapeutic use.

Leiodermatolide, a novel marine natural product, has potent cytotoxic and antimitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits antitumor activity.

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research.

Abstract 1733: Inhibition of embryonic stem cells gene expression in pancreatic cancer: Effects of metformin and indole-3-carbinol

Abstract Pancreatic cancer is among the top five deadliest cancers in developing countries and is rapidly increasing in minority populations, particularly African Americans. Understanding molecular mechanisms involved in the etiology and progression of this cancer can greatly improve patient prognosis. Cancer stem cells have been identified in pancreatic tumors and have been shown to contribute to its progression and resistance to standard treatments. Pancreatic cancer stem cells are subject to regulation by key embryonic stem cell transcription factors that are known to be aberrantly expressed in pancreatic cancer, such as SOX2, OCT4 and NANOG. Overexpression of OCT4, SOX2 and NANOG together or separately led to tumor transformation and tumor metastasis. These stem cell factors are known to promote self-renewal by interacting with other transcription factors. Using a human embryonic stem cell RT2 Profiler gene array, numerous stem cell genes were expressed in pancreatic cancer cell lines (MIAPaca2, Panc1). Both cell lines expressed high levels of SOX2, NANOG, CD44, GATA2, POU5F1 (OCT4) and other genes. Panc1 expressed high levels of SOX 17, while MIAPaCa2 expressed high levels of SOX 3,15 and 17. Treatment of cells with indole-3-carbinol alone at 100 and 200μM inhibited or decreased the expression of SOX2, NANOG, CD44, SOX15 and STAT3. However, treatment with metformin alone increased expression of SOX 15 and STAT3 in Panc1 cells but not in MIAPaCa2. Combination of metformin and indole-3-carbinol inhibited the expression of SOX2, NANOG, STAT3, and CD44. SOX2, POU5F1(OCT4) and NANOG are thought to be important players in various human cancers. OCT4 has been found to be expressed in 69% of PDAC and expression was shown too correlated to status and clinical state. High levels of OCT4 and NANOG has been found to correlate to worse prognosis and furthermore, contribute to multidrug-resistance and metastasis. This study have shown that the dietary agent, indole-3-carbinol alone and in combination with metformin down-regulate critical stem cell genes involved in maintenance of pluripotency and self-renewal of cancer stem cells. Citation Format: Beverly D. Lyn-Cook, Stancy Joseph, Beverly Word, George Hammons. Inhibition of embryonic stem cells gene expression in pancreatic cancer: Effects of metformin and indole-3-carbinol. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1733.

Synthesis of a series of novel dihydroartemisinin monomers and dimers containing chalcone as a linker and their anticancer activity

A new series of monomer and dimer derivatives of dihydroartemisinin (DHA) containing substituted chalcones as a linker were synthesized and investigated for their cytotoxicity in human cancer cell lines HL-60 (leukemia), Mia PaCa-2 (pancreatic cancer), PC-3 (prostate cancer), LS180 (colon cancer) and HEPG2 (hepatocellular carcinoma). Some of these derivatives have greater antiproliferative and cytotoxic effects in tested cell lines than parent compound DHA. The structures of the all compounds were confirmed by IR, 1H NMR and mass spectral data. Among the new derivatives, compounds 8, 14, 15, 20 and 24 were found to be more active than parent DHA against tested human cancer cell lines. DHA derivatives were found to be most active in human leukemia cell lines with compounds 8, 14, 15, 20 and 24 showed IC50 values less than 1 μM for 48 h whereas DHA has IC50 value of 2 μM at same time period. The most potent compounds 8 with IC50 = 0.3 μM (at par with doxorubicin (IC50 = 0.3 μM)) and 15 with IC50 = 0.4 μM, of the series, six and three times active than DHA (with IC50 = 2 μM) respectively were selected for further mechanistic work in human leukemia HL-60 cells.

262. Therapeutic Suppression of the KRAS-MYC Oncogenic Axis in Human Pancreatic Cancer Xenografts with U1 Adaptor Oligonucleotide / RGD Peptide Conjugates

U1 Adaptors are synthetic oligonucleotides that enable the U1 small nuclear ribonucleoprotein (U1 snRNP) complex to stably bind within the terminal exon of a specific pre-mRNA. This interferes with the obligatory polyadenylation step in mRNA maturation, causing selective destruction of the targeted mRNA inside the nucleus. In contrast to siRNA or antisense oligos, U1 Adaptors can accept extensive covalent modifications for nuclease resistance, targeted delivery or in-vivo imaging without loss of silencing activity, offering important advantages as therapeutic agents. A panel of candidate U1 Adaptors targeting human KRAS (KRAS Adaptors) was screened in vitro using the human pancreatic cancer cell line MIA-PaCa2. The best candidates reduced KRAS mRNA expression by up to 76% - as effectively as siRNA controls. Reduced KRAS protein expression was confirmed by western blot. Inhibition of cell growth in vitro and increased apoptosis were seen for both the MIA-PaCa2 (KRASG12C) and Panc1(KRASG12D) cell lines, but not in BxPC3, a KRASwildtype pancreatic cancer cell line. In a parallel project, U1 Adaptors targeting human MYC mRNA were designed and screened in B-cell lymphoma lines, where the best candidates reduced MYC mRNA levels by over 95%. Because of the observed relationship between activating KRAS mutation and MYC overexpression in pancreatic cancers, MYC Adaptors were tested in MIA-PaCa2 cells. MYC Adaptors also inhibited cell growth and increased apoptosis in vitro. U1 Adaptors were tested for efficacy in mice bearing subcutaneous MIA-PaCa2 xenograft tumors. For in-vivo delivery, Adaptor oligos were directly conjugated to a cyclic RGD-motif peptide (cRGD), which is a targeting ligand for specific integrin-family receptors overexpressed on parenchyma and endothelial cells of many solid tumors. Alternately, U1 Adaptor oligos were linked to “internalizing” RGD (iRGD), a variant RGD peptide that also triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin-1. KRAS Adaptors linked to cRGD or iRGD were administered by tail vein injections twice weekly for three to four weeks. Over a series of experiments, tumor growth was inhibited by averages of 68% to 93%. Tumor stasis or regression occurred in some treated mice. In a pilot study, MYC Adaptors conjugated to iRGD peptide were also highly effective in suppressing tumor growth and inducing tumor regression. Excised tumors were analyzed by qPCR and western blot, which confirmed reductions of the targeted mRNAs and proteins. We have shown that U1 Adaptors conjugated to tumor-targeting / tumor-penetrating peptides can effectively target human KRAS and MYC oncogenes in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS, MYC and possibly other oncogene targets in pancreatic cancer.

Inhibition of Cell Proliferation and Growth of Pancreatic Cancer by Silencing of Carbohydrate Sulfotransferase 15 In Vitro and in a Xenograft Model.

Chondroitin sulfate E (CS-E), a highly sulfated glycosaminoglycan, is known to promote tumor invasion and metastasis. Because the presence of CS-E is detected in both tumor and stromal cells in pancreatic ductal adenocarcinoma (PDAC), multistage involvement of CS-E in the development of PDAC has been considered. However, its involvement in the early stage of PDAC progression is still not fully understood. In this study, to clarify the direct role of CS-E in tumor, but not stromal, cells of PDAC, we focused on carbohydrate sulfotransferase 15 (CHST15), a specific enzyme that biosynthesizes CS-E, and investigated the effects of the CHST15 siRNA on tumor cell proliferation in vitro and growth in vivo. CHST15 mRNA is highly expressed in the human pancreatic cancer cell lines PANC-1, MIA PaCa-2, Capan-1 and Capan-2. CHST15 siRNA significantly inhibited the expression of CHST15 mRNA in these four cells in vitro. Silencing of the CHST15 gene in the cells was associated with significant reduction of proliferation and up-regulation of the cell cycle inhibitor-related gene p21CIP1/WAF1. In a subcutaneous xenograft tumor model of PANC-1 in nude mice, a single intratumoral injection of CHST15 siRNA almost completely suppressed tumor growth. Reduced CHST15 protein signals associated with tumor necrosis were observed with the treatment with CHST15 siRNA. These results provide evidence of the direct action of CHST15 on the proliferation of pancreatic tumor cells partly through the p21CIP1/WAF1 pathway. Thus, CHST15-CS-E axis-mediated tumor cell proliferation could be a novel therapeutic target in the early stage of PDAC progression.

Pharmacodynamic modeling of combined chemotherapeutic effects predicts synergistic activity of gemcitabine and trabectedin in pancreatic cancer cells.

This study investigates the combined effects of gemcitabine and trabectedin (ecteinascidin 743) in two pancreatic cancer cell lines and proposes a pharmacodynamic (PD) model to quantify their pharmacological interactions. Effects of gemcitabine and trabectedin upon the pancreatic cancer cell lines MiaPaCa-2 and BxPC-3 were investigated using cell proliferation assays. Cells were exposed to a range of concentrations of the two drugs, alone and in combination. Viable cell numbers were obtained daily over 5 days. A model incorporating nonlinear cytotoxicity, transit compartments, and an interaction parameter ψ was used to quantify the effects of the individual drugs and combinations. Simultaneous fitting of temporal cell growth profiles for all drug concentrations provided reasonable cytotoxicity parameter estimates (the cell killing rate constant K max and the sensitivity constant KC50) for each drug. The interaction parameter ψ was estimated as 0.806 for MiaPaCa-2 and 0.843 for BxPC-3 cells, suggesting that the two drugs exert modestly synergistic effects. The proposed PD model enables quantification of the temporal profiles of drug combinations over a range of concentrations with drug-specific parameters. Based upon these in vitro studies, trabectedin may have augmented benefit in combination with gemcitabine. The PD model may have general relevance for the study of other cytotoxic drug combinations.

A novel triazole derivative of betulinic acid induces extrinsic and intrinsic apoptosis in human leukemia HL-60 cells

In an attempt to arrive at more potent cytotoxic agent than the bioactive natural product betulinic acid, influence of small structural modifications of its 1, 2, 3 triazole derivatives tethered at C-28 and both C3, C-28 using click chemistry approach has been studied. The chemically characterized triazoles have been screened for in vitro cytotoxicity against four human cancer cell lines HL-60, MiaPaCa-2, PC-3 and A549 which has allowed to identify triazole derivative 28{1N (4-fluoro phenyl)-1H-1, 2, 3-triazol-4-yl} methyloxy betulinic ester having better potency profile than the parent compound with IC50 values in the range of 5–7 μM. It caused disruption of mitochondrial membrane potential, rendered Bcl-2 cleavage, Bax translocation and decrease Bcl-2/Bax ratio. These events are accompanied by activation of caspases −9, -3, which cleave the PARP-1. It also induces caspase-8, which is involved in extrinsic apoptotic pathway. Therefore, it induces apoptosis through both intrinsic and extrinsic pathways in human leukemia HL-60 cells.

Evaluation of low-dose proton beam radiation efficiency in MIA PaCa-2 pancreatic cancer cell line vitality and H2AX formation

Background and objectiveThe aim of this study was to evaluate the efficiency of proton beam irradiation in pancreatic cancer cell line MIA PaCa-2 and its role in the cell cycle, apoptosis, and formation of histone γH2AX in different reparation times (72-h follow-up). Material and methodsThe MIA PaCa-2 pancreatic carcinoma cell line was irradiated with 1.6-Gy proton beam. After irradiation, cell viability was measured colorimetrically, and the cell cycle, apoptosis, and γH2AX expression were evaluated on a FACScan cytometer. ResultsLow-dose proton beam irradiation had an effect on the MIA PaCa-2 tumor cell line already 1h after exposure, but maximal lethality was reached after 72h postirradiation with a cell viability rate of 24%. The cell cycle went into partial G1/0 arrest, and was released after 72h. The expression of γH2AX was strong and its levels were significantly elevated as late as 48h post radiation. The apoptosis levels increased with post radiation incubation time to reach 79% after 72h. ConclusionsOur data demonstrate that low-doses proton beam irradiation had an effect on MIA PaCa-2 pancreatic carcinoma cell line. Full extent of irradiation had an impact only 24h postirradiation, triggering DNA arrested cell cycle in G1/0 phase. Formed DNA DSBs were found to be repaired via the NHEJ pathway mechanism within 72h. Unsuccessful repaired DSBs induced apoptotic cell death. After 72h reparation processes were completed, and cell cycle was released from arrest in G1/0 phase.

Synthesis, in vitro, and in vivo evaluation of novel functionalized quaternary ammonium curcuminoids as potential anti-cancer agents.

Novel functionalized quaternary ammonium curcuminoids have been synthesized from piperazinyl curcuminoids and Baylis-Hillman reaction derived allyl bromides. These molecules are found to be highly water soluble with increased cytotoxicity compared to native curcumin against three cancer cell lines MIAPaCa-2, MDA-MB-231, and 4T1. Preliminary in vivo toxicity evaluation of a representative curcuminoid 5a in healthy mice indicates that this molecule is well tolerated based on normal body weight gains compared to control group. Furthermore, the efficacy of 5a has been tested in a pancreatic cancer xenograft model of MIAPaCa-2 and has been found to exhibit good tumor growth inhibition as a single agent and also in combination with clinical pancreatic cancer drug gemcitabine.

Abstract A25: Immunotherapy using LOAd700 armed with CD40 ligand controls experimental pancreatic cancer and activates immune responses

Abstract The aim of this study was to evaluate the efficacy of LOAd immunotherapy to treat pancreatic cancer in a xenograft model as well as to determine the capacity of LOAds to stimulate the immune system in in vitro models. LOAd viruses are oncolytic adenoviruses (5/35) armed with immunostimulatory genes in order to shift the tumor milieu towards immune activation at the same time providing release and spread of tumor antigens due to oncolysis. In this manner, LOAd virus is an antigen-independent immunotherapy for various solid tumors. LOAd armed with CD40 ligand (LOAd700) was constructed from the ICOVIR system by changing the virus shaft and knob to that of serotype 35 to broaden virus binding and entry into cells (pending patent EP14163704). Oncolysis in OCOVIRs is restricted to cells with a disrupted Rb pathway. Viruses were produced using A549 cells. The pancreatic cancer cell lines BxPC3, Panc01, MiaPaCa2 and PaCa3 were used for in vitro evaluation and Panc01 was used in the xenograft model in Nu/Nu mice. Human dendritic cells (DCs) were obtained by differentiation of CD14+ monocytes with GM-CSF and IL4 for 7 days. LOAd viruses infected both the panel of pancreatic tumor cell lines and human DCs with high efficacy and both the cell lines and the DCs expressed the CMV driven transgene/s post transduction. The oncolytic LOAd virus did not kill DCs nor healthy pancreatic cells present in donor islet cell isolation surplus material. In contrast, oncolysis was restricted to the tumor cell lines. The LOAd-transduced DCs increased markers of maturation such as MHC II, CD86, CD70 and CD83 and produced high levels of IL12. LOAd stimulated DCs were able to activate and expand antigen-specific T cells and NK cells as demonstrated in a CMV in vitro system. LOAd viruses could control tumor growth in a xenograft immunodeficient model due to oncolysis alone. In conclusion, LOAd700 can kill pancreatic tumor cells in vitro and control growing tumor in mice due to oncolysis alone. Further, upon DC transduction, LOAd viruses mature DCs to efficient antigen presenters and stimulators of Th1 effector cells such as T cells and NK cells. LOAd is an interesting new immunotherapy for solid malignancies including pancreatic cancer. Citation Format: Emma Svensson, Rafael Moreno, Ioanna Milenova, Lisa Christiansson, Ramon Alemany, Angelica Loskog. Immunotherapy using LOAd700 armed with CD40 ligand controls experimental pancreatic cancer and activates immune responses. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A25.

Hyaluronic acid-conjugated polyamidoamine dendrimers for targeted delivery of 3,4-difluorobenzylidene curcumin to CD44 overexpressing pancreatic cancer cells

The current study was aimed to develop a targeted dendrimer formulation of 3, 4-difluorobenzylidene curcumin (CDF) and evaluate its potential in CD44 targeted therapy for pancreatic cancer. Using amine terminated fourth generation poly(amidoamine) (PAMAM) dendrimer nanocarrier and hyaluronic acid (HA) as a targeting ligand, we engineered a CD44-targeted PAMAM dendrimer (HA-PAMAM) formulation of CDF. The resulting dendrimer nanosystem (HA-PAMAM-CDF) had a particle size and surface charge of 9.3±1.5nm and −7.02±9.53mV, respectively. When CD44 receptor overexpressing MiaPaCa-2 and AsPC-1 human pancreatic cancer cells were treated with HA-PAMAM-CDF, a dose-dependent cytotoxicity was observed. Furthermore, blocking the CD44 receptors present on the MiaPaCa-2 cells using free excess soluble HA prior to treatment with HA-PAMAM-CDF nano-formulation resulted in 1.71 fold increase in the IC50 value compared to non-targeted formulation (PAMAM-CDF), confirming target specificity of HA-PAMAM-CDF. Additionally, HA-PAMAM-CDF formulation when compared to PAMAM-CDF, displayed higher cellular uptake in MiaPaCa-2 cancer cell lines as shown by fluorescence studies. In summary, the novel CD44 targeted dendrimer based nanocarriers appear to be proficient in mediating site-specific delivery of CDF via CD44 receptors, with an improved therapeutic margin and safety.

Glycogen synthase kinase-3 inhibitor AR-A014418 suppresses pancreatic cancer cell growth via inhibition of GSK-3-mediated Notch1 expression

BackgroundGlycogen synthase kinase-3 (GSK-3) can act as either a tumour promoter or suppressor by its inactivation depending on the cell type. There are conflicting reports on the roles of GSK-3 isoforms and their interaction with Notch1 in pancreatic cancer. It was hypothesized that GSK-3α stabilized Notch1 in pancreatic cancer cells thereby promoting cellular proliferation. MethodsThe pancreatic cancer cell lines MiaPaCa2, PANC-1 and BxPC-3, were treated with 0–20 μM of AR-A014418 (AR), a known GSK-3 inhibitor. Cell viability was determined by the MTT assay and Live-Cell Imaging. The levels of Notch pathway members (Notch1, HES-1, survivin and cyclinD1), phosphorylated GSK-3 isoforms, and apoptotic markers were determined by Western blot. Immunoprecipitation was performed to identify the binding of GSK-3 specific isoform to Notch1. ResultsAR-A014418 treatment had a significant dose-dependent growth reduction (P< 0.001) in pancreatic cancer cells compared with the control and the cytotoxic effect is as a result of apoptosis. Importantly, a reduction in GSK-3 phosphorylation lead to a reduction in Notch pathway members. Overexpression of active Notch1 in AR-A014418-treated cells resulted in the negation of growth suppression. Immunoprecipitation analysis revealed that GSK-3α binds to Notch1. ConclusionsThis study demonstrates for the first time that the growth suppressive effect of AR-A014418 on pancreatic cancer cells is mainly mediated by a reduction in phosphorylation of GSK-3α with concomitant Notch1 reduction. GSK-3α appears to stabilize Notch1 by binding and may represent a target for therapeutic development. Furthermore, downregulation of GSK-3 and Notch1 may be a viable strategy for possible chemosensitization of pancreatic cancer cells to standard therapeutics.

Abstract 4200: Novel treatment strategy for pancreatic cancer

Abstract Background: Solid association exists between high expression level of glycogen synthase kinase 3 beta (GSK-3β) and pancreatic cancer (PaCa) progression in humans. GSK-3β inhibition in a mouse orthotopic model of PaCa induced tumor shrinkage. However, GSK-3β inhibition stimulates pro-metastasis epithelial to mesenchymal transition (EMT). HDAC1/2 is involved in regulation of EMT in PaCa cells. We developed a novel strategy based on the combination of GSK-3β inhibition with inhibition of HDAC1 to prevent cancer cell survival, EMT and metastasis in vitro and in vivo. We designed, synthesized and tested a novel dual-inhibitor CSME-357 which inhibits both the proteins. Methods: Pdx-Cre;LSL-Kras (KC) mice were ip injected with GSK-3β inhibitor TDZD-8 and/or HDAC1/2 inhibitor Saha. Pancreatic intraepithelial neoplasia (PanIN) lesions, fibrosis, and inflammation were measured by IHC. Pancreatic cancer cell lines MIA PaCa-2, BxPC3, AsPC1 were cultured in the presence of HDAC and/or GSK-3β inhibitors or with the novel compound CSME-357 which inhibits both proteins. Cell survival and apoptosis were measured by MTT assay and DNA fragmentation, respectively. EMT and cancer stemness markers, histone acetylation and GSK-3β level were measured by IHC and Western in tissue and cells. Invasion of the cells was measured by invasion assay. Ability of the cells to form metastatic niches in nude mice was measured by injecting PaCa cells in mice tails in the presence or absence of CSME-357 and live mice were analyzed by MRI and by tissue analysis after necropsy. Results: Treatment of KC mice with Saha decreased the level of PanIN lesions, fibrosis, inflammation, and EMT. TDZD-8 potentiated the effect of Saha on PanIN prevention. Saha and TDZD-8 decreased EMT in KC mice. Pharmacological and molecular inhibitions of GSK-3β significantly, dose dependently, and synergistically decreased proliferation and at a lesser extent stimulated apoptosis in PaCa cell lines. GSK-3β inhibition stimulated EMT in PaCa cells; whereas, Saha reversed this effect. CSME-357 significantly and more potently (compared to the combination of two inhibitors) decreased proliferation, invasion and EMT and stimulated apoptosis in PaCa cells. Significance was achieved at 300nM. Toxicity assays showed no to very little toxicity of the novel compound. CSME-357 decreased the ability of cancer cells to form metastatic niches in nude mice. Conclusion: Combination of HDAC1 and GSK-3β inhibitions induced a triple beneficial effect by decreasing proliferation, stimulating apoptosis and inhibiting EMT/metastasis. CSME-357 is a novel compound with promising anti-cancer properties. CSME-357 is a very potent inhibitor for both GSK-3β and HDAC1/2; in addition, CSME-357 prevents cancer cell growth, resistance to apoptosis, EMT, invasion and metastasis. Note: This abstract was not presented at the meeting. Citation Format: Chintan Chheda, Ramachandran Murali, Paul Grippo, Dale Uyeminami, Kent Pinkerton, Stephen Pandol, Mouad Edderkaoui. Novel treatment strategy 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 4200. doi:10.1158/1538-7445.AM2015-4200