Cancer Cell BxPC-3 Research Articles

DNA binding studies and in-vitro anticancer studies of novel lanthanide complexes

Pancreatic cancer, is an aggressive type of cancer and the most common malignancy with a poor prognosis regarding metastatic disease (survival < 10 %). The development of Novel chemotherapeutic drugs holds significant prospects for practical applications. Here, this work focuses on the interaction between two lanthanide complexes, Yb-BZA and Er-BZA, with DNA, as well as their anticancer activity against pancreatic cancer. The relationship between complexes and DNA is revealed by fluorescence, absorption spectral titration, cyclic voltammetric (CV) experiments, indicating that the Yb-BZA and Er-BZA interact with FS-DNA by bind groove. Moreover, molecular docking technology was utilized to confirm the binding of Yb-BZA and Er-BZA with 1BNA and 4AV1. The cytotoxic effects of Yb-BZA and Er-BZA on cancer cells BxPC-3 were evaluated, Yb-BZA (IC50 = 6.459 μg/mL) is more effective than oxaliplatin (IC50 = 16.46 μg/mL) evaluated using cytotoxicity assay. Yb-BZA and Er-BZA has the potential to become a chemotherapy drug for pancreatic cancer cells.

A novel chalcone derivative exerts anticancer effects by promoting apoptotic cell death of human pancreatic cancer cells

Aggressive pancreatic cancer is typically treated using chemotherapeutics to reduce the tumor pre-operatively and prevent metastasis post-operatively, as well as surgical approaches. In the present study, we synthesized a hydroxyl group-introduced chalcone derivative (1, IC50 = 32.1 μM) and investigated its potential as an anticancer drug candidate by evaluating its apoptosis-promoting effects on BXPC-3 cancer cells. The viability of BXPC-3 cells treated with 1 was measured using the water-soluble tetrazolium 1 reagent. BXPC-3 cells induced by 1 were stained with diverse probes or antibodies, such as ethidium homodimer-1, Hoechst, anti-Ki67, and MitoTracker. Protein expression was measured using an immunoblotting assay, and mRNA expression was determined using real-time polymerase chain reaction. Apoptotic molecular features, such as lipid accumulation and protein degradation, were monitored directly using stimulated Raman scattering microspectroscopy. Through incubation time- and concentration-dependent studies of 1, we found that it significantly reduced the proliferation and increased the number of apoptotic BXPC-3 cells. Compound 1 induced mitochondrial dysfunction, phosphorylation of p38, and caspase 3 cleavage. These results indicate that 1 is a potential therapeutic agent for pancreatic cancer, providing valuable insights into the development of new anticancer drug candidates.

Abstract 6285: MicroRNA-217 affects tumor microenvironment and enhances the chemosensitivity of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and an inherently chemoresistant tumor. Dense fibrotic stroma is a major obstacle for drug delivery to the tumor cells in PDAC. Pancreatic stellate cells (PSCs) are the major stromal cells responsible for fibrotic microenvironment. MicroRNA-217 (miR-217) is known mainly as a tumor suppressor. However, the detailed roles of miR-217 on pathophysiology of PDAC remain unclarified. Methods: Expressions levels of miR-217 were measured in surgically resected human PDAC samples. Using Lipofectamine RNAiMAX, miR-217 or negative control miR (NC) were transfected in human pancreatic cancer cells, Panc-1, MIApaca-2, BxPC-3 and murine PAN02 cells, and chemosensitivities to gemcitabine and paclitaxel were investigated by MTS assay. Human hPSC-1 (obtained from Riken cell bank, Japan) was cultured in 20%FBS+DMEM and the changes of phenotypes and expression levels of miR-217 after the stimulation with TGF-β1 was measured by fluorescein microscope and digital PCR, respectively. Results: The expression level of miR-217 was markedly downregulated in PDAC tissues than in noncancerous pancreatic tissues (P&amp;lt;0.001, n=9). Overexpression of miR-217 significantly inhibited the proliferation of Panc-1, MIApaca-2 and BxPC-3 (P&amp;lt;0.001) and migration of Panc-1, MIApaca-2 and PAN02 (P&amp;lt;0.05). Lipofection of miR-217 further reduced the number of PAN02 exposed to 1-5nM gemcitabine (P&amp;lt;0.0001) and 0.01-0.1uM paclitaxel (P=0.0085) which resulted in the reduced the IC-50 as compared with NC. Human PSC-1 stimulated with TGF-β1 (10ng/ml) significantly reduced miR-217 expression compared to nascent cells. The stimulation with TGF-β1 upregulated the expression of cancer associated fibroblast (CAF) markers, α-smooth muscle actin (SMA) and vimentin. However, the expression levels of these markers were significantly reduced by the kipofection of miR-217. Conclusion: MiR-217 affects both cancer and stromal cells in PDAC which may regulate chemosensitivity in PDAC. Restoring miR-217 levels in tumor cells and/or PSCs could be a future therapeutic strategy. Citation Format: Yuki Kaneko, HIdeyuki Ohzawa, Yuki Kimura, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Naohiro Sata, Joji Kitayama. MicroRNA-217 affects tumor microenvironment and enhances the chemosensitivity of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6285.

Zebrafish Xenograft Model for Studying Pancreatic Cancer-Instructed Innate Immune Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) has up to half the tumor mass of tumor-associated myeloid cells. Myeloid innate immune cells play important roles in regulating cancer cell recognition and tumor growth. PDAC cells often mold myeloid cells into pro-tumoral state to fuel cancer growth and induce immune suppression. However, how tumor cells educate the innate immune responses remains largely unknown. In this study, we used four different human PDAC cell lines (PANC1, BxPC3, AsPC1, and CFPAC1) to establish the zebrafish xenograft model and investigated the interaction between pancreatic cancer and innate immune cells. The primary tumor-derived cancer cells PANC1 and BxPC3 activated innate immune anti-tumoral responses efficiently, while cancer cells from metastatic tissues AsPC1 and CFPAC1 induced an innate immune suppression and educated innate immune cells towards pro-tumoral state. Chemical conversion of innate immune cells to anti-tumoral state inhibited tumor growth for AsPC1 and CFPAC1. Moreover, genetic and pharmacological inhibition of macrophages also significantly reduced tumor growth, supporting the important roles of macrophages in innate immune suppression. REG4 expression is high in AsPC1 and CFPAC1. Knockdown of REG4 induced innate immune activation and reduced tumor growth in the xenografts, indicating that REG4 is a beneficial target for PDAC therapy. Our study provides a fast in-vivo model to study PDAC-innate immune interaction and their plasticity that could be used to study the related mechanism as well as identify new drugs to enhance immunotherapy.

Cytoglobin attenuates pancreatic cancer growth via scavenging reactive oxygen species

Pancreatic cancer is a highly challenging malignancy with extremely poor prognosis. Cytoglobin (CYGB), a hemeprotein involved in liver fibrosis and cancer development, is expressed in pericytes of all organs. Here, we examined the role of CYGB in the development of pancreatic cancer. CYGB expression appeared predominately in the area surrounding adenocarcinoma and negatively correlated with tumor size in patients with pancreatic cancer. Directly injecting 7, 12-dimethylbenz[a]anthracene into the pancreatic tail in wild-type mice resulted in time-dependent induction of severe pancreatitis, fibrosis, and oxidative damage, which was rescued by Cygb overexpression in transgenic mice. Pancreatic cancer incidence was 93% in wild-type mice but only 55% in transgenic mice. Enhanced CYGB expression in human pancreatic stellate cells in vitro reduced cellular collagen synthesis, inhibited cell activation, increased expression of antioxidant-related genes, and increased CYGB secretion into the medium. Cygb-overexpressing or recombinant human CYGB (rhCYGB) -treated MIA PaCa-2 cancer cells exhibited dose-dependent cell cycle arrest at the G1 phase, diminished cell migration, and reduction in colony formation. RNA sequencing in rhCYGB-treated MIA PaCa-2 cells revealed downregulation of cell cycle and oxidative phosphorylation pathways. An increase in MIA PaCa-2 cell proliferation and reactive oxygen species production by H2O2 challenge was blocked by rhCYGB treatment or Cygb overexpression. PANC-1, OCUP-A2, and BxPC-3 cancer cells showed similar responses to rhCYGB. Known antioxidants N-acetyl cysteine and glutathione also inhibited cancer cell growth. These results demonstrate that CYGB suppresses pancreatic stellate cell activation, pancreatic fibrosis, and tumor growth, suggesting its potential therapeutic application against pancreatic cancer.

Different pancreatic cancer microenvironments convert iPSCs into cancer stem cells exhibiting distinct plasticity with altered gene expression of metabolic pathways

BackgroundCancer stem cells (CSCs) are generated under irregular microenvironment in vivo, of which mimic is quite difficult due to the lack of enough information of the factors responsible for cancer initiation. Here, we demonstrated that mouse induced pluripotent cells (miPSCs) reprogrammed from normal embryonic fibroblasts were susceptible to the microenvironment affected by cancer cells to convert into CSCs in vivo.MethodsThree different pancreatic cancer line cells, BxPC3, PANC1, and PK8 cells were mixed with miPSCs and subcutaneously injected into immunodeficient mice. Tumors were evaluated by histological analysis and cells derived from iPSCs were isolated and selected from tumors. The isolated cells were characterized for cancer stem cell characters in vitro and in vivo as well as their responses to anticancer drugs. The impact of co-injection of iPSCs with cancer cells on transcriptome and signaling pathways of iPSCs was investigated.ResultsThe injection of miPSCs mixed with human pancreatic cancer cells into immunodeficient mice maintained the stemness of miPSCs and changed their phenotype. The miPSCs acquired CSC characteristics of tumorigenicity and self-renewal. The drug responses and the metastatic ability of CSCs converted from miPSCs varied depending on the microenvironment of cancer cells. Interestingly, transcriptome profiles of these cells indicated that the pathways related with aggressiveness and energy production were upregulated from the levels of miPSCs.ConclusionsOur result suggests that cancer-inducing microenvironment in vivo could rewire the cell signaling and metabolic pathways to convert normal stem cells into CSCs.

WITHDRAWN: Molecular Docking, synthesis, anticancer assessment and ADME study of naproxen containing imidazole ring derivatives

Abstract Overexpression of The epidermal growth factor receptor EGFR, a part of the tyrosine kinase family, is linked to drug resistance and poor prognosis in many cancers, like lung and pancreatic cancer. As a result, targeting EGFR and its downstream signaling cascades is now seen as a promising cancer treatment strategy. A new class of Imidazolone-schieff base derivatives has been developed. Before starting the synthesis, docking simulation has been used to insert compounds within the EGFR/TK structure active site using erlotinib as a standard, which exhibit significant tyrosine kinase inhibitor activities because of the hydrogen bonding and short contact with crucial amino acid of EGFR. The identification of newly synthesized compounds was done using IR, 1H NMR, 13CNMR spectroscopic methods as well as some physicochemical properties and in vitroTheir antiproliferative properties were assessed on A549 and BxPC3 cancer cell lines using MTT assay. Notably, compounds 10 and 11 exhibit a valuable cytotoxic effect on A549 cancer cell with an IC50 values (12.3 μM, 9.4 μM) respectively when compared with Erlotinib (IC50 = 25.23 μM) Moreover, compound 11 show significant activity on BxPC3 cancer cell with an IC50 value (18.3 μM). Pharmacokinetic and physicochemical properties of the final compounds predicted by using ADME evaluations. Based on this study, according to the findings, the synthesized compounds have a high oral bioavailability and effective GI absorption. It can be concluded that compound 11 and 10 could be potentially distinct anti-cancer derivatives which would aid in the creation of new therapeutics.

Identification of the Biosynthetic Gene Cluster of Thermoactinoamides and Discovery of New Congeners by Integrated Genome Mining and MS-Based Molecular Networking.

The putative non-ribosomal peptide synthetase (NRPS) gene cluster encoding the biosynthesis of the bioactive cyclohexapeptide thermoactinoamide A (1) was identified in Thermoactinomyces vulgaris DSM 43016. Based on an in silico prediction, the biosynthetic operon was shown to contain two trimodular NRPSs, designated as ThdA and ThdB, respectively. Chemical analysis of a bacterial crude extract showed the presence of thermoactinoamide A (1), thereby supporting this biosynthetic hypothesis. Notably, integrating genome mining with a LC-HRMS/MS molecular networking-based investigation of the microbial metabolome, we succeeded in the identification of 10 structural variants (2–11) of thermoactinoamide A (1), five of them being new compounds (thermoactinoamides G-K, 7–11). As only one thermoactinoamide operon was found in T. vulgaris, it can be assumed that all thermoactinoamide congeners are assembled by the same multimodular NRPS system. In light of these findings, we suggest that the thermoactinoamide synthetase is able to create chemical diversity, combining the relaxed substrate selectivity of some adenylation domains with the iterative and/or alternative use of specific modules. In the frame of our screening program to discover antitumor natural products, thermoactinoamide A (1) was shown to exert a moderate growth-inhibitory effect in BxPC-3 cancer cells in the low micromolar range, while being inactive in PANC-1 and 3AB-OS solid tumor models.

Synthesis and Cytotoxic Activity Evaluation of New Cu(I) Complexes of Bis(pyrazol-1-yl) Acetate Ligands Functionalized with an NMDA Receptor Antagonist.

In the present article, copper(I) complexes of bis(pyrazol-1-yl) carboxylic acid (LH), bis(3,5-dimethylpyrazol-1-yl) carboxylic acid (L2H), and bis(pyrazol-1-yl) acetates conjugated with an N-methyl-d-aspartate (NMDA) receptor antagonist (LNMDA or L2NMDA) and phosphane ligands (triphenylphosphine or 1,3,5-triaza-7-phosphaadamantane) were synthesized. The selection of an NMDA antagonist for the coupling with LH and L2H was suggested by the observation that NMDA receptors are expressed and play a role in different types of cancer models. All the new complexes showed a significant antitumor activity on a panel of human tumor cell lines of different histology, with cisplatin-sensitive, cisplatin-resistant, or multi-drug-resistant phenotype. Their half maximal inhibitory concentration (IC50) values were in the low- and sub-micromolar range and, in general, significantly lower than that of cisplatin. Interestingly, the fact that all the complexes proved to be significantly more active than cisplatin even in three-dimensional (3D) spheroids of H157 and BxPC3 cancer cells increased the relevance of the in vitro results. Finally, morphological analysis revealed that the most representative complex 8 induced a massive swelling of the endoplasmic reticulum (ER) membrane, which is a clear sign of ER stress.

Cytotoxic Activity of Selected Nigerian Medicinal Plants

ABSTRACTThe study investigated eight Nigerian medicinal plants: Artocarpus communis fruit (Ac-F), Cassia sieberiana root bark (Cs-R), Eugenia uniflora leaf (Eu-L), Hibiscus rosa-sinensis flower (Hr-F), Laportea aestuans aerial part (La-A), Massularia acuminata stem bark (Ma-S), Trichilia heudelotii leaf (Th-L), and Xylopia aethiopica fruit (Xa-F), for anticancer activity. The alcoholic extracts of the plants were tested against human prostate DU-145, breast JIMT-1, and pancreatic BxPC-3 cancer cell lines using Methanethiosulfonate (MTS) assay, where chlorambucil was used as positive control. The active fractions were solvent-partitioned and tested against the three cancer cell lines. The results showed a concentration dependent decrease in cancer cell viability and cytotoxicity of four of the extracts, namely Massularia acuminata stem bark (Ma-S), Xylopia aethioipica fruit (Xa-F), Laportea aestuans aerial part (La-A), and Eugenia uniflora leaf (Eu-L). The petroleum ether fraction of X. aethiopica fruit (XP) demonstrated the greatest anticancer activity. At 75 µg mL−1, XP (37.2 ± 4.4%) was comparable to chlorambucil (39.88 ± 2.41%), in its cytotoxicity against breast cancer cell line JIMT-1.

The effects of microRNA-181b on proliferation and invasion of pancreatic cancer cells

Objective To investigate the effects of microRNA-181b (miR-181b) on cell proliferation and invasion of pancreatic cancer cells. Methods MiR-181b expression levels in 3 pancreatic cancer cell lines PANC-1, BXPC-3, ASPC-1 and normal pancreatic HTERT-HPNE cells were observed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). PANC-1 cells were transfected with 50 nmol/L miR-181b inhibitor or negative control using Lipofectamine 2000. After transfection, the expression of miR-181b was measured by RT-qPCR. Cell proliferation was tested by methylthiazol tetrazolium (MTT) assay. Cell invasion was detected by transwell. Bioinformatics prediction and luciferase activity tests were to verify phosphate and tension homology deleted on chromsome ten (PTEN) was the targeted gene of miR-181b. The expression levels of PTEN, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were measured by RT-qPCR. Results Compared with the HTERT-HPNE cells (1.00±0.06), the expression level of miR-181b in PANC-1 (2.84±0.16), BXPC-3 (1.74±0.18), ASPC-1 (2.21±0.20) was significantly increased. After transfection with 50 nmol/L miR-181b inhibitor, there was no obvious change in PANC-1 cell proliferation (P>0.05), and cell invasive ability was inhibited (P<0.01). Bioinformatics predicted PTEN might be the targeted gene of miR-181b. Luciferase experiments showed that luciferase activity of wild-type PTEN group fell by 39% after transfection of miR-181b mimics, but there was no statistical difference in the mutant group. After transfection with miR-181b inhibitor, PTEN was significantly increased (1.85±0.06 vs. 1.01±0.08, P<0.05), and MMP-2 (0.59±0.02 vs. 1.01±0.04) and MMP-9 (0.66±0.02 vs. 0.99±0.07) were significantly decreased (P<0.05). Conclusion These results demonstrate that miR-181b promotes proliferation and invasion in pancreatic cancer cells, which may be partly related to effecting MMP-2 and MMP-9 expression by PTEN. Therefore, it may be a new target for the biologic therapy for pancreatic cancer. Key words: Pancreatic cancer; MicroRNA-181b; Proliferation; Invasion

KRas4B-PDE6\u03b4 complex stabilization by small molecules obtained by virtual screening affects Ras signaling in pancreatic cancer

BackgroundThe GTPase KRas4B has been utilized as a principal target in the development of anticancer drugs. PDE6δ transports KRas4B to the plasma membrane, where it is released to activate various signaling pathways required for the initiation and maintenance of cancer. Therefore, identifying new small molecules that prevent activation of this GTPase by stabilizing the KRas4B-PDE6δ molecular complex is a practical strategy to fight against cancer.MethodsThe crystal structure of the KRas4B-PDE6δ heterodimer was employed to locate possible specific binding sites at the protein-protein interface region. Virtual screening of Enamine-database compounds was performed on the located potential binding sites to identify ligands able to simultaneously bind to the KRas4B-PDE6δ heterodimer. A molecular dynamics approach was used to estimate the binding free-energy of the complex. Cell viability and apoptosis were measured by flow cytometry. G-LISA was used to measure Ras inactivation. Western blot was used to measure AKT and ERK activation. MIA PaCa-2 cells implanted subcutaneously into nude mice were treated with D14 or C22 and tumor volumes were recorded.ResultsAccording to the binding affinity estimation, D14 and C22 stabilized the protein-protein interaction in the KRas4B-PDE6δ complex based on in vitro evaluation of the 38 compounds showing antineoplastic activity against pancreatic MIA PaCa-2 cancer cells. In this work, we further investigated the antineoplastic cellular properties of two of them, termed D14 and C22, which reduced the viability in the human pancreatic cancer cells lines MIA PaCa-2, PanC-1 and BxPC-3, but not in the normal pancreatic cell line hTERT-HPNE. Compounds D14 and C22 induced cellular death via apoptosis. D14 and C22 significantly decreased Ras-GTP activity by 33% in MIA PaCa-2 cells. Moreover, D14 decreased AKT phosphorylation by 70% and ERK phosphorylation by 51%, while compound C22 reduced AKT phosphorylation by 60% and ERK phosphorylation by 36%. In addition, compounds C22 and D14 significantly reduced tumor growth by 88.6 and 65.9%, respectively, in a mouse xenograft model.ConclusionsWe identified two promising compounds, D14 and C22, that might be useful as therapeutic drugs for pancreatic ductal adenocarcinoma treatment.

Docetaxel-polymeric nanoparticle enhances radiotherapeutic efficacy in human pancreatic cancer

Background: Nanoparticle therapeutics is promising platform for cancer treatment. In our previous study, we have developed polymeric nanoparticles (PNP) in which docetaxel was incorporated to reduce the side effects and improve the therapeutic efficacy, and recently finished its phase 1 clinical study in patients with solid tumors. Methods: Radiotherapeutic efficacy of the docetaxel-contained PNP (DTX-PNP) in pancreatic cancer cells was determined by both in vitro and in vivo assay such as clonogenic survival assay with cancer cell lines, western blot for apoptotic cell death and tumor growth inhibition assay using several kinds of xenograft models. The tumors derived from human pancreatic cancer AsPC-1 or BxPC-3 cells were analyzed by immunohistochemistry (IHC) to detect in apoptosis and tubulin polymerization induced by DTX-PNP. The combinational therapeutic effect of DTX-PNP and ionizing radiation (IR) was evaluated in vivo mice models of AsPC-1 or BxPC-3 cell line-derived xenograft models and patient-derived xenograft model, and compared to that of reference drugs. Results: DTX-PNP in combination with IR showed high cytotoxicity to pancreatic cancer cells, and ultimate inhibition of cell proliferation as determined via in vitro assays. In vivo radiotherapeutic efficacy was markedly enhanced by intravenous injection of DTX-PNP comparing to Gemzar, a common chemoradiation therapeutic agent in pancreatic cancer. Conclusions: These results suggested DTX-PNP can hold an invaluable and promising position in treating human pancreatic cancer as a novel and effective radiosensitizing agent.

Hyperglycemia aggravates microenvironment hypoxia and promotes the metastatic ability of pancreatic cancer

BackgroundDiabetes mellitus and pancreatic cancer are intimately related. Our previous studies showed that high levels of blood glucose promote epithelial-mesenchymal transition of pancreatic cancer. In this study, we evaluated the relationship between hyperglycemia and hypoxic tumor microenvironments. MethodsHIF-1α expression was evaluated by immunohistochemistry in clinical pancreatic cancer tissues with or without diabetes mellitus. Statistcal analysis was performed to explore the relationship between HIF-1α expression and pathological features of patients with pancreatic cancer. In vivo and in vitro models was established to detect whether a hyperglycemia environment could cause hypoxia in the pancreatic parenchyma and promote pancreatic cancer. In addition, we also tested the effect of HIF-1α siRNA on the high glucose-induced invasive and migratory abilities of BxPC-3 cells in culture. ResultOur data showed that pancreatic cancer patients with diabetes had a higher level of HIF-1α expression as well as biliary duct invasion and larger tumor volumes than individuals in the euglycemic group. Diabetic nude mice treated with streptozotocin (STZ) exhibited larger tumors and were more likely to develop liver metastasis than control mice. Acinar cells of the pancreas in diabetic mice showed an obvious expansion of the endoplasmic reticulum and increased nuclear gaps as well as chromatin close to the cellular membrane in some acinar cells. The expression area for Hypoxyprobe-1 and HIF-1α in the diabetic orthotopic xenograft group was larger than that in the control group. The expression level of HIF-1α in the BxPC-3 cancer cell line increased in response to high glucose and CoCl2 concentrations. The high glucose-induced invasive ability, migratory capacity and MMP-9 expression were counter-balanced by siRNA specific to HIF-1α. ConclusionOur results demonstrate that the association between hyperglycemia and poor prognosis can be attributed to microenvironment hypoxia in pancreatic cancer.

The effects of miRNAs-107 on cell proliferation, senescence and invasion of pancreatic cancer PANC-1 cells

Objective To investigate the effects of miRNAs-107(miR-107) on pancreatic cancer proliferation, senescence and invasion. Methods MiR-107 expression levels in 3 pancreatic cancer cell lines PANC-1, ASPC-1, BXPC-3 and normal pancreatic HTERT-HPNE cells were studied by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). PANC-1 cells were transfected with 50 nmol/L anti-miR-107 or negative control using Lipofectamine 2000. After transfection, the miR-107 expression was measured by qRT-PCR. Cell proliferation was tested by methylthiazol tetrazolium (MTT) assay. Cell senescence was detected by β-galactosidase staining. The expression levels of PCNA, P16INK4A and MMP2 were measured by qRT-PCR. Results Compared with the HTERT-HPNE cells, the expression level of miR-107 in 3 pancreatic cancer cell lines was significantly increased (P<0.01). After transfected with 50 nmol/L anti-miR-107, cell proliferation was inhibited, and cell senescence were increased in PANC-1 cells (P<0.05), and there was no obvious change in cell invasion. Compared with the HTERT-HPNE cells, after transfected with anti-miR-107, the PCNA expression was significantly decreased and P16INK4A was significantly increased, but expression of MMP2 didn’t change significantly. Conclusions These results demonstrate that miR-107 promotes the proliferation and escapes cell senescence in PANC-1 cells by targeting PCNA and P16INK4A. But it has no obvious effects on cell invasion. Therefore, it may be a new target for the biologic therapy for pancreatic cancer. Key words: Pancreatic cancer; MiRNAs-107(MiR-107); Proliferation; Senescence; Invasion

Abstract 2698: IL-17A alone or in combination with IL-4/IFN-γ induces Duox2/DuoxA2 expression and reactive oxygen production in human colon and pancreatic cancer cells

Abstract NADPH oxidase (NOX)-derived reactive oxygen species (ROS) contribute significantly to inflammation-associated carcinogenesis. Expression of dual oxidase 2 (Duox2), one of seven members of the NOX gene family, is up-regulated in inflammatory bowel disease, chronic pancreatitis, and in many human malignancies including carcinomas of the colon, pancreas, prostate, lung, and breast. Previously, we demonstrated that Stat1 and/or NF-kB play a critical role in modulating the enhanced expression of Duox2, and its cognate maturation factor DuoxA2, by the pro-inflammatory mediators IFN-γ and lipopolysaccharide in human pancreatic cancer cells. This cytokine-mediated increase in Duox2 expression was responsible for a significant increase in tumor cell H2O2 production, DNA damage, and HIF-1α and VEGFA upregulation in pancreatic cancer cell lines. Using the T84 human colon cancer line, and BxPC-3, AsPC-1, and HTB134 pancreatic cancer cell lines, we now report that the pro-inflammatory cytokine IL-17A, pivotal in the adaptive immune response against various pathogens, and in the promotion of colon and pancreatic cancer progression, strongly induces Duox2/DuoxA2 mRNA and protein expression (alone or in combination with IL-4 or IFN-γ). In BxPC-3 cells, IL-17A enhances the expression of Duox2/DuoxA2 in a time- and concentration-dependent manner, which leads to a significant increase in the generation of extracellular H2O2. When IL-17A is combined with IL-4 in either T-84 or AsPc-1 lines, Duox2-mediated ROS formation is sufficient to produce a DNA damage response, indicated by the strong induction of γH2AX expression; moreover, in AsPc-1 cells, Duox2-specific siRNA significantly attenuated cytokine-induced ROS production and γH2AX phosphorylation. IL-17A in combination with IL-4 also significantly increases Stat3 phosphorylation and promotes proliferation in AsPC-1 cells. Ongoing cellular experiments are focused on whether the cytokine-mediated upregulation of Duox2/DuoxA2 is responsible for Stat3 activation and cell growth promotion in the K-ras mutated AsPC-1 cells. Bio-informatic analysis with the TCGA data set, as well as RT-PCR using surgically-resected human colon cancer samples compared to adjacent, uninvolved colonic epithelium revealed that Duox2 was significantly upregulated in human colon cancers. These studies, in concert with our previous data demonstrating high-level Duox protein expression in pancreatitis, pancreatic intra-epithelial neoplasia (PanIN), and frank pancreatic cancer, strongly suggest that ROS, produced as a consequence of cytokine-related Duox2 up-regulation, may be associated with the development of inflammation-related gastrointestinal malignancies. Citation Format: James H. Doroshow, Yongzhong Wu, Jiamo Lu, Smitha Antony, Jennifer Meitzler, Agnes Juhasz, Guojian Jiang, Iris Dahan, Mariam Konate, Krishnendu Roy. IL-17A alone or in combination with IL-4/IFN-γ induces Duox2/DuoxA2 expression and reactive oxygen production in human colon and pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2698. doi:10.1158/1538-7445.AM2017-2698

Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism.

There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS) that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach.

Impact of down-regulation of histone methyltransferase enhancer of zeste homolog 2 on RUNX3 expressions, proliferation and apoptosis in human pancreatic cancer

Objective To investigate the impact of down-regulation of histone methyltransferase enhancer of zeste homolog 2 (EZH2) on RUNX3 expressions, proliferation and apoptosis in human pancreatic cancer. Methods The expressions of EZH2 and RUNX3 in 38 pancreatic cancer patients and human pancreatic cancer AsPC-1, PANC-1 and BxPC-3 cells were detected by immunohistochemistry and western blot, respectively. Cells were transfected with siEZH2 by lipofectamin 2000. Real time-PCR and western blot were used to detect EZH2 and RUNX3 expressions. Cell growth and apoptosis in vitro and vivo were assessed by MTT, flow cytometry and nude mice experiments, respectively. The correlation among the expressions of EZH2, clinical pathological features and overall survival rate were analyzed. Results Elevated EZH2 and decreased RUNX3 expressions were observed in human pancreatic cancer tissues and cells (P<0.05). Knockdown of EZH2 reduced cell growth and induced apoptosis in vitro and vivo by upregulating RUNX3 protein expression (P<0.05). In addition, the EZH2 expressions were correlated with tumor stage, lymph node metastasis and poor prognosis (P<0.05). Conclusions EZH2 expressions were correlated with malignancy and poor prognosis in pancreatic cancer. Tumor cell proliferation was promoted by EZH2 through down-regulation of RUNX3. EZH2 may be a potential therapeutic target for pancreatic cancer. Key words: Pancreatic cancer; Gene, EZH2; Cancer suppressor gene, RUNX3; Apoptosis; Prognosis

Preliminary study on the effects of pancreatic cancer-derived microvesicles on glycometabolism

Objective To explore the role of pancreatic cancer-derived microvesicles (MV) and their enclosed microRNAs (miRNA) in the pathogenesis of pancreatic cancer induced diabetes mellitus (DM). Methods The supernatants of three pancreatic cancer cell lines SW1990, BxPC3 and PANC1 were collected, and MV were isolated with gradient centrifugation.The entrance of MV into pancreatic islet cell line MIN6 was proved by Western blot assay and fluorescence-label method. The miRNA-19a levels were measured in MV and MV-free supernatants of three pancreatic cancer cells lines. The three experimental groups were MIN6 cells separately treated by MV derive from SW1990, BxPC3 and PANC1, and untreated MIN6 cells were assigned to the control group. The miRNA-19a levels as well as changes of glucose stimulated insulin secretion (GSIS) were measured. Afterward, pre-miRNA-19a and anti-miRNA-19a were transfected into MIN6 cells by liposome, and the effects of them on GSIS were observed. Results CD63 and AGO2 as the protein markers of MV and the entrance of MV from pancreatic cancer into pancreatic islet cell line MIN6 were detected by Western blotting.The miRNA-19a levels in MV and MV-free supernatants of SW1990, BxPC3 and PANC1 were (132.7±16.0), (32.8±4.3), (78.4±8.9), (22.6±3.3), (63.3±12.0) and (23.3±3.3) pmol/L, respectively, and the differences were statistically significant (t=10.44, 10.12 and 5.56, all P<0.01). Compared to the MIN6 control group, the miRNA-19a levels of MIN6 treated by MV from SW1990, BxPC3 and PANC1 significantly increased, and the 2-△△Ctvalue was 2.02±0.50, 1.80±0.41 and 2.11±0.59, respectively, and the differences were statistically significant (t=2.97, 2.77, 2.84; all P<0.05). Stimulated with high glucose, the GSIS of pancreatic islet cells treated by SW1990, BxPC3 and PANC1 in three groups decreased, which were (103.73±16.49), (141.17±11.26), and (138.24±13.97) ng·mg protein-1·h-1 MV, respectively, and that of control group was (256.24±33.05) ng·mg protein-1·h-1. The differences were statistically significant (t=4.13, 3.30 and 3.29, all P<0.05). Compared with control group, GSIS of pre-miRNA-19a treated MIN6 remarkably decreased, which was (126.17±62.87) ng·mg protein-1·h-1 and (316.72±91.87) ng·mg protein-1·h-1, and the difference was statistically significant (t=2.97, P<0.05). GSIS of MIN6 cells transfected with anti-miRNA-19a was higher than that of control group, which was (697.47±77.62) ng·mg protein-1·h-1and (355.33±84.77) ng·mg protein-1·h-1, and the difference was statistically significant (t=-2.97, P<0.05). Conclusion The entrance of MV derived from pancreatic cancer into pancreatic islet cell line MIN6 may cause the dysfunction of insulin secretion an important signaling molecules, miRNA-19a. Key words: Pancreatic neoplasms; Diabetes mellitus; MicroRNAs; Microvesicle

Cancer cells BXPC3 and MCF7 differentially reverse the inhibition of thrombin generation by apixaban, fondaparinux and enoxaparin

IntroductionCancer cells may alter the efficiency of the antithrombotic agents. To explore this possibility, the present study compared the capacity of the LMWH enoxaparin and the specific inhibitors of Xa (apixaban and fondaparinux) to inhibit thrombin generation triggered by pancreas adenocarcinoma cells (BXPC3) and human breast carcinoma cells (MCF7). Materials and methodsSamples of platelet poor (PPP) or platelet rich plasma (PRP) spiked with apixaban, fondaparinux or enoxaparin were added in micro wells carrying cancer cells and assessed for thrombin generation. In the control experiment thrombin generation was triggered with tissue factor reagent. ResultsThe three antithrombotics inhibited thrombin generation in a concentration dependent manner. The BXPC3 and MCF7 cells reversed in a different intensity the effect of the studied agents. According to the histological type of the cancer the antithrombotic efficiency of apixaban was preserved or partially reversed. Fondaparinux, was more vulnerable to the presence of cancer cells as compared to apixaban. The effect of BXCP3 or MCF7 cells on the antithrombotic potency of enoxaparin was of similar magnitude as that on apixaban. ConclusionsThe type of cancer cells is determinant for the antithrombotic efficiency of the specific factor Xa inhibitors. In contrast it does not significantly influence the potency of enoxaparin. The present study shows that the impact of the type of cancer cells on the antithrombotic activity of the specific Xa inhibitors should not be neglected. This has to be taken into consideration for the design of dose-finding studies of the direct orally active FXa inhibitors in patients with different histological types of cancer.