Human Gastric Tumor Cells Research Articles

Abstract 2478: Inhibition of CD39 by AB598 increases extracellular ATP resulting in activation of myeloid cells and T cells to enhance anti-tumor immunity

Abstract Extracellular ATP (eATP) is a potent immunostimulatory signal in the tumor microenvironment (TME), but it is rapidly degraded into AMP and immunosuppressive adenosine. Therefore, ATP metabolism is an attractive anti-tumor target. Several enzymes are involved in the immunosuppressive process used by the TME to convert ATP to AMP and adenosine. Among them, CD39 (gene: ENTPD1), an extracellular ATPase, is a promising target. Analyzing mRNA expression from the TCGA, we show that CD39 is consistently expressed at high levels in comparison to the other extracellular ATP-converting enzymes, ENPP1 and TNAP. We further demonstrate that CD39 has greater enzymatic activity at the slightly acidic physiological pH (pH = 6.8) of the TME than either ENPP1 or TNAP. Flow cytometry profiling of dissociated primary human gastric tumors showed that CD39 was highly expressed on both myeloid and lymphoid cells, including several populations of T cells present in the tumors. This is distinct from findings in healthy primary human PBMCs that show high expression of CD39 on myeloid subsets and relatively few subsets of T cells with high CD39 expression. AB598 is a novel, humanized, Fc-silent anti-CD39 therapeutic antibody that potently binds to CD39 and inhibits its enzymatic activity with sub-nanomolar potency. Treatment of dissociated primary human gastric tumor cells with AB598 resulted in a decrease in the rate of ATP degradation, indicating both the effectiveness of AB598 and the relevance of CD39 as a main contributor to the ATPase activity of the TME. Although eATP is low in healthy tissues, high levels of ATP can be released when cancer cells are treated with immunogenic cell death (ICD)-inducing agents, increasing the eATP in the TME, providing a rationale for the combination of ICD-inducing chemotherapy and AB598. Thus, cancer cells were co-cultured with monocyte-derived dendritic cells (moDCs) and treated with chemotherapy and AB598 to preserve the eATP released by dying cancer cells. The increased expression of CD83 and CD86 on moDCs demonstrated that the combination treatment enhanced the maturation of moDCs, compared to single or no treatment. To further elucidate the anti-tumor effects of the combination treatment, autogenous T cells were introduced into this system. AB598 significantly enhanced T cell activation in the presence of chemotherapy. These data demonstrate that using AB598 results in elevated eATP, which boosts immune cell activation to promote anti-tumor immunity. Citation Format: Ke Jin, Julie Clor, Kaustubh Parashar, Rebecca Fuchs, LC Stetson, Sean Cho, Matthew J. Walters, Ester Fernandez-Salas, Christine E. Bowman. Inhibition of CD39 by AB598 increases extracellular ATP resulting in activation of myeloid cells and T cells to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2478.

Quantitative and amplification-free detection of SOCS-1 CpG methylation percentage analyses in gastric cancer by fiber optic nanoplasmonic biosensor

A new innovative approach is essential for early and effective diagnosis of gastric cancer, using promoter hypermethylation of the tumor suppressor, SOCS-1, that is frequently inactivated in human cancers. We have developed an amplification-free fiber optic nanoplasmonic biosensor for detecting DNA methylation of the SOCS-1 human genome. The method is based on the fiber optic nanogold-linked sorbent assay of PCR-free DNA from human gastric tumor tissue and cell lines. We designed a specific DNA probe fabricated on the fiber core surface while the other probe is bioconjugated with gold nanoparticles in free form to allow percentage determination and differentiating the methylated and unmethylated cell lines, further demonstrating the SOCS-1 methylation occurs in cancer patients but not in normal cell lines. The observed detection limit is 0.81 fM for methylated DNA, and the detection time is within 15 min. In addition, our data were significantly correlated to the data obtained from PCR-based pyrosequencing, and yet with superior accuracy. Hence our results provide new insight to the quantitative evaluation of methylation status of the human genome and can act as an alternative to PCR with a great potential.

Corilagin exhibits differential anticancer effects through the modulation of STAT3/5 and MAPKs in human gastric cancer cells.

Corilagin (CLG) is a hydrolyzable tannin and possesses various pharmacological activities. Here, we investigated the impact of CLG as an anti-tumor agent against human gastric tumor cells. We observed that CLG could cause negative regulation of JAKs-Src-STAT3/5 signaling axis in SNU-1 cells, but did not affect these pathways in SNU-16 cells. Interestingly, CLG promoted the induction of mitogen-activated protein kinases (MAPKs) signaling pathways in only SNU-16 cells, but not in the SNU-1 cells. CLG exhibited apoptotic effects that caused an increased accumulation of the cells in sub-G1 phase and caspase-3 activation in both SNU-1 and SNU-16 cell lines. We also noticed that CLG and docetaxel co-treatment could exhibit significantly enhanced apoptotic effects against SNU-1 cells. Moreover, the combinations treatment of CLG and docetaxel markedly inhibited cell growth, phosphorylation of JAK-Src-STAT3 and induced substantial apoptosis. Additionally, pharmacological inhibition of JNK, p38, and ERK substantially blocked CLG-induced activation of MAPKs, cell viability, and apoptosis, thereby implicating the pivotal role of MAPKs in the observed anti-cancer effects of CLG. Taken together, our data suggest that CLG could effectively block constitutive STAT3/5 activation in SNU-1 cells but induce sustained MAPKs activation in SNU-16 cells.

Astringent Gallic Acid in Red Wine Regulates Mechanisms of Gastric Acid Secretion via Activation of Bitter Taste Sensing Receptor TAS2R4

Red wine is rich in phenolic compounds, which chiefly determine its characteristic taste. One of its major phenolic acid constituents for which an astringency, yet no clear contribution to bitter taste has been reported, is gallic acid (GA). In previous studies, we have demonstrated bitter-tasting constituents to regulate cellular proton secretion (PS) as a key mechanism of gastric acid secretion via activation of bitter taste sensing receptors (TAS2Rs). Here, we hypothesized a contributing role of GA to the red wine-stimulated effect on PS in human gastric tumor cells (HGT-1 cells). Sensory analyses revealed that 10 μM GA as the lowest concentration tested more bitter than tap water, with increasing bitter ratings up to 1000 μM. In HGT-1 cells, the concentration of 10 μM GA evoked the most pronounced effect on PS secretion, either when added to cells as in-water solution or when spiked to a red wine matrix. GA-spiking of Zweigelt and Blaufränkisch red wine samples up to a concentration of 10 μM resulted in an equally stimulated PS, whereas the non-GA-spiked wine samples demonstrated contrary effects on PS, indicating a functional role of GA on PS. Involvement of TAS2R4 in the GA-induced PS was verified by means of an HGT-1 homozygote CRISPR-Cas9 TAS2R4 knockout approach. Moreover, gene expression analyses revealed GA to increase TAS2R4. These results demonstrate a functional role of TAS2R4 in GA-evoked PS as a key mechanism of gastric acid secretion aiding digestion. Moreover, our data provide mechanistic insights, which will help to produce stomach-friendly red wines.

ASN004, A 5T4-targeting scFv-Fc Antibody-Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models.

The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors but shows very limited expression in normal adult tissues. ASN004 is a 5T4-targeted antibody-drug conjugate (ADC) that incorporates a novel single-chain Fv-Fc antibody and Dolaflexin drug-linker technology, with an Auristatin F hydroxypropylamide payload drug-to-antibody ratio of approximately 10-12. The pharmacology, toxicology, and pharmacokinetic properties of ASN004 and its components were investigated in vitro and in vivo ASN004 showed high affinity for the 5T4 antigen and was selectively bound to and internalized into 5T4-expressing tumor cells, and potent cytotoxicity was demonstrated for a diverse panel of solid tumor cell lines. ASN004 induced complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a wide range of 5T4 expression levels. A single dose of ASN004, as low as 1 mg/kg i.v., achieved complete tumor regression leading to tumor-free survivors in the A431 cervical cancer model. In head-to-head studies, superior activity of ASN004 was demonstrated against trastuzumab-DM1, in a low-5T4/high-HER2 expressing gastric tumor model, and 10-fold greater potency was found for ASN004 against the 5T4-targeted ADC PF-06263507 in a lung tumor model. In marmoset monkeys, ASN004 was well tolerated at doses up to 1.5 mg/kg Q3W i.v., and showed dose-dependent exposure, linear pharmacokinetics, and markedly low exposure of free payload drug. Taken together, these findings identify ASN004 as a promising new ADC therapeutic for clinical evaluation in a broad range of solid tumor types.

Tumor-specific overexpression of histone gene, H3C14 in gastric cancer is mediated through EGFR-FOXC1 axis

Incorporation of different H3 histone isoforms/variants have been reported to differentially regulate gene expression via alteration in chromatin organization during diverse cellular processes. However, the differential expression of highly conserved histone H3.2 genes, H3C14 and H3C13 in human cancer has not been delineated. In this study, we investigated the expression of H3.2 genes in primary human gastric, brain, breast, colon, liver, and head and neck cancer tissues and tumor cell lines. The data showed overexpression of H3.2 transcripts in tumor samples and cell lines with respect to normal counterparts. Furthermore, TCGA data of individual and TCGA PANCAN cohort also showed significant up-regulation of H3.2 genes. Further, overexpressed H3C14 gene coding for H3.2 protein was regulated by FOXC1 transcription factor and G4-cassette in gastric cancer cell lines. Elevated expression of FOXC1 protein and transcripts were also observed in human gastric cancer samples and cell lines. Further, FOXC1 protein was predominantly localized in the nuclei of neoplastic gastric cells compared to normal counterpart. In continuation, studies with EGF induction, FOXC1 knockdown, and ChIP-qPCR for the first time identified a novel axis, EGFR-FOXC1-H3C14 for regulation of H3C14 gene overexpression in gastric cancer. Therefore, the changes the epigenomic landscape due to incorporation of differential expression H3 variant contributes to change in gene expression pattern and thereby contributing to pathogenesis of cancer.

Monoamine Oxidase A is a Major Mediator of Mitochondrial Homeostasis and Glycolysis in Gastric Cancer Progression.

ObjectiveMonoamine oxidase A (MAO-A) is a mitochondrial protein involved in tumourigenesis in different types of cancer. However, the biological function of MAO-A in gastric cancer development remains unknown.MethodsWe examined MAO-A expression in gastric cancer tissues and in gastric cancer cell lines by immunohistochemistry and Western blot analyses. CCK8, FACS and bromodeoxyuridine incorporation assays were performed to assess the effects of MAO-A on gastric cancer cell proliferation. The role of MAO-A in mitochondrial function was determined through MitoSOX Red staining, ATP generation and glycolysis assays.ResultsIn the present study, we observed that MAO-A was significantly upregulated in gastric cancer tissues and in AGS and MGC803 cells. The observed MAO-A inhibition indicated decreased cell cycle progression and proliferation. Silencing MAO-A expression was associated with suppressed migration and invasion of gastric cancer cells in vitro. Moreover, alleviated mitochondrial damage in these cells was demonstrated by decreased levels of mitochondrial reactive oxygen species and increased ATP generation. MAO-A knockdown also regulated the expression of the glycolysis rate-limiting enzymes hexokinase 2 and pyruvate dehydrogenase. Finally, we observed that the glycolysis-mediated effect was weakened in AGS and MGC803 cells when MAO-A was blocked.ConclusionThe findings of the present study indicate that MAO-A is responsible for mitochondrial dysfunction and aerobic glycolysis, which in turn leads to the proliferation and metastasis of human gastric tumour cells.

In Vitro Digestion of Grape Seed Oil Inhibits Phospholipid-Regulating Effects of Oxidized Lipids.

The intake of dietary lipids is known to affect the composition of phospholipids in gastrointestinal cells, thereby influencing passive lipid absorption. However, dietary lipids rich in polyunsaturated fatty acids, such as vegetable oils, are prone to oxidation. Studies investigating the phospholipid-regulating effect of oxidized lipids are lacking. We aimed at identifying the effects of oxidized lipids from moderately (18.8 ± 0.39 meq O2/kg oil) and highly (28.2 ± 0.39 meq O2/kg oil) oxidized and in vitro digested cold-pressed grape seed oils on phospholipids in human gastric tumor cells (HGT-1). The oils were analyzed for their antioxidant constituents as well as their oxidized triacylglycerol profile by LC-MS/MS before and after a simulated digestion. The HGT-1 cells were treated with polar oil fractions containing epoxidized and hydroperoxidized triacylglycerols for up to six hours. Oxidized triacylglycerols from grape seed oil were shown to decrease during the in vitro digestion up to 40% in moderately and highly oxidized oil. The incubation of HGT-1 cells with oxidized lipids from non-digested oils induced the formation of cellular phospholipids consisting of unsaturated fatty acids, such as phosphocholines PC (18:1/22:6), PC (18:2/0:0), phosphoserine PS (42:8) and phosphoinositol PI (20:4/0:0), by about 40%–60%, whereas the incubation with the in vitro digested oils did not affect the phospholipid metabolism. Hence, the gastric conditions inhibited the phospholipid-regulating effect of oxidized triacylglycerols (oxTAGs), with potential implications in lipid absorption.

Inhibition of Autophagy via Lysosomal Impairment Enhances Cytotoxicity of Fullerenol under Starvation Condition.

Autophagy is well-known as a common cellular response to nanomaterials. As one of the most comprehensively studied carbon-based nanomaterials, fullerene and its derivatives have been reported to bring about autophagic features in various cell lines, but little is known about the role of fullerenol (C60(OH)44) on the modulation of autophagy in human gastric tumor cell line SGC-7901. Fullerenol treatment led to the accumulation of autophagosomes, as evidenced by the increased fluorescent intensity of monodansylcadaverine (MDC) staining cells, an elevated level of LC3 protein, and the observation of auotphagosomes in cytoplasm. Subsequent results of the p62 level demonstrated that the accumulation of autophagosomes resulted from the blockade of autophagic flux rather than the activation of autophagy. Fullerenol disrupted autophagic flux by impairing lysosomal function, including lysosome membrane permeabilization (LMP), alkaline of lysosomes, and reduced activity of capthesin B. Interestingly, fullerenol treatment was noncytotoxic under a nutrient-rich condition. When serum was deprived, cytotoxicity occurred in a concentration- and time-dependent manner, along with massive vacuoles in cytoplasm, a large amount of ROS generation, and finally cell death, which can be ascribed to the disruption of essential autophagy in cells. Taken together, understanding this autophagy-lysosome pathway will shed light on the potential anticancer application of fullerenol.

Epigenetic Suppression of Interferon Lambda Receptor Expression Leads to Enhanced Human Norovirus Replication In Vitro.

Human norovirus (HuNoV) is the main cause of gastroenteritis worldwide, yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an interferon (IFN)-cured population of replicon-harboring HGT cells (HGT-Cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-Cured cells created an environment favoring norovirus replication. Microarrays were used to identify genes differentially regulated in HGT-NV and HGT-Cured compared to parental cells. We found that IFN lambda receptor (IFNLR1) expression was highly reduced in HGT-NV and HGT-Cured cells. While all three cell lines responded to exogenous IFN-β by inducing interferon-stimulated genes, HGT-NV and HGT-Cured cells failed to respond to exogenous IFN-λ. Methylation-sensitive PCR showed that an increased methylation of the IFNLR1 promoter and inhibition of DNA methyltransferase activity partially reactivated IFNLR1 expression in HGT-NV and HGT-Cured cells, indicating that host adaptation occurred via epigenetic reprogramming. Moreover, IFNLR1 ectopic expression rescued response to IFN-λ and restricted HuNoV replication in HGT-NV cells. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. This study unravels for the first time epigenetic reprogramming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signaling is able to control human norovirus replication.IMPORTANCE Noroviruses are one of the most widespread causes of gastroenteritis, yet no suitable therapeutics are available for their control. Moreover, to date, knowledge of the precise cellular processes that control the replication of the human norovirus remains ill defined. Recent work has highlighted the importance of type III interferon (IFN) responses in the restriction of viruses that infect the intestine. Here, we analyzed the adaptive changes required to support long-term replication of noroviruses in cell culture and found that the receptor for type III IFN is decreased in its expression. We confirmed that this decreased expression was driven by epigenetic modifications and that cells lacking the type III IFN receptor are more permissive for norovirus replication. This work provides new insights into key host-virus interactions required for the control of noroviruses and opens potential novel avenues for their therapeutic control.

Oleanolic acid reduces aerobic glycolysis-associated proliferation by inhibiting yes-associated protein in gastric cancer cells

Gastric cancer represents a common malignancy of digestive tract with high incidence and mortality. Increasing evidence suggests that the growth of gastric tumor cells relies largely on aerobic glycolysis. Currently, many potential anti-cancer candidates are derived from natural products. Here, we evaluated the effects of oleanolic acid (OA), a triterpenoid component widely found in the plants of Oleaceae family, on aerobic glycolysis and proliferation in human MKN-45 and SGC-7901 gastric cancer cells. Our results demonstrated that OA reduced the viability and proliferation of gastric cancer cells and inhibited the expression of cyclin A and cyclin-dependent kinase 2. OA blocked glycolysis in these cells evidenced by decreases in the uptake and consumption of glucose, intracellular lactate levels and extracellular acidification rate. Glycolysis inhibitor 2-deoxy-d-glucose, similar to OA, suppressed gastric cancer cell proliferation. OA also decreased the expression and intracellular activities of glycolysis rate-limiting enzymes hexokinase 2 (HK2) and phosphofructokinase 1 (PFK1). Moreover, OA downregulated the expression of hypoxia inducible factor-1α (HIF-1α) and decreased its nuclear abundance. Upregulation of HIF-1α by deferoxamine rescued OA-inhibited HK2 and PFK1. Furthermore, OA reduced the nuclear abundance of yes-associated protein (YAP) in gastric tumor cells. YAP inhibitor verteporfin, similar to OA, downregulated the expression of HIF-1α and glycolytic enzymes in gastric cancer cells; whereas overexpression of YAP abrogated all these effects of OA. Collectively, inhibition of YAP was responsible for OA blockade of HIF-1α-mediated aerobic glycolysis and proliferation in human gastric tumor cells. OA could be developed as a promising candidate for gastric cancer treatment.

Cycloastragenol can negate constitutive STAT3 activation and promote paclitaxel-induced apoptosis in human gastric cancer cells

BackgroundCycloastragenol (CAG), a triterpene aglycone is commonly prescribed for treating hypertension, cardiovascular disease, diabetic nephropathy, viral hepatitis, and various inflammatory-linked diseases. HypothesisWe investigated CAG for its action on signal transducer and activator of transcription 3 (STAT3) activation cascades, and its potential to sensitize gastric cancer cells to paclitaxel-induced apoptosis. MethodsThe effect of CAG on STAT3 phosphorylation and other hallmarks of cancer was deciphered using diverse assays in both SNU-1 and SNU-16 cells. ResultsWe observed that CAG exhibited cytotoxic activity against SNU-1 and SNU-16 cells to a greater extent as compared to normal GES-1 cells. CAG predominantly caused negative regulation of STAT3 phosphorylation at tyrosine 705 through the abrogation of Src and Janus-activated kinases (JAK1/2) activation. We noted that CAG impaired translocation of STAT3 protein as well as its DNA binding activity. It further decreased cellular proliferation and mediated its anticancer effects predominantly by causing substantial apoptosis rather than autophagy. In addition, CAG potentiated paclitaxel-induced anti-oncogenic effects in gastric tumor cells. ConclusionsOur results indicate that CAG can function to impede STAT3 activation in human gastric tumor cells and therefore it may be a suitable candidate agent for therapy of gastric cancer.

Noncaloric Sweeteners Induce Peripheral Serotonin Secretion via the T1R3-Dependent Pathway in Human Gastric Parietal Tumor Cells (HGT-1).

The role of sweet taste in energy intake and satiety regulation is still controversial. Noncaloric artificial sweeteners (NCSs) are thought to help reduce energy intake, although little is known about their impact on the satiating neurotransmitter serotonin (5-HT). In the gastrointestinal (GI) tract, 5-HT regulates gastric acid secretion and gastric motility, both part of the complex network of mechanisms regulating food intake and satiety. This study demonstrated a stimulating impact compared to controls (100%) on 5-HT release in human gastric tumor cells (HGT-1) by the NCSs cyclamate (50 mM, 157% ± 6.3%), acesulfame potassium (Ace K, 50 mM, 197% ± 8.6%), saccharin (50 mM, 147% ± 6.7%), sucralose (50 mM, 194% ± 11%), and neohesperidin dihydrochalcone (NHDC, 1 mM, 201% ± 13%). Although these effects were not associated with the sweet taste intensity of the NCSs tested, involvement of the sweet receptor subunit T1R3 in the NCS-evoked response was demonstrated by mRNA expression of TAS1R3, co-incubation experiments using the T1R3 receptor antagonist lactisole, and a TAS1R3 siRNA knockdown approach. Analysis of the downstream signaling revealed activation of the cAMP/ERK/Ca2+ cascade. Co-treatment experiments with 10 mM glucose enhanced the 5-HT release induced by cyclamate, Ace K, saccharin, and sucralose, thereby supporting the enhancing effect of glucose on a NCS-mediated response. Overall, the results obtained identify NCSs as potent inducers of 5-HT release via T1R3 in human gastric parietal cells in culture and warrant in vivo studies to demonstrate their efficacy.

Human Sweet Receptor T1R3 is Functional in Human Gastric Parietal Tumor Cells (HGT-1) and Modulates Cyclamate and Acesulfame K-Induced Mechanisms of Gastric Acid Secretion.

The noncaloric sweeteners (NCSs) cyclamate (Cycl) and acesulfame K (AceK) are widely added to foods and beverages. Little is known about their impact on gastric acid secretion (GAS), which is stimulated by dietary protein and bitter-tasting compounds. Since Cycl and AceK have a bitter off taste in addition to their sweet taste, we hypothesized they modulate mechanisms of GAS in human gastric parietal cells (HGT-1). HGT-1 cells were exposed to sweet tastants (50 mM of glucose, d-threonine, Cycl, or AceK) and analyzed for their intracellular pH index (IPX), as an indicator of proton secretion by means of a pH-sensitive dye, and for mRNA levels of GAS-associated genes by RT-qPCR. Since the NCSs act via the sweet taste-sensing receptor T1R2/T1R3, mRNA expression of the corresponding genes was analyzed in addition to immunocytochemical localization of the T1R2 and T1R3 receptor proteins. Exposure of HGT-1 cells to AceK or d-threonine increased the IPX to 0.60 ± 0.05 and 0.80 ± 0.04 ( P ≤ 0.05), respectively, thereby indicating a reduced secretion of protons, whereas Cycl demonstrated the opposite effect with IPX values of -0.69 ± 0.08 ( P ≤ 0.05) compared to controls (IPX = 0). Cotreatment with the T1R3-inhibitor lactisole as well as a TAS1R3 siRNA knock-down approach reduced the impact of Cycl, AceK, and d-thr on proton release ( P ≤ 0.05), whereas cotreatment with 10 mM glucose enhanced the NCS-induced effect ( P ≤ 0.05). Overall, we demonstrated Cycl and AceK as modulators of proton secretion in HGT-1 cells and identified T1R3 as a key element in this response.

Impact of free Nε-carboxymethyllysine, its precursor glyoxal and AGE-modified BSA on serotonin release from human parietal cells in culture.

Advanced glycation end products (AGEs) are frequently encountered in a western diet, in addition to their formation in vivo. N-Epsilon-carboxymethyllysine (CML), one of the chemically diverse compounds formed in the reaction between reducing carbohydrates and amines, is often used as a marker of advanced glycation, and has been shown to stimulate serotonin release from cells representing the central (SH-SY5Y cells) and the peripheral (Caco-2 cells) serotonin system in vitro. Here, we investigated the effect of glyoxal, free CML, and protein-linked AGE-BSA on serotonin release from human gastric tumour cells, which originate from an adenocarcinoma of the stomach and have recently been shown to be capable of serotonin synthesis and release. Microarray experiments showed both CML and glyoxal to alter genes associated with serotonin receptors. Furthermore, treatment with glyoxal resulted in a small change in RAGE expression while CML did not alter its expression. On a functional level, treatment with 500 μM CML increased extracellular serotonin content by 341 ± 241%, while treatment with 1 mg mL-1 AGE-BSA led to a reduction by 49 ± 11% compared to non-treated cells. The CML-induced serotonin release was reduced by the HTR3 antagonist granisetron. Incubation with the RAGE antagonist FPS-ZM1 abolished the effect of AGE-BSA on serotonin release, while no impact on CML-induced serotonin release was observed. Furthermore, treatment with 5 mM CML stimulated proton secretion as a functional outcome measure, assessed using a pH sensitive dye. Taken together, these results indicate a likely HTR3-mediated, RAGE-independent effect of free CML on serotonin release and a RAGE-dependent mechanism for the protein linked AGE-BSA.

Abstract B109: ASN004, a novel 5T4-targeted Dolaflexin ADC, causes complete and durable tumor regression in a variety of tumor xenograft models

Abstract The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors, while showing very limited expression in normal adult tissues. This selective pattern of expression establishes 5T4 as an attractive therapeutic target for the Antibody-Drug Conjugate (ADC) strategy. ASN004 is an anti-5T4 ADC that incorporates a novel single-chain homo-dimer antibody, Fleximer® linker technology (Mersana Therapeutics), and cytotoxic dolastatin (auristatin) analog warheads, with a drug/antibody ratio of ~15:1. ASN004 shows high affinity for the 5T4 antigen (Kd < 30 pM), with selective binding and internalization to 5T4-expressing tumor cells. Potent, selective cytotoxicity has been determined for a diverse panel of tumor cell lines. ASN004 provides complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a range of 5T4 expression levels. Tumor-free survivors could be achieved in tumor models with a single dose of ASN004, as low as 1 mg/kg iv. The broad, superior activity of ASN004 was demonstrated in a head-to-head study against trastuzumab-DM1, in a low-5T4/high-HER2 expressing tumor model. Finally, efficacy of ASN004 in patient-derived tumor xenograft (PDX) models will be presented. In summary, ASN004 has been established as a promising ADC therapeutic for multiple tumor types, encompassing a wide range of 5T4-expression levels. IND-enabling studies are ongoing in preparation for advancement to clinical development. Citation Format: Roger A. Smith, David J. Zammit, Sanjeeva P. Reddy. ASN004, a novel 5T4-targeted Dolaflexin ADC, causes complete and durable tumor regression in a variety of tumor xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B109.

Design of theranostic nanomedicine (II): synthesis and physicochemical properties of a biocompatible polyphosphazene–docetaxel conjugate

To prepare an efficient theranostic polyphosphazene–docetaxel (DTX) conjugate, a new drug delivery system was designed by grafting a multifunctional lysine ethylester (LysOEt) as a spacer group along with methoxy poly(ethylene glycol) (MPEG) to the polyphosphazene backbone ([NP]n), and then DTX was conjugated to the carrier polymer using acid-cleavable cis-aconitic acid (AA) as a linker. The resultant polyphosphazene–DTX conjugate, formulated as [NP(MPEG550)3(Lys-OEt)(AA)(DTX)]n and named “Polytaxel”, exhibited high water solubility and stability by forming stable polymeric micelles as shown in its transmission electron microscopy image and dynamic light scattering measurements. Another important aspect of Polytaxel is that it can easily be labeled with various imaging agents using the lysine amino group, enabling studies on various aspects, such as its organ distribution, tumor-targeting properties, pharmacokinetics, toxicity, and excretion. The pharmacokinetics of Polytaxel was remarkably improved, with prolonged elimination half-life and enhanced area under the curve. Ex vivo imaging study of cyanine dye-labeled Polytaxel showed that intravenously injected Polytaxel is long circulating in the blood stream and selectively accumulates in tumor tissues. Polytaxel distributed in other organs was cleared from all major organs at ~6 weeks after injection. The in vitro study of DTX release from the carrier polymer showed that >95% of conjugated DTX was released at pH 5.4 over a period of 7 days. Xenograft trials of Polytaxel using nude mice against the human gastric tumor cell line MKN-28 showed complete tumor regression, with low systemic toxicity. Polytaxel is currently in preclinical study.

PPeOp from Omphalia lapidescens Schroet induces cell cycle arrest and inhibits the migration of MC-4 gastric tumor cells.

The aim of the present study was to investigate the effect of purified Omphalia lapidescens protein (pPeOp) extracted by polyvinylpyrrolidone from the fungus Omphalia lapidescens Schroet on the proliferation and cell cycle progression of MC-4 human gastric tumor cells. Using polyvinylpyrrolidone, pPeOp was extracted from O. lapidescens Schroet. MC-4 cells were cultured with 30, 60 or 90 µg/ml pPeOp, with 5-fluorouracil used as a positive control. Survival rates of treated cells were significantly decreased compared with those of the untreated control group in a dose-dependent manner. Using flow cytometric analysis, cells treated with pPeOp were demonstrated to arrest in S phase and exhibit abnormal G0/G1 and G2/M phase cell cycle distribution. In addition, a wound healing assay demonstrated that pPeOp significantly inhibited the migration of MC-4 cells. The mRNA and protein expression levels of cyclin D1/cyclin-dependent kinase (CDK) 4, cyclin B/CDK1, cyclin A/CDK2, matrix metalloproteinase (MMP)-2 and MMP-9 were determined using reverse transcription-quantitative polymerase chain reaction analysis and western blotting. The mRNA expression level of CDK4 and cyclin A was significantly increased compared with the untreated control; however, cyclin D1, CDK1, CDK2, cyclin B, MMP-2, and MMP-9 exhibited a significantly decreased mRNA expression level, indicating that there is a negative association between concentration and cyclin D1 expression levels. The expression of the cycle arrest-associated proteins and migration-associated proteins examined were similar to the observed mRNA expression levels. In conclusion, pPeOp was identified to inhibit migration of and cause S phase cell cycle arrest in MC-4 cells.

Characterization of Bitter Compounds via Modulation of Proton Secretion in Human Gastric Parietal Cells in Culture.

Humans perceive bitterness via around 25 different bitter receptors. Therefore, the identification of antagonists remains a complex challenge. We previously demonstrated several bitter-tasting compounds such as caffeine to induce acid secretion in the stomach and in a human gastric tumor cell line (HGT-1). Here, the results of a fluorescent-based in vitro assay using HGT-1 cells and a human sensory panel testing nine selected potential bitter modulators, with or without the bitter compounds caffeine or theobromine, were compared. Of the bitter-modulating compounds tested, eriodictyol, matairesinol, enterolacton, lariciresinol, and homoeriodictyol reduced the effect of caffeine on proton secretion by -163 ± 14.0, -152 ± 12.4, -74 ± 16.4, -58 ± 7.2, and -44.6 ± 16.5%, respectively, and reduced the bitter intensity of caffeine in the human sensory panel. In contrast, naringenin and 5,7-dihydroxy-4(4-hydroxyphenyl)chroman-2-one neither reduced the caffeine-induced proton secretion in HGT-1 cells nor showed an effect on bitter intensity perceived by the sensory panel. Results for theobromine were not as pronounced as those for caffeine, but followed a similar trend. The results demonstrate that the HGT-1 in vitro assay is a useful tool to identify potential bitter-masking compounds. Nevertheless, a sensory human panel is necessary to quantify the bitter-masking potency.

Increased nuclear localization of substance P in human gastric tumor cells

Gastric cancer (GC) is an aggressive disease that remains the fourth most common type of cancer and is the second leading cause of cancer-related death worldwide. Treatment of advanced or metastatic GC has seen little progress and median overall survival in this group remains <1 year. It is urgent to investigate new mechanisms to understand GC progression. It is known that substance P (SP), after binding to the neurokinin-1 (NK-1) receptor, elicits GC proliferation; that GC cells and samples express NK-1 receptors; that NK-1 receptor antagonists, in a concentration dependent manner, inhibit the proliferation of GC cells and that these cells die by apoptosis. However, the presence of SP in GC and normal gastric cells is unknown. In order to know more on the involvement of the SP/NK-1 receptor system in GC, we studied in thirty human GC and normal gastric samples the immunolocalization of SP after using an immunohistochemical technique. SP was observed in the cytoplasm and in the nucleus of GC and normal gastric cells. The nuclear expression of SP was higher in GC cells than in normal cells. No significant difference was observed when the cytoplasmatic expression of SP in normal and GC cells was compared. The findings suggest that SP plays an important role in both nuclear function and GC.