Cell Lines OE33 Research Articles

Fluorescent analogs of peptoid-based HDAC inhibitors: Synthesis, biological activity and cellular uptake kinetics

Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.

Abstract 2863: Long non-coding RNA LINC00152 plays an oncogenic role via targeting MET, STAT3 signaling in esophagus adenocarcinoma

Abstract Esophageal cancer remains a threat to public health with an increasing incidence and low survival rate world wide. Long non-coding RNA, LINC00152 has been linked to several human cancers and promotes cell proliferation in lung, gastric, hepatocellular, colorectal, and clear cell renal carcinoma. The expression of LINC00152 and its roles in esophagus adenocarcinomas (EAC), however, are unknown. The objective of this study is to investigate the role of LINC00152 in EAC. We used the esophageal cancer cell lines Flo-1, OE19, and OE33 for in vitro study. siRNA of specific to LINC00152 was used to knockdown LINC00152 followed by cell proliferation, colony formation, invasion and migration assays. Real-time PCR assay was used for detecting mRNA expression and Western blot for examining altered proteins expression affected by LINC00152 knockdown. Data analysis were performed using excel and Prism. The difference of variables was analyzed using the Student’s T-test. We found that LINC00152 was increased in EAC as compared to Barrett, low-grad displasia or high-grad dysplasia. The LINC00152 expression was decreased more than 90% after LINC00152 knockdown with siRNA on Flo, OE19 and OE33 cells measured by RT-PCR. Cell proliferation, colony and invasion were decreased significantly after LINC00152 siRNA transfection on these EAC cell lines. Apoptosis was also induced. Mechanistic study indicated that MET protein was significantly decreased in OE33 cells after LINC00152 knockdown, while STAT3 decreased in Flo cells. P21, p27 and Myc was decreased in all 3 cell lines. In this study we found that LINC00152 knockdown could inhibit cell proliferation, colony formation, cell migration and invasion, as well as trigger apoptosis via targeting multiple oncogenic signaling. We also found LINC00152 was increased in EAC tumors. Our study indicates that LINC00152 plays an important role in EAC and is potentially a diagnostic marker. Further characterization of LINC00152 as a novel therapeutic target of EAC is warranted. Citation Format: Matthew Xiao, Zhuwen Wang, Jules Lin, David G. Beer, Andrew C. Chang, Guoan Chen. Long non-coding RNA LINC00152 plays an oncogenic role via targeting MET, STAT3 signaling in esophagus adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2863.

Abstract 3738: A STING independent type-1 interferon response induced by fractionated radiotherapy coincides with altered tumor growth and clonogenicity

Abstract Introduction To improve radiotherapy efficacy, insight in the dynamic biological responses that occur during clinical radiation schedules is vital. In the current study we explored gene expression changes during the course of fractionated radiotherapy (FrRT) that might be targeted with combination treatment strategies. Methods Human cell lines HT29, SW480, HCT116, COLO320, RKO (colorectal carcinoma), D384 (glioblastoma) and OE19 (esophageal adenocarcinoma) as well as HT29 xenograft tumors in nude mice were irradiated up to 3 to 5 weeks with daily fractions of 2 Gy for 5 days per week. In vivo tumor growth was monitored daily and in vitro clonogenic survival was determined every other day. At the end of each treatment week tumors and cells were harvested for RNA isolation. Subsequently, gene expression analysis was performed by RNA sequencing and micro-array analysis. Altered gene expression levels were validated by qPCR. Protein expression was determined by ELISA and Western blot. Results Tumor growth of the HT29 xenografts declined after 2 weeks of FrRT compared to non-irradiated tumors. In irradiated cell lines, clonogenic survival analyses revealed a log-linear decline in survival in the first 2 weeks, after which a steady-state-like phase was reached up to 5 treatment weeks. Gene expression analysis of both xenograft tumors as well as irradiated cell lines at this 2 week time point identified a type-1 interferon (IFN) mediated signaling pathway as the most significantly enriched biological process. The induction of a selection of IFN-stimulated genes (ISGs) was confirmed by qPCR. Comparable responses were observed in both STING positive (HT29, SW480, HCT116, D384) and negative (COLO320, RKO, OE19) cell lines. To get more insight in the dynamics of the IFN response we analyzed ISGs expression at the end of each treatment week. This revealed a peak in ISGs expression after 10 to 15 fractions, both in vivo and in vitro. The ISGs expression levels decreased with continuation of treatment but always remained above the basal level of non-irradiated cells. Single dose irradiation up to 10 Gy also induced dose-dependent ISGs expression, albeit typically 5 to 10 fold lower as compared to FrRT. Subsequent protein and mRNA expression analyses consistently revealed induction of IFN beta expression, with peak levels measured after 10 to 15 fractions. In addition, occasional induction of IFN lambda 2/3 protein and mRNA expression was observed. Conclusion FrRT induces an intrinsic type-1 IFN response which occurs independent of STING. This response is associated with induction of IFN beta expression, which peaks after 2 to 3 weeks of treatment and coincides with a shift in in vivo tumor growth and in vitro clonogenic survival. Targeting this response during FrRT might be exploited as a novel combination treatment. Citation Format: Ruben S. Goedegebuure, Esther A. Kleibeuker, Kitty C. Castricum, Jaap van den Berg, Sarah Derks, Henk M. Verheul, Ben J. Slotman, Adrian Harris, Victor L. Thijssen. A STING independent type-1 interferon response induced by fractionated radiotherapy coincides with altered tumor growth and clonogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3738.

Capsaicin in Hot Chili Peppers: In Vitro Evaluation of Its Antiradical, Antiproliferative and Apoptotic Activities.

Capsaicin is a spicy capsaicinoid, produced as secondary metabolite by Capsicum fruits. This alkaloid has been used for years in folk medicine for its analgesic and antinflammatory properties although most data is referred to the raw fruit. In this study, the antiradical activity of the pure capsaicin has been studied using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays as well as its antiproliferative activity, using MTT assay, against two human tumour cell lines, the colorectal Caco-2 and the oesophageal OE19 cells. Furthermore, the antiproliferative activity observed on tumoral cells was compared with that of the human normal-like fibroblast cell line TelCOFS02MA. In addition, the apoptotic activity was evaluated using TUNEL assay. A higher radical scavenging activity was observed against ABTS radical cation than DPPH. Capsaicin showed also a higher cytotoxicity against cancer cells than normal-like cells with Selectivity index values greater than 2 at 72h. Capsaicin induced apoptosis especially in OE19 cell line.

PS02.057: INDUCTION OF APOPTOSIS IN ESOPHAGEAL ADENOCARCINOMA CELLS BY CURCUMIN

Abstract Background Curcumin naturally occurring in curry powder from Curcuma longa is an anti-inflammatory and anti-proliferative agent. It represses NFκB activation and induces apoptosis in cancer cells. Because, inflammation favours the onset of an intestinal metaplasia in esophageal squamous epithelial cells and esophageal carcinoma cells, a reduction of such processes may contribute to establish a stable disease and/or sensitization for chemotherapeutic approaches. Methods Curcumin receptivity was investigated in metaplastic (CP-A), dysplastic (CP-B), adenocarcinoma (OE19, OE33), and esophageal fibroblast (FFE3) cell lines, which were treated with 10 or 25 μM Curcumin for 48h or 72h. Response to Curcumin was measured by proliferation assays as well as by induction of apoptosis and Akt activation by western blot analyses. Results The EAC cell lines OE33 and OE19 show a decrease of proliferation with raising curcumin concentration with an IC50 of 9.6 and 14.8 μM, respectively. While the metaplastic and dysplastic cell lines showed a comparable IC50 of 7.7 and 11.6 μM to the EAC cell lines, the FEF3 cell showed a higher IC50 of approx. 20μM. The phosphorylation of Akt was decreased and apoptosis was induced, showing cleaved PARP, when treated with 25μM curcumin after 48h and 72h. Conclusion Metaplastic, dysplastic and EAC cells show a higher receptivity to curcumin than esophageal fibroblasts cells. This constrains the NFκB activation and its contribution in the manifestation of the Barrett's esophagus. The usage of curcumin to generate an anti-inflammatory microenvironment will potentially help to develop a stable disease or to reverse the development of the Barrett's esophagus. Disclosure All authors have declared no conflicts of interest.

PS02.044: A BARRETT’S CELL CULTURE MODEL, SHOWING AN INCREASED ACID TOLERABILITY OF ESOPHAGEAL ADENOCARCINOMA CELLS

Abstract Background Inflammation is a common in esophageal adenocarcinoma (EAC). The response of EAC to common chemotherapeutic regimes is relatively low and the 5-years-survival is poor. The influence of acidic reflux on the viability of esophageal adenocarcinoma cells is unknown. Methods We investigated the influence of acidified medium conditions (pH3; pH3.5; pH4) in a Barrett's cell culture model, using six cell lines representing the Barrett's sequence from normal esophageal epithelium (EPC1 and EPC2), metaplasia (CP-A), dysplasia (CP-B) and EAC (OE33 and OE19) for their tolerability against an acid pulse of 15 and 30 min as well as expression profile of the Nrf2-Keap1-signalling pathway and their downstream targets. Results The Barrett's sequence was characterized by an increase of acid tolerability in the more advanced cells of this sequence. The squamous epithelium cell line EPC1 has shown the highest susceptibility with a decrease of 75% of cell viability, when treated with pH4 medium for 15min, while dysplastic and EAC cells had shown only a decrease of 10–25%. The lowest susceptibility was observed in the EAC cell line OE33, which had also 90% living cell in pH3 medium after 15min and had almost 30% living cells after 30min, when all other investigated cell lines showed no more living cells. Additionally, treatment with acidified medium was accompanied with an increase of the Nrf2 target gene hemeoxygensae-1. Conclusion We could show an increased tolerability against acidified medium (pH3) in cells representing advanced Barrett's esophagus stages, accompanied by an activation of Keap1-Nrf2-signalling pathway. However, acidic reflux may influence inflammation and chemo-sensitivity of the EAC. Disclosure All authors have declared no conflicts of interest.

Secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro inhibitory activities of Nardostachys jatamansi on key enzymes linked to hyperglycemia, hypertension and cognitive disorders

Ethnopharmacological relevanceNardostachys jatamansi (D. Don) DC., ‘Spikenard’ or ‘Jatamansi’, a highly valued, aromatic herb from alpine Himalayas has a long history of use as ethnomedicine and dietary supplements in Ayurveda, Unani and Chinese system of medicine since Vedic ages (1000–800 BC). In Ayurveda and traditional system of medicine, the species is used as stimulant, sedative, brain tonic or mind rejuvenator, antidiabetic, cardio tonic, and in the treatment of various neurological disorders such as insomnia, epilepsy, hysteria, anxiety and depression. It is considered as Sattvic herb in Ayurveda and is now commercially marketed either as single or poly-herbal formulations by many companies in national and international markets. Aim of the studyThe species has become threatened in its natural habitats due to over exploitation and illegal trade of its rhizomes for drug preparation in herbal and pharmaceutical industries. Considering the increasing demand and tremendous medicinal importance of this threatened plant species, a detailed study was undertaken to evaluate its antioxidant potential, secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro enzyme inhibitory activities on key enzymes linked to hyperglycemia, hypertension and cognitive disorders in different plant parts of wild and in vitro-raised plants with respect to different solvent systems for its sustainable utilization. Materials and methodsAnti-cholinesterase activity of leaves and rhizome of wild and cultured plant extracts was investigated against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. In vitro anti-hyperglycemic (α-amylase and PTP1B), anti-hypertensive (angiotensin-converting enzyme), anti-tyrosinase and anti-inflammatory potential (5-lipoxygenase and hyaluronidase) of different plant parts of wild and in vitro-raised plants with respect to different solvent systems were also evaluated. In vitro cytotoxic effect of rootstock extracts of wild and in vitro-derived plants were against cancer (HCT-116, MCF-7 and OE33) and two normal (HEK and MEF) cell lines. Secondary metabolite profiling of rhizome segments of wild and in vitro-derived plants was carried out by quantitative gas chromatography-mass spectrometry (GC–MS). ResultsIn vitro-raised plantlets showed comparative higher yield of various secondary metabolites with a significantly high antioxidant activity as compared to the wild plants. Methanolic rootstock extracts of both wild and in vitro-derived plants of N. jatamansi exhibited significant AChE (IC50 36.46 ± 2.1 and 31.18 ± 2.6 µg/ml, respectively) and BuChE (IC50 64.6 ± 3.5 and 60.12 ± 3.6 µg/ml, respectively) inhibitory potential as compared to standard inhibitor galanthamine (IC50 0.94 ± 0.03 and 4.45 ± 0.5 µg/ml). Methanolic rootstock extract of in vitro-derived plants showed significant α-amylase (IC50 90.69 ± 2.1 µg/ml), PTP1B (IC50 24.56 ± 0.8 µg/ml), angiotensin-converting enzyme (IC50 42.5 ± 3.6 µg/ml) and tyrosinase (IC50 168.12 ± 3.6 µg/ml) inhibitory potential as compared to standard acarbose (IC50 52.36 ± 3.1 µg/ml), ursolic acid (IC50 5.24 ± 0.8 µg/ml), captopril (IC50 32.36 ± 2.5 µg/ml) and kojic acid (IC50 = 54.44 ± 2.3 µg/ml). Both the methanolic rootstock and leaf extracts of tissue culture-derived plants exhibited promising anti-5-LOX and anti-hyaluronidase activities against the known inhibitor of 5-LOX and hyaluronidase. Furthermore, methanolic rootstock extracts of both wild and in vitro-derived plants exhibited promising cytotoxic effects to HCT-116, MCF-7 and OE33 cell lines as compared to the normal HEK and MEF after 12 h of treatment. Secondary metabolite profiling of wild and in vitro-derived plants by quantitative GC–MS analysis revealed the presence of different classes of terpenoids and phenolic acids might be responsible for its effective biological activities. ConclusionIn vitro-derived plants revealed a substantial anti-cholinesterases, anti-hyperglycemic anti-inflammatory, anti-hypertensive and anti-tyrosinase potential with higher yield of various bioactive metabolites and significantly higher antioxidant activity which substantially explain medicinal importance of N. jatamansi in traditional medicine, used for centuries in different Ayurvedic formulations. The present findings suggest that cultured plants could be a promising alternative for the production of bioactive metabolites with comparative biological activities to the wild plants.

Esophageal cancer cells resistant to T-DM1 display alterations in cell adhesion and the prostaglandin pathway.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate that specifically targets HER2 thanks to its antibody component trastuzumab. In spite of responses to this novel agent, acquired resistance to treatment remains a major obstacle. Prolonged in vitro exposure of the gastroesophageal junction cancer cell line OE-19 to T-DM1, in the absence or presence of ciclosporin A resulted in the selection of two resistant cell lines to T-DM1. T-DM1-resistant cells presented an increased expression of adhesion genes, altered spreading and higher sensitivity to anoikis than parental cells. A resistant cell line showed decreased adhesion strength, increased migration speed and increased sensitivity to RhoA inhibition. Genes involved in the prostaglandin pathway were deregulated in resistant models. Addition of prostaglandin E2 to T-DM1 partially restored its cytotoxic activity in resistant models. This work demonstrates that T-DM1-resistance may be associated with alterations of cell adhesion and the prostaglandin pathway, which might constitute novel therapeutic targets.

Selective oestrogen receptor antagonists inhibit oesophageal cancer cell proliferation in vitro

BackgroundOestrogen receptors (ER) have a well-established role to the initiation, progression and regulation of responses to treatment of breast, prostate, and lung cancers. Previous data indicates altered ER expression in oesophageal cancers (OC). However the role of ER subtypes and ER specific inhibitors in the regulation of OC progression remains unclear. This study sought to assess levels of ERα and ERβ in OC. The effects of highly selective ER antagonists on cell proliferation and apoptosis in two OC adenocarcinoma cell lines was also studied.MethodsERα and ERβ expression profiling in paired normal oesophageal mucosa and tumour tissues (n = 34; adenocarcinoma n = 28; squamous cell carcinoma n = 6) was performed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Correlation between levels of ER with the clinico-pathological features for OC was determined. The effect of selective ER antagonists on proliferation of OE33 and OE19 OC cell lines was studied.ResultsERα and ERβ mRNA expression was significantly higher (p < 0.05) in tumour tissues relative to their paired normal mucosa and correlated inversely with survival outcome (p < 0.05). Upregulation of ERα mRNA correlated with higher pathological T-stage (p < 0.05) and lymph node metastasis (p < 0.05) while ERβ mRNA upregulation correlated with positive vascular invasion (p < 0.05). A significant concentration-dependent inhibition of proliferation in OE33 and OE19 cell lines was induced by a highly-selective ERα antagonist (MPP) and an ERβ specific antagonist (PHTPP) (p < 0.05). Moreover, anti-oestrogens induced cell death through stimulation of apoptotic caspase activity.ConclusionThese findings indicate that the ER system is involved in OC progression and thus may provide a novel target for the treatment of OC.

Low Expression of Transforming Growth Factor β in the Epithelium of Barrett's Esophagus.

BackgroundThe aim of this study was to investigate the expression of transforming growth factor β (TGF-β) in the different stages of Barrett’s esophagus (BE).MethodsPaired endoscopic esophageal biopsy samples were obtained from patients with BE prospectively. Subjects were classified into three groups: BE, BE with dysplasia, and adenocarcinoma (AC) arising from BE. Biopsy specimens over normal esophageal epithelium and gastric cardiac epithelium of limited cases were done. Four cell lines, HETA1 (human esophageal epithelium), CA-A and CP-C (non-dysplastic metaplasia), and OE33 (AC) were analyzed for quantitative mRNA and Western blotting of TGF-β.ResultsAll 30 subjects with BE were enrolled. Expression of TGF-β mRNA in BE were significantly (P < 0.01) lower than that in the normal esophagus and cardiac epithelium. The BE tissue showed a lower positive ratio of TGF-β immunohistochemical (IHC) stain than the cardiac epithelium. The expression of TGF-β mRNA in the cell lines CA-A, CP-3, OE-33, was significantly (P < 0.05) lower than that in the cell line HETA-1. The Western blotting result showed lower TGF-β protein expression of the cell lines CA-A, CP-3, and OE-33.ConclusionsThe expression of TGF-β was lower in the tissue of BE.

HER2 Confers Resistance to Foretinib Inhibition of MET-Amplified Esophageal Adenocarcinoma Cells

Recent genomic studies indicated that esophageal adenocarcinoma (EAC) is driven by amplification of c-MET or HER2 or both in a subset of patients. We studied the effect of MET targeting by the small molecule inhibitor foretinib in EAC cells and the interplay between MET and HER2 signaling. We measured the expression levels and phosphorylation status of MET and HER2 proteins in EAC cell lines using Western blot analysis. The expression levels of MET and HER2 were manipulated by transfecting cells with specific siRNA or a plasmid expressing HER2. The small molecule inhibitors of c-MET and ERBB1/2 (foretinib and lapatinib, respectively) were tested for effect on growth, apoptosis, and downstream signaling pathways of EAC cells as single agents or in combination. The response to inhibitors was correlated to the levels of MET, HER2 expression, and amplification status. Foretinib inhibits phosphorylation of MET, which correlated with reduced EAC cell growth and inhibition of AKT and ERK phosphorylation. Cell growth inhibition by foretinib is most profound in the ESO51 cell line, which has MET gene amplification and overexpression. Inhibition of MET signaling by foretinib or siRNA-specific knock down of MET expression induces apoptosis in ESO51 cells. Ectopic expression of HER2 reduces foretinib-mediated growth inhibition and downstream ERK phosphorylation in ESO51-HER2 cells. The EAC OE33 cell line, with amplification and overexpression of both MET and HER2, demonstrated reduced sensitivity to foretinib or lapatinib and had a transient effect on downstream inhibition of phosphorylated AKT and ERK (p-AKT, p-ERK). The coadministration of foretinib and lapatinib effectively blocked both MET and HER2 signaling through the p-AKT and p-ERK pathways, dramatically inhibited growth, and induced apoptosis to overcome single-agent resistance in OE33 cells. The mechanism for foretinib growth inhibition in MET-amplified EAC tumor cells is demonstrated. The interplay of dual MET/HER2 overexpression in the AKT and ERK pathways for esophageal cancer is described. Therefore, combination therapy could be a novel strategy for EAC with amplification of both MET and HER2.

Combination of duligotuzumab, anti HER3 antibody or taselisib, PI3K INhibitor with trastuzumab shows synergetic antitumoral activity in HER2 positive gastric cancer cells

Introduction: The anti-HER2 monoclonal antibody trastuzumab is central to the treatment of HER2-positive gastric cancer (GC); however, its responses are limited due to some poorly understood mechanisms of resistance. The aim of this study was to asses the antitumortal activity of Duligotuzumab, an anti HER3 antibody or Taselisib, a Pi3k inhibitor combined with Trastuzumab in a panel of HER2 positive human gastric cancer cell lines (GCG), to improve anti HER2 treatment efficacy. Methods: We evaluated in vitro the effect of Duligotuzumab, Taselisib and Trastuzumabin single agent and in combination treatments in HER2-positive GCG (NCI-N87, KATOIII, OE19) and in negative HER2 GCC (MKN28), throughproliferation, migration and apoptosis assays. We also investigated the effect of combined treatment on downstream intracellular signaling, by western blot analysis. Results: After establishing, through a dose response curve, the IC50 for each drug used (≈ 0.5 µM), a significant synergistic effect of Duligotuzumab, Taselisib and Trastuzumab treatments in HER2-positive GCG was observed by reduction of proliferation and migration in KATOIII, OE19 and N87 cell lines; the same effect was found analyzing the apoptotic rate. At cellular level, in particular in KATOIII and OE19 cell lines, the combined treatment with Duligotuzumab or Taselisib plusTrastuzumabcompletely inhibited the activation of proteins downstream of HER3, PI3K and MAPK pathways. Conclusions: Targeting both HER2 and HER3 or HER2 and PI3K with the combination of anti-HE3 antibody or pi3k inhibitor with Trastuzumab may result in an improved treatment effects on HER2-positive GCG. These important findings can be utilized to facilitate the design of future clinical trials.

Interleukin-17A promotes esophageal adenocarcinoma cell invasiveness through ROS-dependent, NF-κB-mediated MMP-2/9 activation.

Interleukin-17A (IL-17A), a pro-inflammatory cytokine secreted primarily by Th17 cells, has been proved to be involved in the microenvironment of certain inflammation-related tumors. However, the role of IL-17A in cancer development has always been controversial. In this study, we investigated the effect of IL-17A on the regulation of esophageal adenocarcinoma (EAC) cell invasiveness and related molecular mechanism. Surface IL-17 receptor (IL-17R) expression on human EAC cell line OE19 was examined using flow cytometry. The effect of IL-17A on cell proliferation was measured by MTT assay. Cell migration and invasive ability invitro were assessed by wound-healing and Matrigel-coated Transwell invasion assay. Intracellular reactive oxygen species (ROS) levels were determined by flow cytometry and fluorescence microscope. The protein expression levels of MMP-2, MMP-9, NF-κB and p-IκB-α were detected by western blotting. Our results showed that IL-17A promoted migration and invasion of OE19 cells in a dose-dependent manner, however it had less effect on OE19 cell proliferation. Furthermore, IL-17A treatment significantly upregulated the expression of MMP-2 and MMP-9, stimulated intracellular ROS production, increased IκB-α phosphorylation and NF-κB nuclear translocation. Nevertheless, IL-17A-induced expression of MMP-2/9 and OE19 cell invasiveness were both inhibited by pretreatment with N-acetyl-L-cysteine (NAC, a ROS scavenger) or pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor). In conclusion, these findings demonstrate that IL-17A can promote the migration and invasiveness of EAC cells through ROS/NF-κB/MMP-2/9 signaling pathway activation, indicating that IL-17A may be a potential therapeutic target for EAC.

Effects of galectin-9 on apoptosis, cell cycle and autophagy in human esophageal adenocarcinoma cells

The incidence of esophageal adenocarcinoma (EAC) is rapidly increasing in western countries. The overall mortality of this disease remains high with a 5-year survival rate of less than 20%, despite remarkable advances in the care of patients with EAC. Galectin-9(Gal-9) is a tandem-repeat type galectin that exerts anti-proliferative effects on various cancer cell types. The aim of the present study was to evaluate the effects of Gal-9 on human EAC cells and to assess the expression of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 invitro. Gal-9 suppressed the proliferation of the EAC cell lines OE19, OE33, SK-GT4, and OACM 5.1C. Additionally, Gal-9 treatment induced apoptosis and increased the expression levels of caspase-cleaved cytokeratin 18, activated caspase-3 and activated caspase-9. However, it did not promote cell cycle arrest by reducing cell cycle-related protein levels. Furthermore, Gal-9 increased the level of the angiogenesis-related protein interleukin-8(IL-8) and markedly altered miRNA expression. Based on these findings, Gal-9 may be of clinical use for the treatment of EAC.

The angiotensin II type 1 receptor antagonist telmisartan inhibits cell proliferation and tumor growth of esophageal adenocarcinoma via the AMPKα/mTOR pathway in vitro and in vivo.

Telmisartan, a widely used antihypertensive drug, is an angiotensin II type 1 (AT1) receptor blocker (ARB). This drug inhibits cancer cell proliferation, but the underlying mechanisms in various cancers, including esophageal cancer, remain unknown. The aim of the present study was to evaluate the effects of telmisartan on human esophageal cancer cell proliferation in vitro and in vivo. We assessed the effects of telmisartan on human esophageal adenocarcinoma (EAC) cells using the cell lines OE19, OE33, and SKGT-4. Telmisartan inhibited the proliferation of these three cell lines via blockade of the G0 to G1 cell cycle transition. This blockade was accompanied by a strong decrease in cyclin D1, cyclin E, and other cell cycle-related proteins. Notably, the AMP-activated protein kinase (AMPK) pathway, a fuel sensor signaling pathway, was enhanced by telmisartan. Compound C, which inhibits the two catalytic subunits of AMPK, enhanced the expression of cyclin E, leading to G0/G1 arrest in human EAC cells. In addition, telmisartan reduced the phosphorylation of epidermal growth factor receptor (p-EGFR) and ERBB2 in vitro. In our in vivo study, intraperitoneal injection of telmisartan led to a 73.2% reduction in tumor growth in mice bearing xenografts derived from OE19 cells. Furthermore, miRNA expression was significantly altered by telmisartan in vitro and in vivo. In conclusion, telmisartan suppressed human EAC cell proliferation and tumor growth by inducing cell cycle arrest via the AMPK/mTOR pathway.

Low-dose non-targeted radiation effects in human esophageal adenocarcinoma cell lines

Purpose: To investigate non-targeted radiation effects in esophageal adenocarcinoma cell lines (OE19 and OE33) using human keratinocyte and colorectal cancer cell reporters following γ-ray exposure.Materials and methods: Both clonogenic assays and ratiometric calcium endpoints were used to check for the occurrence of bystander signals in reporter cells.Results: We report data suggesting that γ-irradiation increases cell killing over the expected linear quadratic (LQ) model levels in the OE19 cell line exposed to doses below 1 Gy, i.e. which may be suggestive to be a low hyper-radiosensitive (HRS) response to direct irradiation. Both EAC cell lines (OE19 and OE33) have the ability to produce bystander signals when irradiated cell conditioned medium (ICCM) is placed onto human keratinocyte reporters, but do not seem to be capable of responding to bystander signals when placed on their autologous reporters. Further work with human keratinocyte reporter models showed statistically significant intracellular calcium fluxes following exposure of the reporters to ICCM harvested from both EAC cell lines exposed to 0.5 Gy.Conclusion: These experiments suggest that the OE19 and OE33 cell lines produce bystander signals in human keratinocyte reporter cells. However, the radiosensitivity of the EAC cell lines used in this study cannot be enhanced by the bystander response since both cell lines could not respond to bystander signals.

Silencer-of-Death Domain Mediates Acid-Induced Decrease in Cell Apoptosis in Barrett's Associated Esophageal Adenocarcinoma Cells.

We have shown that NADPH oxidase (NOX)5-S may mediate the acid-induced decrease in cell apoptosis. However, mechanisms of NOX5-S-dependent decrease in cell apoptosis are not fully understood. In this study, we found that silencer-of-death domain (SODD) was significantly increased in esophageal adenocarcinoma (EA) tissues, EA cell lines FLO and OE33, and a dysplastic cell line CP-B. Strong SODD immunostaining was significantly higher in low-grade dysplasia (66.7%), high-grade dysplasia (81.2%), and EA (71.2%) than in Barrett's mucosa (10.5%). Acid treatment significantly increased SODD protein and mRNA expression and promoter activity in FLO cells, an increase that was significantly decreased by the knockdown of NOX5-S and nuclear factor κB (NF-κB)1 p50 with their small interfering RNAs. Similarly, acid-induced increase of SODD mRNA was blocked by knockdown of NOX5-S and p50 in a BE cell line CP-A. Overexpression of NOX5-S significantly increased SODD protein expression in FLO cells. Moreover, overexpression of NOX5-S or p50 significantly increased the SODD promoter activity and decreased the caspase 9 activity or apoptosis. NOX5-S overexpression-induced increase in SODD promoter activity was significantly decreased by knockdown of p50. In addition, acid treatment significantly decreased the caspase 9 activity, a decrease that was significantly inhibited by knockdown of SODD. Furthermore, chromatin immunoprecipitation assay showed that NF-κB1 p50 bound to SODD genomic DNA containing a NF-κB-binding element GGGGACACCCT. This binding element was further confirmed by a gel mobility shift assay. We conclude that acid-induced increase in SODD expression and decrease in cell apoptosis may depend on the activation of NOX5-S and NF-κB1 p50 in FLO cells.

Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.

New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC: TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project.

Abstract 3749: Interaction of 5FU chemotherapy and oncolytic adenovirus in combined cancer treatment

Abstract Previous studies have shown that Interferon(IFN)-based chemoradiation therapy can improve survival after resection of pancreas cancer. However, its clinical utility to this point has been limited due to the severe toxicity related to its use of systemic IFN. Our aim in this study is to evaluate our group's novel oncolytic adenovirus (OAd) which allows targeting IFN treatment to cancer cells while sparing healthy tissue. We have previously shown the ability of IFN to sensitize cancer cells to chemotherapy as well as demonstrated an increased therapeutic effect of the drug in immunocompetent models. This study was conducted to analyze the combination of 5FU chemotherapy and our OAd in vitro in order to assess the interaction of treatments and determine the optimum combined treatment regimen. Treatments were analyzed in pancreatic cancer and esophageal adenocarcinoma cell lines S2013 and OE19. Recombinant OAds expressing luciferase rather than IFN were used to isolate the combination of 5FU and the virus. Two viral models were evaluated: our therapeutic virus (Cox2) selectively replicative in Cox2 expressing cancer cells and a nearly identical but universally replicative virus (wild type). Cells were treated with 0, 1 or 10 viral particles per cell and 0, 5, 10, or 20 uM 5FU. Three timing regimens were used: simultaneous administration, 5FU 4 hours before virus, and virus 48 hours before 5FU. Crystal Violet and MTS Assays were used to measure cell death. Viral Copy number to assess viral replication was measured using qPCR. Cell death analysis showed time dependent killing of each virus, with a 2 day delay for the Cox2 virus. 5FU and each virus produced dose dependent cell death independently. There was a significant additive effect seen in cell death from combining treatments when using 5FU before virus in both S2013 and OE19. Simultaneous treatment showed an additive killing effect with the combination in S2013, but a reduced killing by the combination compared to virus alone in OE19. There was also reduced killing when using virus before 5FU in S2013. Viral copy experiments are in progress. Our Cox2 OAd shows a killing effect similar to wild type in multiple cancer cell lines. When combined with 5FU treatment the expected addition in overall cell death from the independent treatments varies by timing of administrations as well as by type of cancer cell line. The reduced killing of the combination treatment in the simultaneous and virus before 5FU regimens may suggest an inhibition of the virus by 5FU under certain conditions. This also suggests the possibility of a treatment regimen with optimal therapeutic effect, which appears to be 5FU before virus based on the results collected. Further studies investigating different chemotherapeutic drugs and regimens should be conducted to examine these trends. Citation Format: Jordan Sell, Amanda Oliviera, Eric Jensen, Masato Yamamoto, Julia Davydova. Interaction of 5FU chemotherapy and oncolytic adenovirus in combined cancer treatment. [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 3749.

Abstract 309: Mechanisms of resistance to trastuzumab emtansine in gastric cancer

Abstract Background: Overexpression of Human Epidermal growth factor Receptor 2 (HER2) in cancer was associated with poor outcome and high chances of recurrence before the development of anti-HER2 agents. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate targeting HER2 based on its antibody component trastuzumab. After internalization of T-DM1/HER2, Lys-MCC-DM1, a tubulin binding agent is released in the cytoplasm. In spite of therapeutic advances, acquired resistance to treatment remains a major obstacle. A better understanding of the mechanisms of resistance to T-DM1 is necessary to improve treatment regimens for HER2-overexpressing cancer patients. We have developed and characterized resistance models to T-DM1 in order to describe underlying resistance mechanisms and develop alternative strategies. Methods: To develop in vitro T-DM1 resistance models, HER2-overexpressing esophageal adenocarcinoma cell line OE-19 was exposed to increasing doses of T-DM1 several months in the absence or presence of cyclosporin A (CsA, an inhibitor of MDR1 transporter). Resistance models were called OE-19 TR (exposed to T-DM1) and OE-19 TCR (exposed to T-DM1 and CsA). To characterize the resistance models, sensitivity to T-DM1 was assessed by MTT assay, Annexin V-PI staining and xCELLigence. Cell cycle distribution was assessed by propidium iodide using flow cytometry. Cross-resistance was studied by MTT assay. Expression of HER2, ABC transporters and tubulin was studied by rt-qPCR, flow cytometry and/or Western Blot. An expression profile of sensitive and resistant cells to T-DM1 was performed using a pan-genomic Illumina cDNA array. Results: After prolonged exposure of OE-19 cells to T-DM1, IC50 value is 18-fold higher in TR cells and 21-fold higher in TCR cells and both models become less sensitive to T-DM1-induced apoptosis. T-DM1 induced G2/M arrest in OE-19 sensitive cells but not in resistant cells. We found that resistant cells become less sensitive to trastuzumab but remain sensitive to other HER2-targeted therapies such as lapatinib, as well as to a variety of chemotherapy agents. Among the resistant cells, a subpopulation with increased expression MDR1 was identified in OE-19 TR and TCR. Concerning the expression of the main targets of T-DM1 (HER2 and tubulin); we found that HER2 expression and total α and β tubulin expression and content remained unchanged. However the isoforms β2 and β3 were overexpressed in both resistant models. A transcriptomic approach of T-DM1 resistant cells showed modification in adhesion junctions and focal adhesions. Conclusion: Prolonged exposure of the OE-19 cell line to T-DM1 resulted in resistance to this immunoconjugate. We are working on describing the resistance mechanisms to T-DM1, which are important to understand cancer progression in patients receiving this therapy. In the long term, we hope this study will allow proposing agents that benefit T-DM1-refractory cancer patients. Citation Format: Juliette Sauveur, Abdelkamel Chettab, Eva matera, Aurore Cleret, Ariel Savina, Charles Dumontet. Mechanisms of resistance to trastuzumab emtansine in gastric cancer. [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 309.