Pancreatic Cancer Cell Lines Capan-1 Research Articles

A Single Talent Immunogenic Membrane Antigen and Novel Prognostic Predictor: voltage-dependent anion channel 1 (VDAC1) in Pancreatic Cancer.

Immunogenic membrane antigens associated with multiple biological functions of human cancer cells, have significant value in molecule diagnosis and targeted therapy. Here we screened immunogenic membrane antigens in pancreatic cancer by immunobloting IgG purified from sera of 66 pancreatic cancer patients with membrane proteins separated from two-dimensional PAGE of human pancreatic cancer cell line SWl990, and identified voltage-dependent anion channel 1 (VDAC1) as one of the potential immunogenic membrane antigens. Further studies focusing on VDAC1 demonstrated that VDAC1 mRNA and protein were significantly expressed in the tested pancreatic cancer cell lines. VDAC1 silencing with RNAi significantly decreased cell growth, invasion and migration in the pancreatic cancer cell line Capan-1. Additionally, VDAC1 expression was upregulated in pancreatic cancer tissue compared with normal pancreas samples and patients with low VDAC1 expression had a significantly greater median survival compared to those with high expression (27.0 months vs. 17.8 months, P = 0.039). In multivariable analysis, VDAC1 staining was an independent prognostic factor for survival [(Hazard-Ratio) HR = 1.544, 95% CI = 0.794–3.0, P = 0.021]. These results demonstrated that VDAC1 may be a candidate immunogenic membrane antigen for pancreatic cancer, a potential independent prognostic marker, and an ideal drug target.

Investigating the role of mucin in the delivery of nanoparticles to cellular models of human cancer disease: an in vitro study

Mucin, a glycosylated protein, is aberrantly overexpressed in a variety of tumor cells. The glycoprotein mesh decreases the rate of intracellular drug uptake and effectiveness. We investigated the influence of the mucin mesh on the cellular uptake of anti-MUC1 antibody and nanoparticles by fluorescence spectroscopy and microscopy. A glycosylation inhibitor (benzyl-α-GalNAc) was employed to regulate mucin glycosylation events. In our panel of pancreatic cell lines, only PANC-1 cells exhibited a significant increase in the uptake of liposomes following glycosylation inhibition, resulting in improved cytotoxicity of gemcitabine-loaded liposomes. Interestingly, areas devoid of liposome uptake were observed for pancreatic cancer cell lines PANC-1, Capan-1, and Capan-2; however, these restricted regions could be diminished for PANC1 cells only. In conclusion, investigating the reason(s) for differential cellular uptake of nanoparticles, in association with the production of mucin glycosylation mesh, should provide valuable leads to the future development of nanomedicine for cancer treatment.

Hypoxia regulates ABCG2 activity through the activivation of ERK1/2/HIF-1α and contributes to chemoresistance in pancreatic cancer cells

ABSTRACTPancreatic cancer is a drug resistant hypovascular tumor. Although there are many studies on the mechanism of chemoresistance in pancreatic cancers, studies on the relationship between ABCG2 and chemoresistance during hypoxia of pancreatic cancer are rare. Hypoxia-inducible factor-1 (HIF-1α) is a master regulator of the transcriptional response to oxygen deprivation in cancer cells. The aim of this study was to examine the role of ABCG2 and HIF-1α in mediating chemoresistance during hypoxia in pancreatic cancer. In this study, we detected the expression levels of ABCG2, ERK/phosphorylated-ERK (p-ERK) and HIF-1α by immunohistochemistry in fresh pancreatic cancer and paracarcinoma tissues obtained from 25 patients. The mechanism by which p-ERK1/2 and HIF-1α affect ABCG2s expression was analyzed in the hypoxic cultured human pancreatic cancer cell line Capan-2. ABCG2-mediatedregulation of gemcitabine response under hypoxic conditions in pancreatic cancer cells was observed. It was found that ABCG2, ERK/p-ERK and HIF-1α were overexpressed in cancer tissues. ABCG2, HIF-1α and p-ERK levels were demonstrated to be high during hypoxic conditions in pancreatic cancer cells. Hypoxia induced phosphorylation of ERK1/2 to activate HIF-1α and contribute the ABCG2 expression and mediated gemcitabine chemoresistance in pancreatic cancer cells. Hypoxic conditions induced HIF-1α binding to target gene sequences in the ABCG2 promoter, resulting in increased transcription in pancreatic cancer cells. We demonstrated that hypoxia-induced chemoresistance is due to the regulation of ABCG2 through the activation of ERK1/2/HIF-1α. ABCG2 could serve as a predictor of gemcitabine response and, potentially, as a chemotherapeutic target in pancreatic cancer. Inhibition of ERK1/2 and HIF-1αcould result in increased gemcitabine sensitization in pancreatic cancer with highly expressed ABCG2 cell member protein.

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.

Abstract 5071: Inhibition of Porcupine reduces Wnt-dependent colony formation and/or proliferation in both autocrine and paracrine cell models

Abstract Aberrant activation of the pro-survival Wnt signalling pathway is a key determinant in numerous cancers (Nusse and Varmus, 2012) and therefore constituents of this pathway are potentially promising therapeutic targets. The membrane-bound o-acyltransferase porcupine (PORCN) is required for post-translational modification of all Wnts, and is vital for Wnt secretion (Herr et. al., 2012). Due to the complex and varied oncogenic roles of the known Wnt ligands, prevention of active ligand secretion provides an effective mechanism for inhibiting canonical and non-canonical Wnt signalling in both paracrine and autocrine systems. Here we show that a novel inhibitor of porcupine potently inhibits both autocrine and paracrine models of Wnt/β-catenin signalling. Treatment of the pancreatic cancer cell line CAPAN-2 with REDX05562 resulted in a significant decrease of axin 2 mRNA expression by qPCR (10.4 fold, 10nM). This reduction in expression was partially reversed by the addition of recombinant human Wnt-3A (100ng.ml−1). The decrease in axin 2 expression was concomitant with an inhibition of CAPAN-2 proliferation (IC50 1.4nM +/- 0.8nM). Conversely treatment of MCF-7 mammospheres with REDX05562 did not inhibit sphere forming efficiency (SFE). However, when stimulated with recombinant Wnt-3A an increase in anoikis resistance was observed suggesting a potential paracrine influence. In vivo the presence of cancer associated fibroblasts (CAFs) are a likely source of paracrine Wnt (Green et. al., 2013). Indeed, conditioned media taken from primary CAFs (isolated from fresh mastectomies) increased anoikis resistance in MCF-7 cells without any discernible effect on CAF proliferation. This effect was significantly inhibited when MCF-7 cells were incubated with conditioned media taken from primary CAFs treated with REDX05562. Moreover, immunofluorescent labelling of PORCN revealed that PORCN expression is markedly higher in CAFs than in MCF-7s. This is consistent with the hypothesis that different mechanisms underpin Wnt-signalling in breast and pancreatic cancer. The above data show that the novel PORCN inhibitor REDX05562 effectively reduces the impact of pro-survival Wnt ligand-mediated signalling in both paracrine and autocrine models. This further emphasises the broad therapeutic potential of PORCN inhibition in Wnt-dependent cancers. Citation Format: Ben A. Thompson, Kate J. Messenger, Emily Linnane, Sarah E. Coupland, Peter J. Calcraft. Inhibition of Porcupine reduces Wnt-dependent colony formation and/or proliferation in both autocrine and paracrine cell models. [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 5071. doi:10.1158/1538-7445.AM2015-5071

The flavonoid beverage Haelan 951 induces growth arrest and apoptosis in pancreatic carcinoma cell lines in vitro

BackgroundA major challenge in pancreatic cancer treatment is the resistance of human pancreatic cancer cells to apoptosis. Soy isoflavones and calpain inhibition have been suggested to exert inhibitory effects on cancer development and progression. We investigated the effects of the isoflavone containing beverage Haelan 951 and the calpain inhibitor PD150606 on the viability, growth and apoptosis of the human pancreatic cancer cell lines CAPAN-1 and BxPC-3, on the rat pancreatic cancer cell line AR42J, and on human fibroblasts as the control cell line.MethodsCellular viability and proliferation were determined using the LDH cytotoxicity and WST-1 assay, respectively. Apoptosis was detected by flow cytometric analyses of Annexin V-FITC labeled-cells, TUNEL assay and caspase activation. Student’s t test or Mann–Whitney Rank Sum test were used to compare the data.ResultsHaelan concentrations lower than 8 % showed no cytotoxic effects, whereas higher concentrations led to necrosis. Eight percent Haelan induced significant growth inhibition of CAPAN-1 and BxPC-3 cell lines by 30 % and 35 %, respectively, compared with the control. The proliferation rate of AR42J cells decreased by 50 %, whereas the fibroblasts remained unaffected. An 1.1-fold increase in apoptosis was found in CAPAN-1 cells, whereas the number of apoptotic BxPC-3 cells was elevated 2-fold. The number of apoptotic AR42J cells and fibroblasts was elevated 1.5-fold, each. Inhibition of calpain activity amplified the Haelan-induced growth inhibition of CAPAN-1 and BxPC-3 cells, but failed to amplify the growth inhibition of Haelan-treated AR42J cells. In fibroblasts, calpain inhibition induced Haelan-independent growth inhibition. Calpain inhibition also amplified the Haelan-induced apoptotic activity in all cancer cell lines, but exerted no further effect in fibroblasts.ConclusionsThe proliferation-inhibiting and apoptosis-inducing effects of Haelan are highly dependent on cell type and concentration administered. The results show for the first time that Haelan may be a promising candidate in the treatment of human pancreatic cancer, and its anticancer activity may be potentiated when administered with calpain inhibitors.

MicroRNA-222 Controls Human Pancreatic Cancer Cell Line Capan-2 Proliferation by P57 Targeting.

Pancreatic cancer (PC) is one of the most common cancers and has a poor prognosis due to late diagnosis and ineffective therapeutic multimodality. MicroRNAs (miRNAs, miRs) are a group of non-coding, small RNAs with active biological activities. In our investigation, human pancreatic cancer cell line Capan-2 were transfected with miR-222 mimics, inhibitors or their negative controls. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8), EdU incorporation assay and cell cycle determination by flow cytometry. MiR-222 and putative target gene expression levels including p27, p57 and PTEN were determined using quantitative reverse transcription polymerase chain reactions and Western blotting. Our results showed that miR-222 could lead to increased vitality and proliferative rate of Capan-2 cells, and also higher S-phase and lower G1-phase of cell cycle. Further, we found p57 at protein level, but not p27 nor PTEN, was regulated by miR-222 in Capan-2 cells. Finally, we co-transfected miR-222 inhibitor and p57 si-RNA into Capan-2 cells, and found that proliferation-suppressing effects of miR-222 inhibitor on Capan-2 cells could be partially reversed by silencing p57. Our results indicate that miR-222 controls Capan-2 cell proliferation by targeting p57. This study provides a novel idea for developing effective therapeutic strategy for PC patients through inhibiting miR-222.

Differential Gene Expression of Pancreatic Cancer Cell Line Capan-2 in Response to Gemcitabine plus BVDU (RP101) Combinatorial Treatment

Every year worldwide approximately 3 million cancer patients receive chemotherapy. Unfortunately in many cases the treatment does not prolong patient’s survival time. Chemotherapy and radiation therapy still remain the major strategies with or without surgery. One of the main obstacles in chemoand radiation therapy is the rapid development of resistance by the cancer cells. In recent years, the Heat Shock Protein HSP27 has been identified as one of the key players driving resistance development. HSP27 is overexpressed in many kinds of cancer and influences cellular processes like apoptosis (programmed cell death), DNA repair, recombination, and metastasis. As a result, cancer cells are able to suppress apoptosis and develop resistance towards cytostatic drugs such as Gemcitabine which is used as standard therapy in pancreatic cancer. To address this problem the HSP27-inhibitor BVDU ((E)-5-(2-Bromovinyl)-2′-deoxyuridine, RP101) was administered in combination with Gemcitabine to prevent resistance development. In the study design, the human pancreatic adenocarcinoma cell line Capan-2 was exposed to Gemcitabine alone and in combination with BVDU. Differential gene expression was displayed using DNA microarrays. This paper presents the resulting dataset, with pairwise comparison of control (untreated), Gemcitabine-, BVDU-, and Gemcitabine+BVDU treated probes. The five spreadsheet files are openly accessible for validation, re-use and aggregation in future research. Creative Commons BY-NC-SA 4.0 © 2015 Hosting by Procon Ltd. All rights reserved. Heinrich J-C & Fahrig F. Biomed Data J. 2015; 1(2): 7-9

The negative interplay between Aurora A/B and BRCA1/2 controls cancer cell growth and tumorigenesis via distinct regulation of cell cycle progression, cytokinesis, and tetraploidy.

It is well known that the activation of Aurora A/B (Aur A/B) or inactivation of BRCA1/2 induces tumor formation. Others and we have reported that the mutual suppression between Aur A/B and BRCA1/2 may manipulate cancer cell growth and tumorigenesis, however, the interactive regulation and mechanism between these molecules are still elusive. In this study, by consecutive silencing of Aur A/B or/and BRCA1/2 with specific shRNAs, we showed that, in BRCA2-deficient pancreatic cancer cell line Capan-1 and in ovarian cancer cell line OVCA433, Aur A/B and BRCA1/2 inversely regulated the expression of each other likely through proteasome-mediated proteolysis but not through gene transcription. Aur A/B and BRCA1/2 conversely regulated cell cycle progression mainly through control of p53 and cyclin A. Moreover, the disruption of Aur A/B blocked abnormal cytokinesis and decreased cell multinuclearity and chromosome tetraploidy, whereas the deprivation of BRCA1/2 promoted the abnormal cytokinesis and enhanced the cell multinuclearity and tetraploidy. Furthermore, we showed by animal assays that the depletion of Aur A/B inhibited tumor growth of both cell lines, while the knockdown of BRCA1/2 promoted the tumor growth. However, the concurrent silencing of Aur A/B and BRCA1/2 diminished the effects of these molecules on the regulation of cell cycle, cytokinesis, and tetraploidy, leading to the burdened tumor sizes similar to those induced by scrambled shRNA-treated control cells. In summary, our study revealed that the negative interplay between Aur A/B and BRCA1/2 inversely controls the cell proliferation, cell cycle progression, cell multinuclearity, and tetraploidization to modulate tumorigenesis.

Resveratrol plays dual roles in pancreatic cancer cells

Although the potential anticancer effect of resveratrol (RSV) on pancreatic cancer has been reported, its mechanism was not fully understood. The role of vascular endothelial growth factor B (VEGF-B) in cancer remains controversial. Herein, we aimed to examine whether the anticancer effect of RSV was related to the VEGF-B. The effect of RSV on pancreatic cancer cell line (capan-2 cells) was evaluated by CCK-8 assay, Hoechst 33342 staining, and flow cytometry. The mRNA level of VEGF-B was measured by real-time PCR. VEGF-B expression was knockdown by small interfering RNA (siRNA).The protein levels of VEGF-B, glycogen synthase kinase-3 beta (GSK-3β), and Bax were measured by Western blot. Resveratrol treatment inhibited tumor growth, induced apoptosis, and up-regulated Bax expression in capan-2 cells. The mRNA and protein levels of VEGF-B were up-regulated after RSV treatment. However, VEGF-B siRNA treatment increased the apoptotic rate, and inhibited tumor activator GSK-3β, while Bax expression was not affected. The combination of RSV and VEGF-B siRNA showed significantly higher apoptotic rate in comparison with RSV or VEGF-B siRNA mono-treatment group. Resveratrol plays dual roles in pancreatic cancer: as a tumor suppressor via the up-regulation of Bax; as a tumor activator via the up-regulation of VEGF-B; and the anticancer effect of RSV is much stronger than the cancer promotion effect. The combination of RSV with pharmacological inhibitor of VEGF-B might, therefore, be a promising modality for clinical pancreatic cancer therapy.

Glycan Elongation Beyond the Mucin Associated Tn Antigen Protects Tumor Cells from Immune-Mediated Killing

Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr), STn (NeuAcα2-6GalNAc-Ser/Thr), T (Galβ1–3GalNAc-Ser/Thr), and ST (NeuAcα2-6Galβ1–3GalNAc-Ser/Thr) antigens are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing only the shortest possible mucin-like glycans (Tn and STn). Glyco-engineering was performed by zinc finger nuclease (ZFN) knockout (KO) of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast and pancreatic cancer cell lines T47D and Capan-1 increases sensitivity to both NK cell mediated antibody-dependent cellular-cytotoxicity (ADCC) and cytotoxic T lymphocyte (CTL)-mediated killing. In addition, we investigated the association between total cell surface expression of MUC1/MUC16 and NK or CTL mediated killing, and observed an inverse correlation between MUC16/MUC1 expression and the sensitivity to ADCC and CTL-mediated killing. Together, these data suggest that up-regulation of membrane bound mucins protects cells from immune mediated killing, and that particular glycosylation steps, as demonstrated for glycan elongation beyond Tn and STn, can be important for fine tuning of the immune escape mechanisms in cancer cells.

Effects of KAP-1 in promoting the epithelial-mesenchymal transition of pancreatic cancer cell line Capan-2

Objective To investigate the effects of KAP-1 in promoting the epithelial-mesenchymal transition (EMT) of pancreatic cancer cell line Capan-2.Methods The lentiviral vector of LV-plenti-GFP-KAP-1 was constructed.Capan-2 cells were divided into the experimental group (cells transfected by lentiviral vector of LVplenti-GFP-KAP-1),negative control group (cells transfected by empty vector) and blank control group 1 (cells cultured in 1640 medium plus 10％ fetal calf serum).Capan-2 cells in the experimental group were subdivided into the miR-100-5p inhibitor transfection group (cells transfected with miR-100-5p inhibitor),empty vector control group (cells transfected with microRNAs inhibitor),blank control group 2 (cells cultured in 1640 medium plus 10％ fetal calf serum).The lentivirus was identified by double endonuclease restriction and sequencing,and the virus titer was detected.The morphological changes of the cells were observed after transfecting lentiviral vector of LV-plenti-GFP-KAP-1 to the Capan-2 cells.The expressions of KAP-1,genes of EMT proteins and mRNA of miR-100-5p were detected by real-time quantitative polymerase chain reaction.The protein expressions of KAP-1,EMT proteins in all the groups were detected by Western blot.The measurement data were presented by mean ± standard deviation,and were analyzed using the analysis of variance.Results The lentiviral vector of LV-plentiGFP-KAP-1 was successfully constructed,and the virus titer was 2 × 108 TU/ml.Compared with the control group,the mesenchymal transition of the Capan-2 cells was detected in the experimental group after transfecting the Capan-2 cells with lentiviral vector of LV-plenti-GFP-KAP-1 for 48 hours.The relative mRNA expressions of KAP-1,N-cadherin,vimentin,E-cadherin,miR-100-5p were 1.77 ± 0.83,2.62 ± 0.71,2.50 ± 0.21,7.20 ± 1.17 and 1.81 ±0.40 in the experimental group,5.03 ±0.29,5.07 ±1.53,3.83 ±0.57,7.83 ±0.78,7.01 ± 0.96 in the negative control group,5.13 ± 1.14,5.81 ± 1.49,4.92 ± 0.90,3.07 ± 0.36,6.87 ± 0.35 in the blank control group 1,with significant difference among the 3 groups (F =5.99,7.62,7.88,6.62,4.64,P ＜0.05).The relative mRNA expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 1.56 ± 0.42,4.89 ± 0.61,5.20 ± 0.38,with significant difference among the 3 groups (F =5.14,P ＜ 0.05).The relative mRNA expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.10 ± 1.37,3.44 ± 0.94,3.08 ±1.16,with no significant difference among the 3 groups (F =0.49,P ＞ 0.05).The results of western blot showed that the relative protein expressions of KAP-1,N-cadherin,vimentin,E-cadherin were 2.77 ± 1.99,1.31 ±0.38,4.25 ± 0.63,0.62 ± 0.06 in the experimental group,0.83 ± 0.46,0.41 ± 0.37,1.03 ± 0.33,1.17 ± 0.45 in the negative control group,0.71 ± 0.26,0.08 ± 0.04,1.37 ± 0.92,3.04 ± 0.65 in the blank control group 1,with significant difference among the 3 groups (F =5.54,4.68,3.19,8.18,P ＜ 0.05).The relative protein expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 2.27 ±0.71,0.56 ±0.43,0.61 ±0.39,with significant difference among the 3 groups (F =4.81,P ＜0.05).The relative protein expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.19 ± 0.55,3.93 ± 0.06,3.61 ± 0.73,with no significant difference among the 3 groups (F =0.04,P ＞ 0.05).Conclusion KAP-1 promotes the EMT of Capan-2 cells by specifically down-regulating the miR-100-5p expression. Key words: Pancreatic neoplasms; KAP-1 ; Epithelial-mesenchymal transition ; microRNAs

MiR-99b-targeted mTOR induction contributes to irradiation resistance in pancreatic cancer

BackgroundRadiation exerts direct antitumor effects and is widely used in clinics, but the efficacy is severely compromised by tumor resistance. Therefore uncovering the mechanism of radioresistance might promote the development of new strategies to overcome radioresistance by manipulating activity of the key molecules.MethodsImmunohistochemistry were used to find whether mTOR were over-activated in radioresistant patients’ biopsies. Then Western blot, real-time PCR and transfection were used to find whether radiotherapy regulates the expression and activity of mTOR by modulating its targeting microRNA in human pancreatic cancer cell lines PANC-1, Capan-2 and BxPC-3. Finally efficacy of radiation combined with mTOR dual inhibitor AZD8055 was assessed in vitro and in vivo.ResultsIonizing radiation promoted mTOR expression and activation in pancreatic cancer cells through reducing miR-99b expression, which negatively regulated mTOR. Novel mTOR inhibitor, AZD8055 (10 nM, 100 nM, 500 nM) synergistically promoted radiation (0–10 Gy) induced cell growth inhibition and apoptosis. In human pancreatic cancer xenografts, fractionated radiation combined with AZD8055 treatment further increased the anti-tumor effect, the tumor volume was shrinked to 278 mm3 after combination treatment for 3 weeks compared with single radiation (678 mm3) or AZD8055 (708 mm3) treatment (P < 0.01).ConclusionsOur data provide a rationale for overcoming radio-resistance by combined with mTOR inhibitor AZD8055 in pancreatic cancer therapy.

Abstract 1660: Therapeutic targeting cross talk between ROS/autophagy and apoptosis for pancreatic cancer by a herbal extract.

Abstract 5-year survival for pancreatic cancer (PanCA) continuous to be lower than any other solid tumor due to late stage diagnosis combined with lack of effective therapeutic options. Therefore developing novel strategies possibly based on molecular targets is critical for successful management of PanCA. Recent studies from our laboratory show potential anti-pancreatic cancer activity for NexrutineR (Nx), a bark extract isolated from traditional Chinese medicine Phellodendron amurense. These studies also show that Nx reduces pancreatic fibrosis in preclinical animal model through targeting two critical oncogenic transcription factors including NF-κB and Stat3. Nonetheless, the precise molecular mechanism associated with the observed biological activity is unclear. We investigated whether Nx inhibits pancreatic cell proliferation and pancreatic fibrosis via reactive oxygen species (ROS)-mediated activation of autophagy. Our data show that Nx can induce autophagy at earlier time points (4h) but inhibit autophagy at later time points (24h) in human pancreatic cancer cell lines Capan-2 and BxPC-3 as evidenced by autophagosome formation, alteration in the expression and levels of various autophagy related genes. Interestingly Nx also induced apoptosis at 24h time point but not at 4h suggesting that autophagy precedes apoptosis. Further under these experimental conditions, treatment of pancreatic cancer cells with Nx enhanced intracellular production of ROS implicating a cross talk between Nx-induced ROS, autophagy and apoptosis. Overall, our findings divulge an important role for ROS/autophagy/apoptosis in Nx-mediated anti-pancreatic cancer effects. Supported by NCCAM (APK). Citation Format: Gong Jingjing, Addanki P. Kumar. Therapeutic targeting cross talk between ROS/autophagy and apoptosis for pancreatic cancer by a herbal extract. [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 1660. doi:10.1158/1538-7445.AM2013-1660

Effects of 5-Aza-CdR in combination with gemcitabine on the expression of protocadherin 8 gene in pancreatic cancer cell lines

Objective To investigate the effects of 5-Aza-CdR on the transcriptional regulation through methylation of the DNA promoter protocadherin 8(PCDHg) gene in pancreatic cancer cell line Capan-2.The Capan-2 retardation in growth rate and apoptosis were assessed in when administered 5-Aza-CdR and the chemotherapy agent,gemcitabine.Methods MTT and flow cytometry were used to analyze the cell growth inhibition and apoptosis when treated with 5-Aza-CdR or in combination with gemcitabine.Methylation-specific PCR,RT-PCR and western blot were performed to detect methylation state,mRNA and protein respectively of PCDH8 gene in 5-Aza-CdR-treated Capan-2cells.Results Capan-2 cells treated with 5-Aza-CdR showed a slower growth rate,and a significant growth inhibition when given both 5-Aza-CdR in combination with gemcitabine.Compared with single drug administration and control,5-Aza-CdR together with gemcitabine can induce a stronger apoptosis signal.Different concentrations 5-Aza-CdR of were able to reverse methylation,restore mRNA and protein levels of PCDH8 in Capan-2.Conclusion 5 Aza-CdR may demethylate the PCDH8 gene,which would effectively remove the gene silencing caused by high methylation,and thus induce gene mRNA transcription and protein expression to inhibit cell growth and have collaborative antitumor functions with gemcitabine. Key words: Pancreatic neoplasms; Methylation; Gene expression regulation; 5-Aza-CdR

Increased E-selectin in hepatic ischemia-reperfusion injury mediates liver metastasis of pancreatic cancer

Several recent studies have reported that selectins are produced during ischemia-reperfusion injury, and that selectin ligands play an important role in cell binding to the endothelium and in liver metastasis. Portal clamping during pancreaticoduodenectomy with vessel resection for pancreatic head cancer causes hepatic ischemia-reperfusion injury, which might promote liver metastasis. We investigated the liver colonization of pancreatic cancer cells under hepatic ischemia-reperfusion and examined the involvement of E-selectin and its ligands. A human pancreatic cancer cell line (Capan-1) was injected into the spleen of mice after hepatic ischemia-reperfusion (I/R group). In addition, to investigate the effect of an anti-E-selectin antibody on liver colonization in the IR group, mice received an intraperitoneal injection of the anti-E-selectin antibody following hepatic ischemia-reperfusion and tumor inoculation (IR+Ab group). Four weeks later, mice were sacrificed and the number of tumor nodules on the liver was compared to mice without hepatic ischemia-reperfusion (control group). The incidence of liver metastasis in the I/R group was significantly higher (16 of 20, 80%) than that in the control group (6 of 20, 30%) (P<0.01). Moreover, mice in the I/R group had significantly more tumor nodules compared to those in the control group (median, 9.9 vs. 2.7 nodules) (P<0.01). In the I/R+Ab group, only 2 of 5 (40%) mice developed liver metastases. RT-PCR and southern blotting of the liver extracts showed that the expression of IL-1 and E-selectin mRNA after hepatic ischemia-reperfusion was significantly higher than the basal levels. Hepatic ischemia-reperfusion increases liver metastases and E-selectin expression in pancreatic cancer. These results suggest that E-selectin produced due to hepatic ischemia-reperfusion is involved in liver metastasis.

Induction of Pancreatic Cancer Cell Apoptosis and Enhancement of Gemcitabine Sensitivity by RAP80 siRNA

Receptor-associated protein 80 (RAP80) increases substantially in pancreatic cancer. The involvement of RAP80 in the chemoresistance of pancreatic cancer should be elucidated. We investigated the effects of inhibiting RAP80 expression on the sensitivity of pancreatic cancer cells to gemcitabine chemotherapy by using small interfering RNA (siRNA). Chemically synthesized siRNA RAP80 was transfected into human pancreatic cancer cell lines SW1990 and Capan-2. The IC(50) of gemcitabine was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell apoptosis was detected by using flow cytometry. Expression of apoptosis-related genes, Bax, Bcl-2, TRAIL, survivin, and caspase-8 was detected by using reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Gemcitabine inhibits proliferation of SW1990 and Capan-2 cells in a concentration-dependent manner. Inhibition of RAP80 expression significantly reduced the IC(50) of gemcitabine (P < 0.01). RAP80 siRNA combined with gemcitabine significantly increased (P < 0.01) apoptosis of pancreatic cancer cell lines SW1990 and Capan-2, increased expression of Bax mRNA, reduced Bcl-2 mRNA expression (P < 0.01), and slightly increased TRAIL mRNA expression (P < 0.01). Correspondingly, in the RAP80 siRNA combined with gemcitabine group, both Bax and cleaved caspase-8 protein levels were increased (P < 0.01), whereas Bcl-2 protein decreased significantly (P < 0.01). No change in survivin mRNA expression was observed (P < 0.01). Inhibition of RAP80 expression can induce apoptosis in pancreatic cancer cells and improve chemosensitivity to gemcitabine.

Nicotine, IFN-γ and retinoic acid mediated induction of MUC4 in pancreatic cancer requires E2F1 and STAT-1 transcription factors and utilize different signaling cascades

BackgroundThe membrane-bound mucins are thought to play an important biological role in cell–cell and cell–matrix interactions, in cell signaling and in modulating biological properties of cancer cell. MUC4, a transmembrane mucin is overexpressed in pancreatic tumors, while remaining undetectable in the normal pancreas, thus indicating a potential role in pancreatic cancer pathogenesis. The molecular mechanisms involved in the regulation of MUC4 gene are not yet fully understood. Smoking is strongly correlated with pancreatic cancer and in the present study; we elucidate the molecular mechanisms by which nicotine as well as agents like retinoic acid (RA) and interferon-γ (IFN-γ) induce the expression of MUC4 in pancreatic cancer cell lines CD18, CAPAN2, AsPC1 and BxPC3.ResultsChromatin immunoprecipitation assays and real-time PCR showed that transcription factors E2F1 and STAT1 can positively regulate MUC4 expression at the transcriptional level. IFN-γ and RA could collaborate with nicotine in elevating the expression of MUC4, utilizing E2F1 and STAT1 transcription factors. Depletion of STAT1 or E2F1 abrogated the induction of MUC4; nicotine-mediated induction of MUC4 appeared to require α7-nicotinic acetylcholine receptor subunit. Further, Src and ERK family kinases also mediated the induction of MUC4, since inhibiting these signaling molecules prevented the induction of MUC4. MUC4 was also found to be necessary for the nicotine-mediated invasion of pancreatic cancer cells, suggesting that induction of MUC4 by nicotine and other agents might contribute to the genesis and progression of pancreatic cancer.ConclusionsOur studies show that agents that can promote the growth and invasion of pancreatic cancer cells induce the MUC4 gene through multiple pathways and this induction requires the transcriptional activity of E2F1 and STAT1. Further, the Src as well as ERK signaling pathways appear to be involved in the induction of this gene. It appears that targeting these signaling pathways might inhibit the expression of MUC4 and prevent the proliferation and invasion of pancreatic cancer cells.

Mucin production determines sensitivity to bortezomib and gemcitabine in pancreatic cancer cells

The prognosis of pancreatic cancer remains disappointing due to a high intrinsic resistance against chemotherapeutic agents. Standard gemcitabine therapies have improved overall survival only marginally and recently, inhibition of the proteasome by the boronic acid derivative bortezomib has been introduced as a novel therapeutic strategy for solid and hematological malignancies including pancreatic cancer. The mucus-producing pancreatic cancer cell line Capan-1 was cultured under standard conditions and treated with different concentrations of gemcitabine or bortezomib. Mucus production was suppressed by siRNA-mediated silencing of apomucin genes. Cell proliferation was determined by 3H-thymidine incorporation and apoptosis was quantified after propidium iodide staining by flow cytometry. Apoptotic cell death was confirmed by TUNEL staining, determination of mitochondrial transmembrane potential and assessment of caspase 3/7 activity. NFκB-activity was determined by EMSA. The unfolded protein response (UPR) was further investigated by PCR, Western blotting and caspase 12 activity assays. Silencing of MUC4 significantly reduced expression of mucins for up to 5 days after transfection. While native cells showed an increased sensitivity to bortezomib treatment, silenced cells were more sensitive to gemcitabine treatment. Bortezomib induces mitochondrial damage in native cells and also activates the UPR by splicing of Xbp-1 and induction of CHOP, which is significantly reduced by silencing of MUC4. Our data suggest that mucinous pancreatic cancers are more sensitive towards proteasome inhibition by bortezomib and that alternative pathways of apoptosis are involved in cell death induction, while tumor cells with a low secretory activity show a better response to gemcitabine.

Anti Reverse Transcriptase and Anticancer activity of stem ethanol extracts of &lt;i&gt;Excoecaria agallocha&lt;/i&gt; (Euphorbiaceae)

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