Gastric Cancer Cell Lines NCI-N87 Research Articles

Dual targeting non-overlapping epitopes in HER2 domain IV substantially enhanced HER2/HER2 homodimers and HER2/EGFR heterodimers internalization leading to potent antitumor activity in HER2-positive human gastric cancer

BackgroundTrastuzumab and pertuzumab combination has been approved for the treatment of patients with HER2-positive metastatic breast cancer. However, trastuzumab and pertuzumab combination did not show improvement in overall survival in patients with HER2-positive metastatic gastric cancer.MethodsWe developed a new HER2-targeted monoclonal antibody, HLX22, targeting HER2 subdomain IV as trastuzumab but with non-overlapping epitopes. We examined the antitumor effects of this novel HER2-antibody in gastric cell lines and cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models.ResultsHLX22 in combination with HLX02 (trastuzumab biosimilar) induced enhancement of HER2/HER2 homodimers and HER2/EGFR heterodimers internalization, which ultimately led to the reduction in signal transductions involving STAT3, P70 S6, and AKT; gene expressions of FGF-FGFR-PI3K-MTOR, EGF-EGFR-RAS, TGF-β-SMAD, PLCG and cell cycle progression related pathways that favor tumor development, proliferation, progression, migration and survival in gastric cancer cell line NCI-N87 were also reduced. These differing but complementary actions contributed to the synergistic antitumor efficacy of the HLX22 and HLX02 combination in gastric cancer cell lines, CDX and PDX. In addition, HLX22 in combination with HLX02 demonstrated stronger antitumor efficacy than HLX02 and HLX11 (a potential pertuzumab biosimilar) combination treatment both in vitro and in vivo.ConclusionsThese results suggested that the application of non-competing antibodies HLX22 and HLX02 targeting HER2 subdomain IV together may be of substantial benefit to gastric cancer patients who currently respond suboptimal to trastuzumab therapy.

Gefitinib Induces Apoptosis of Gastric Cancer Cells by Promoting Glutaminolysis Through the MEK/ERK Signaling Pathway

We aimed to investigate the effect of gefitinib on the proliferation and apoptosis of gastric cancer (GC) cells through the MEK/ERK signaling pathway. The GC cell line NCI-N87 was cultured in vitro and divided into a control group and a gefitinib group. Cell viability for NCI-N87 was determined using the MTT test. The amount of apoptosis in NCI-N87 cells was measured using flow cytometry. Real-time quantitative PCR was used to measure GDH1 mRNA expression in NCI-N87 cells. P-MEK1/2, MEK1/2, P-ERK1/2, and ERK1/2 protein expression levels in NCI-N87 cells were determined using Western blotting. An assay kit for measuring glutamine was used to determine the intracellular glutamine concentration of NCI-N87 cells. The proliferative activity of NCI-N87 cells was significantly inhibited in the gefitinib group compared to the control group, along with the transcription level of GDH1, intracellular glutamine content, intracellular glutamine content reduction, and p-MEK1/2 and p-ERK1/2 protein expression levels. Gefitinib inhibits proliferation and promotes apoptosis in the GC cell line NCI-N87 by downregulating the EGFR/MEK/ERK signaling pathway. Mechanistically, it is achieved by promoting the expression of GDH1 to promote glutaminolysis in NCI-N87 cells.

Lysine-specific Demethylase 6A Upregulates Cadherin-1 and Accelerates Gastric Cancer Growth.

Gastric cancer (GC) ranks fifth among all common malignancies globally. Genetic research has revealed several genes that are frequently dis-regulated in GC, such as lysine-specific demethylase 6A (KDM6A) and cadherin-1 (CDH1). This study aimed to examine the expression profile and role of KDM6A in GC, as well as the molecular pathway involved. The expression profile and overall survival data of KDM6A were retrieved from the TCGA database. Expression levels of KDM6A were also measured in GC patient samples and compared with those of healthy controls. Furthermore, stable silencing of KDM6A was introduced into the GC cell line NCI-N87, followed by assessments of cell proliferation, migration and invasion, in the xenograft mouse model. The metastatic status of mice injected with NCI-N87 cells was also analyzed. In patients diagnosed with GC, KDM6A was upregulated. Silencing KDM6A reduced the proliferation, migration and invasion of cells, as well as the growth of xenograft tumors. KDM6A knockdown also inhibited metastatic behaviors of injected NCI-N87 cells, as well as elevated CDH1 expression, leading to reversed epithelial-mesenchymal transition. KDM6A serves as an oncogene in GC and exerts its pro-tumor functions by repressing the expression of CDH1.

Wnt/β-catenin pathway is a key signaling pathway to trastuzumab resistance in gastric cancer cells

BackgroundTrastuzumab is the only approved target agent for the first-line treatment of human epidermal growth factor receptor-2 (HER-2) positive gastric cancer; however, trastuzumab resistance is a major problem in clinical practice. To comprehend the mechanism of trastuzumab resistance, we focused on the Wnt/β-catenin signaling pathway and its influence on the phenotypes and behavior of trastuzumab-resistant gastric cancer cells.MethodsTrastuzumab-resistant NCI-N87R cells were established in vitro from the human gastric cancer cell line NCI-N87 by dose-escalating repeated trastuzumab treatment. We investigated the phenotypes of NCI-N87R cells, including Wnt signaling pathway activity. Gastric cancer organoid cells were incubated with complete medium and Wnt3a-depletion medium, and their resistance to trastuzumab was compared.ResultsNCI-N87R exhibited stemness and epithelial-mesenchymal transition (EMT)-like phenotypes, along with decreased levels of the epithelial marker E-cadherin and increased levels of the mesenchymal markers Vimentin and Snail along with an increased Wnt signaling pathway activity. When gastric cancer cells were incubated in Wnt3a-conditioned medium. Wnt signaling pathway activity and resistance to trastuzumab increased. Gastric cancer patient-derived organoids incubated in Wnt3a-depletion medium were more susceptible to dose-dependent inhibition of cell viability by trastuzumab than those incubated in complete medium.ConclusionsTrastuzumab-resistant gastric cancer cells exhibited EMT-like phenotype, and trastuzumab resistance was promoted by the Wnt/β-catenin signaling pathway. The Wnt/β-catenin pathway is a key signaling pathway for trastuzumab resistance in gastric cancer cells.

PVT1/miR-16/CCND1 axis regulates gastric cancer progression.

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) has been reported to be a vital modulator in tumorigenesis of gastric cancer (GC). However, the detailed regulatory mechanism of PVT1 in GC remains largely unclear. In this work, the expressions of PVT1 and microRNA-16 (miR-16) were detected by quantitative real-time PCR (qRT-PCR) in GC tissues and cell lines. GC cell lines NCI-N87 and MKN45 cell lines were chosen for the following assays. After PVT1 was overexpressed or depleted, CCK-8 and Transwell assays were performed to examine the cell viability and invasive capacity. Cell cycle was analyzed by flow cytometry. The expression of cyclin D1 (CCND1) at mRNA and protein levels was measured by qRT-PCR and western blot. The competitive endogenous RNA molecular mechanism among PVT1, miR-16 and CCND1 was verified by bioinformatics analysis, luciferase-reporter gene assay and RNA immunoprecipitation assay. In the present study, it was revealed that PVT1 expression was remarkably evaluated in GC tissues and cell lines than that in the corresponding control group. PVT1 positively regulated the proliferation, migration and cell cycle progression of GC cells. Besides, miR-16 was identified as a target of PVT1, and CCND1 was identified as a target of miR-16. The depletion of PVT1 promoted the expression of miR-16 and suppressed CCND1 expression. Moreover, either miR-16 inhibitor or CCND1 overexpression plasmid could reverse the promoting effects of PVT1 on the malignant biological behaviors of GC cells. In conclusion, PVT1 promoted CCND1 expression by negatively regulating miR-16 expression to enhance the viability, invasion and cell cycle progression of GC cells.

Human gastric fibroblasts ameliorate A20-dependent cell survival in co-cultured gastric epithelial cells infected by Helicobacter pylori

Crosstalk within the gastric epithelium, which is closely in contact with stromal fibroblasts in the gastric mucosa, has a pivotal impact in proliferation, differentiation and transformation of the gastric epithelium. The human pathogen Helicobacter pylori colonises the gastric epithelium and represents a risk factor for gastric pathophysiology. Infection of H. pylori induces the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is involved in the pro-inflammatory response but also in cell survival. In co-cultures with human gastric fibroblasts (HGF), we found that apoptotic cell death is reduced in the polarised human gastric cancer cell line NCI-N87 or in gastric mucosoids during H. pylori infection. Interestingly, suppression of apoptotic cell death in NCI-N87 cells involved an enhanced A20 expression regulated by NF-κB activity in response to H. pylori infection. Moreover, A20 acts as an important negative regulator of caspase-8 activity, which was suppressed in NCI-N87 cells during co-culture with gastric fibroblasts. Our results provide evidence for NF-κB-dependent regulation of apoptotic cell death in cellular crosstalk and highlight the protective role of gastric fibroblasts in gastric epithelial cell death during H. pylori infection.

Isoquinoline Alkaloids from Coptis chinensis Franch: Focus on Coptisine as a Potential Therapeutic Candidate against Gastric Cancer Cells.

Gastric cancer (GC) has high incidence rates and constitutes a common cause of cancer mortality. Despite advances in treatment, GC remains a challenge in cancer therapy which is why novel treatment strategies are needed. The interest in natural compounds has increased significantly in recent years because of their numerous biological activities, including anti-cancer action. The isolation of the bioactive compounds from Coptis chinensis Franch was carried out with the Centrifugal Partition Chromatography (CPC) technique, using a biphasic solvent system composed of chloroform (CHCl3)—methanol (MeOH)—water (H2O) (4:3:3, v/v) with an addition of hydrochloric acid and trietylamine. The identity of the isolated alkaloids was confirmed using a high resolution HPLC-MS chromatograph. The phytochemical constituents of Coptis chinensis such as berberine, jatrorrhizine, palmatine and coptisine significantly inhibited the viability and growth of gastric cancer cell lines ACC-201 and NCI-N87 in a dose-dependent manner, with coptisine showing the highest efficacy as revealed using MTT and BrdU assays, respectively. Flow cytometry analysis confirmed the coptisine-induced population of gastric cancer cells in sub-G1 phase and apoptosis. The combination of coptisine with cisplatin at the fixed-ratio of 1:1 exerted synergistic and additive interactions in ACC-201 and NCI-N87, respectively, as determined by means of isobolographic analysis. In in vivo assay, coptisine was safe for developing zebrafish at the dose equivalent to the highest dose active in vitro, but higher doses (greater than 10 times) caused morphological abnormalities in larvae. Our findings provide a theoretical foundation to further studies on more detailed mechanisms of the bioactive compounds from Coptis chinensis Franch anti-cancer action that inhibit GC cell survival in in vitro settings.

Abstract P2-13-15: VRN101396, a brain-permeable HER2 inhibitor, shows the anti-tumor activity in preclinical HER2-positive cancer models

Abstract Breast cancer (BC) is the second most common cancer associated with brain metastasis (BM) and among patients with breast cancer brain metastasis (BCBM), human epidermal growth factor receptor 2 (HER2)-positive breast cancer is most likely to develop BM. The overall prognosis of HER2-positive BCBM remains dismal with median survival time after BM of less than a year. Drugs like T-DM1, T-DXd and Tucatinib are reported to deliver a progression-free survival benefit in HER2-positive BCBM patients, but there are still unmet needs for a drug that crosses the blood brain barrier more effectively. We develop VRN101396, a brain penetrant, orally available, and irreversible small molecule inhibitor targeting HER2 to offer a new therapeutic option for the treatment of HER2-positive BCBM. VRN101396 potently inhibits catalytic activity of HER2 but spares wild-type EGFR. In HER2-positive breast cancer cell lines BT474 and SK-BR3 and gastric cancer cell line NCI-N87, VRN101396 inhibited the cell proliferation and phosphorylation of HER2 with single- or double-digit nanomolar IC50 values while inhibiting wild type EGFR with an IC50 value of >100 nM. In both subcutaneous and intracranial efficacy mouse models, once-daily oral dose of VRN101396 significantly inhibited tumor growth and achieved tumor regression. In N87 subcutaneous xenograft model, VRN101396 alone showed superior efficacy to the combination of Tucatinib and Trastuzumab. Additionally, in intracranial xenograft model, VRN101396 showed superior efficacy to Tucatinib. Pharmacokinetics of VRN101396 displayed higher brain exposure than tucatinib and lapatinib, indicating better target engagement in the brain. Pharmacodynamic analysis revealed that VRN101396 reduced phosphorylation of HER2, HER3, AKT, and ERK compared to vehicle group after administration up to 24 hours. In conclusion, VRN101396 is a therapeutic candidate for the treatment of HER2-positive BC, BCBM and gastric cancer. Citation Format: Hong-ryul Jung, Sunghwan Kim, Jihye Yoo, Chan mi Park, Somi Lee, Hyoju Lee, Youngyi Lee, Jinhee Park, Kyungah Seo, Dong-Hyuk Seo, Eunhwa Ko, Jung Beom Son, Deakwon Kim, Hwan Geun Choi, Nam Doo Kim. VRN101396, a brain-permeable HER2 inhibitor, shows the anti-tumor activity in preclinical HER2-positive cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-15.

Inhibition of gastric cancer cell growth, invasion and metastasis by tocotrienolic amide

Purpose: To investigate the effect of tocotrienolic amide on gastric cancer (GC) cell growth and metastasis, and the underlying mechanism of action.Methods: Gastric cancer (GC) cell lines MKN28 and NCI-N87 were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin solution at 37 ˚C in a humidified atmosphere of 5 % CO2 and 95 % air. Cell invasion and migration were determined using Transwell and wound healing assays, respectively. Realtime quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used for the determination of changes in the levels of expression of Snail, E-cadherin, vimentin, and microRNA-195-5p (miR-195-5p). In vivo tumor growth inhibition was determined 45 days after establishment of GC xenografts in nude mice.Results: Treatment of MKN28 and NCI-N87 cells with tocotrienolic amide significantly and dosedependently reduced their invasiveness and migratory capacity (p < 0.05). It also significantly and dosedependently downregulated the mRNA and protein expressions of Snail and vimentin, but significantly upregulated E-cadherin expression (p < 0.05). The mRNA expression of miR-195-5p was significantly and dose-dependently upregulated in MKN28 and NCI-N87 cells treated with tocotrienolic amide, but was downregulated after transfection with miR-195-5p inhibitor (p < 0.05). Transfection of MKN28 and NCI-N87 cells with miR-195-5p inhibitor significantly and dose-dependently upregulated mRNA and protein expressions of Snail (p < 0.05). Moreover, treatment of GC mice with TCTA led to significant and dose-dependent reduction in tumor weight and volume (p < 0.05).Conclusion: These results suggest that tocotrienolic amide inhibits the growth and metastasis of GC cells by directly targeting Snail and vimentin genes, and thus can potentially be developed for the management of gastric cancer.
 Keywords: Gastric cancer, Metastasis, Protein expression, Tocotrienolic amide, Tumor volume

Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines

BackgroundGastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide. The molecular mechanisms of action for anti-HER-family drugs in gastric cancer cells are incompletely understood. We compared the molecular effects of trastuzumab and the other HER-family targeting drugs cetuximab and afatinib on phosphoprotein and gene expression level to gain insights into the regulated pathways. Moreover, we intended to identify genes involved in phenotypic effects of anti-HER therapies.MethodsA time-resolved analysis of downstream intracellular kinases following EGF, cetuximab, trastuzumab and afatinib treatment was performed by Luminex analysis in the gastric cancer cell lines Hs746T, MKN1, MKN7 and NCI-N87. The changes in gene expression after treatment of the gastric cancer cell lines with EGF, cetuximab, trastuzumab or afatinib for 4 or 24 h were analyzed by RNA sequencing. Significantly enriched pathways and gene ontology terms were identified by functional enrichment analysis. Furthermore, effects of trastuzumab and afatinib on cell motility and apoptosis were analyzed by time-lapse microscopy and western blot for cleaved caspase 3.ResultsThe Luminex analysis of kinase activity revealed no effects of trastuzumab, while alterations of AKT1, MAPK3, MEK1 and p70S6K1 activations were observed under cetuximab and afatinib treatment. On gene expression level, cetuximab mainly affected the signaling pathways, whereas afatinib had an effect on both signaling and cell cycle pathways. In contrast, trastuzumab had little effects on gene expression. Afatinib reduced average speed in MKN1 and MKN7 cells and induced apoptosis in NCI-N87 cells. Following treatment with afatinib, a list of 14 genes that might be involved in the decrease of cell motility and a list of 44 genes that might have a potential role in induction of apoptosis was suggested. The importance of one of these genes (HBEGF) as regulator of motility was confirmed by knockdown experiments.ConclusionsTaken together, we described the different molecular effects of trastuzumab, cetuximab and afatinib on kinase activity and gene expression. The phenotypic changes following afatinib treatment were reflected by altered biological functions indicated by overrepresentation of gene ontology terms. The importance of identified genes for cell motility was validated in case of HBEGF.

Abstract 2218: High affinity and cleavage resistant CD16 enhances Trastuzumab mediated killing of gastric cancer targets by human placental CD34+-derived natural killer cells

Abstract Introduction: When targeting tumors with monoclonal antibodies, natural killer (NK) cells are key effectors of antibody dependent cellular cytotoxicity (ADCC) following recognition of antibody Fc by CD16. Celularity has developed a novel proprietary technology platform that enables production of an allogeneic, off-the-shelf, placental CD34+-derived NK cell therapy for the treatment of hematologic and solid tumor malignancies. Here we report the genetic modification of CD16 in NK cells for high binding affinity to IgG and resistance to protease cleavage, together with functional evaluation of anti-tumor ADCC activity against epidermal growth factor receptor 2 (HER2) positive gastric cancer cells in combination with Trastuzumab (anti-Her2). Methods: Human placental CD34+ cells were transduced with lentivirus expressing a high binding affinity (158Val) and protease resistant (197Pro) CD16 variant (CD16VP) and cultured for 35 days in the presence of cytokines, including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, to generate CD16VP cells. The tumorigenic activity of CD16VP cells against HER2-positive gastric cancer cell lines NCI-N87 and OE19 was assessed in combination with Trastuzumab. ADCC activity was measured by real-time xCELLigence, and cytokine secretion by Luminex. The kinase inhibitors PD98059 (MEK/ERK), SB203580 (p38 MAPK), SP600125 (JNK), and Wortmannin (PI3K) were used to study the molecular mechanisms underlying cytotoxicity of CD16VP cells against gastric cancer. Results and conclusion: High transduction efficiency was achieved in multiple placental CD34+ donors with an average of 64.6 ± 10.3% (n=8) CD16 expression on NK cells compared to 12.1 ± 3.3% (n=8) on non-transduced cells. These cells expanded [7095 ± 2998-fold (n=8)] and differentiated into NK cells with >90% purity (CD56+CD3-). While treatment of PMA/ionomycin for 4 hours resulted in >89% CD16 cleavage on non-transduced cells, <11% cleavage was observed on CD16VP cells, demonstrating shedding resistance. CD16VP cells in the presence of Trastuzumab compared to IgG control, showed enhanced lysis of NCI-N87 and OE19 targets (97 ± 7% and 95 ± 2% vs. 55 ± 19% and 42 ± 14% respectively, p<0.01), secreted higher levels of GM-CSF, IFN-γ, and TNF-α, and demonstrated increased degranulation against OE19. The ADCC mediated cytotoxicity and degranulation were PI3K and JNK pathway-dependent as Wortmannin decreased ADCC by 82.2 ± 17.7% and degranulation by 43.9 ± 1.9%, and SP600125 decreased degranulation by 72.3 ± 1.2%. Our results demonstrate that CD16VP cells have enhanced Trastuzumab-mediated ADCC activity against gastric cancer tumor cell lines with resistance to CD16 cleavage. Development of CD16VP cells provides a combination therapeutic option by harnessing the anti-tumor activities of both targeted biologics and innate cytotoxicity of NK cells. Citation Format: Irene Raitman, Salvatore Rotondo, Tanel Mahlakõiv, Xuan Guo, Andrea DiFiglia, Hemlata Rana, Qian Ye, Srinivas Somanchi, William van der Touw, Lin Kang, Robert Hariri, Xiaokui Zhang. High affinity and cleavage resistant CD16 enhances Trastuzumab mediated killing of gastric cancer targets by human placental CD34+-derived natural killer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2218.

Abstract 74: Co-targeting BCL-xL and HER2 high expression to overcome apoptosis blockade in gastric cancer

Abstract Background: Gastric cancer is one of the most prevalent cancers in the East Asia population. The five-year survival rate is approximately 20% globally. The analysis of gene expression data suggests that anti-apoptotic protein BCL-xL may be the oncogenic driver in gastric cancer as its expression levels are much higher than BCL-2 (Pan Cancer Atlas). In this study, we investigated if a clinical stage BCL-2/BCL-xL dual inhibitor APG-1252 would elicit therapeutic activity and associated mechanisms using a panel of gastric cancer patient-derived xenograft (PDX) models. Additionally, we explored if the combination with HER2 inhibition could enhance the antitumor activity in HER2+ gastric cancer models. Methods and Experiments: Eighteen PDX models of gastric cancer that bore high levels of BCL-xL expression (BCL-xLhigh) based on the gene expression profiles were selected for a mouse trial (n=2) with a dosage regimen of APG-1252 (100 mg/kg, BIW, IV) for three weeks. In a combination study (n=5), APG-1252 (50 mg/kg, BIW, IV) and lapatinib (100 mg/kg, QD, PO) were concurrently administered for three weeks. An advanced ELISA (MSD) and Western blot analysis were performed to detect BCL-xL complex and the protein level, respectively, in tumor samples. Results: APG-1252 treatment resulted in tumor growth inhibition (TGI greater than 50%) in four PDXs (20%), including three exhibiting TGI greater than 80%. PD study confirmed that BCL-xL protein levels were high, and bound with pro-apoptotic proteins PUMA (major binding partner) and BIM in the xenograft tumors of these models. Generally, in comparison with hematologic cancer cells which exhibit dominant BCL-2 complexes (i.e., Toledo), the levels of BCL-xL complexes were consistently higher than those of BCL-2 complexes in the gastric cancer PDXs, implicating a crucial role of BCL-xL in the solid tumors. After treatment with APG-1252, BCL-xL:PUMA, were disrupted and, interestingly, such a decrease in BCL-xL complex signal correlated with TGI. Additionally, the baseline levels of BCL-xL complexes also correlated with TGI. Furthermore, a consequent increase in MCL-1 complexes after APG-1252 treatment indicated that such a compensatory mechanism among anti-apoptotic proteins may confer the drug resistance. Finally, in mouse tumor models derived from HER2+ gastric cancer cell line NCI-N87 or patient-derived xenografts (PDXs), combined treatment with AGP-1252 and HER2 inhibitor lapatinib synergistically inhibited tumor growth, suggesting that lapatinib may be able to increase mitochondrial priming, thus sensitizing cancer cells to APG-1252. Conclusion: Our results demonstrate the on-target antitumor activity of APG-1252, the potential of BCL-xLhigh as a predictive biomarker, and the resistance mechanism conferred by MCL-1. Furthermore, the data provide a scientific rationale for the combined therapy with BCL-xL and HER2 inhibitors to achieve better clinical outcomes in a subset of HER2+ gastric cancers. Citation Format: Jing Deng, Xiaojing Huang, Guoqin Zhai, Kaixiang Zhang, Jiaxing Gu, Tingting Mao, Yan Yin, Ran Tao, Douglas D. Fang, Dajun Yang, Yifan Zhai. Co-targeting BCL-xL and HER2 high expression to overcome apoptosis blockade in gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 74.

Abstract 4170: Decursin inhibits gastric cancer cell growth and autophagic flux by suppressing cathepsin C

Abstract Purpose: Autophagy is a series of processes in which biomolecules are digested and recycled, and it also provides energy for the development and progression of cancer. Thus, autophagy inhibitors can be used to suppress the progression of cancer. In this study, we investigated whether decursin, which is derived from natural products, inhibits the growth and autophagy of cancer cells. Methods: The gastric cancer cell lines SNU216 and NCI-N87 were treated with or without decursin. Cell growth was evaluated by Cell-Counting Kit-8, colony formation, and BrdU assays, as well as by propidium iodide staining, and autophagic flux was evaluated by western blotting, LC3 puncta, GFP-RFP-LC3, and transmission electron microscopy. Finally, tumor spheroids and patient-derived gastric cancer organoids were used to confirm the effect of decursin in three-dimensional culture. Results: Decursin reduced the growth and anchorage-dependent viability of gastric cancer cells. LC3 cleavage and LC3-GFP puncta were increased by decursin in a time- and concentration-dependent manner. However, the increase in autophagosome formation did not lead to an increase in autophagic flux. Decursin downregulated cathepsin C (CTSC), a lysosomal protease, irrespective of cellular pH. In addition, knockdown of CTSC reduced the mRNA and protein levels of E2F3, resulting in cell-cycle arrest. Decursin also reduced cell growth in both tumor spheroids and patient-derived gastric cancer organoids. Conclusion: The results demonstrated that decursin inhibited tumor growth and progression by suppressing CTSC-mediated autophagic flux and cell-cycle progression. Therefore, decursin is a novel inhibitor of autophagic flux, with potential for the treatment of gastric cancer. Citation Format: Solbi Kim, Mina Joo, Nayoung KIM, Myung-Won Lee, Heung Jin Jeon, Hyewon Ryu, Ik-Chan Song, Hyo Jin Lee. Decursin inhibits gastric cancer cell growth and autophagic flux by suppressing cathepsin C [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4170.

Antiproliferative Acylated Glycerols from New Zealand Propolis.

Previous work has shown that a number of phenolic components of NZ propolis possess antiproliferative activity against certain human gastrointestinal cancer cell lines. Here we report on a series of acylglycerols isolated from the nonpolar fraction of propolis resin, which represent further bioactive constituents unrelated to the more usual phenolic compounds generally found in propolis. NZ propolis is sourced from poplar trees, and the acylglycerols have been shown to be present in the leaves and buds of some common poplars. The compounds are a series of monoglycerides containing 3,8-dihydroxy fatty acids, many of which are further acylated with acetic acid residues. The dihydroxy fatty acids are C18 to C24, with the most abundant being C20 and C22. These acylglycerols were found to have strong antiproliferative activity against three human gastrointestinal cell lines, particularly gastric cancer cell line NCI-N87, where one example shows an IC50 of less than 50 μM.

Effects of neddylation modification inhibitor MLN4924 on proliferation and migration of human gastric cancer cells

Objective To explore the effects of NEDD8 activating enzyme (NAE) inhibitor MLN4924 on gastric cancer cell proliferation and migration. Methods Human gastric cancer cell lines NCI-N87 and SGC-7901 were treated with different concentrations of MLN4924, and the inhibitory concentrations were detected by cell counting kit-8 (CCK-8) method. 20% maximal inhibitory concentration (IC20) was used to treat gastric cancer cells. The cell proliferation was measured by CCK-8 and clone formation assay, and cell migration was determined by cell scratch test. Results When human gastric cancer cell lines NCI-N87 and SGC-7901 were treated with 0.528 and 0.375 μmol/L respectively, the inhibitory rate reached to 20%. CCK-8 method showed that MLN4924 inhibited the proliferation of human gastric cells in a dose- and time-dependent manner. In the clone formation assay, the clone number of NCI-N87 cells was 71.00±10.19 in MLN4924 treated group and 215.33±9.56 in the control group. The clone number of SGC-7901 cells was 23.66±18.66 in MLN4924-treated group, and 32.33±34.12 in the control group.In vitro wound scratch assay revealed that the healing rate of MLN4924-treated NCI-N87 cells was (5.30±0.78)%, and (51.05±6.35)% in the control group, and that of MLN4924-treated SGC-7901 cells was (3.12±0.52)%, and (23.05±2.01)% in the control group. Conclusion Neddylation modification inhibitor MLN4924 suppresses the growth and migration of gastric cancer cells. Key words: Gastric cancer; Neddylation modification inhibitor; Proliferation; Migration

Expression of pleomorphic adenoma gene like-2 in gastric carcinoma and its possible mechanism on invasion and epithelial-mesenchymal transition of gastric carcinoma

Objective To detect the expression of pleomorphic adenoma gene like-2 (PLAGL2) in human gastric cancer and cell lines, and its role in cell invasion and epithelial-mesenchymal transition (EMT) and the molecular mechanism involved. Methods Fifteen cases of pathologically confirmed gastric cancer tissues and their adjacent normal tissues were collected during operation in our hospital from Jan. 10, 2016 to Mar. 20, 2018. Gastric cancer cell lines MKN-28, BGC-823, NCI-N87 and SGC-7901 were cultured. The expression levels of PLAGL2 in gastric cancer tissue samples and MKN-28, BGC-823, NCI-N87 and SGC-7901 cells were determined by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. After successful transfection with PLAGL2-small interfering RNA (siRNA) in SGC-7901 cells, the expression levels of PLAGL2 mRNA and protein were detected by Real-time PCR and Western blotting respectively. Cell invasion was detected by Transwell assay. The expression levels of N-cadherin, Vimentin and E-cadherin, which were EMT related markers, and Wnt/β-catenin related proteins were detected by Western blotting. After PLAGL2-siRNA SGC-7901 cells were treated with Wnt pathway agonist SKL2001 and inhibitor XAV939, the changes of cell invasion ability were retested by Transwell assay. Results The expression levels of PLAGL2 mRNA (1.11±0.27) and protein (0.98±0.26) in gastric cancer tissues were significantly higher than those in the adjacent tissues (mRNA: 0.37±0.10; protein: 0.28±0.11; t=-20.941, P<0.01; t=-20.186, P<0.01). The expression levels of PLAGL2 mRNA and protein in MKN-28, BGC-823, NCI-N87 and SGC-7901 were significantly higher than GES-1 cells (mRNA: t=-12.327, P<0.01; t=-19.812, P<0.01; t=-13.080, P<0.01; t=-15.218, P<0.01; protein: t=-11.816, P<0.01; t=-21.162, P<0.01; t=-14.517, P<0.01; t=-15.899, P<0.01). The expression levels of PLAGL2 mRNA and protein in SGC-7901 cells were significantly decreased after PLAGL2-siRNA transfection (mRNA: 2.10±0.29; protein: 1.96±0.27) as compared with control siRNA group (mRNA: 6.61±0.73; protein: 6.46±0.71; t=-1.000, P<0.01; t=-1.000, P<0.01). The number of invasive cells at 24, 48, 72 hin PLAGL2-siRNA transfection group [(30.75±5.74), (48.75±6.90) and (64.50±4.80) cells] was significantly less than in control siRNA group [(51.00±4.97), ( 75.75±4.92) and (132.50±5.57) cells; t=-1.000, P<0.05; t=-1.000, P<0.01; t=-1.000, P<0.01]. The expression of N-cadherin, Vimentin, β-catenin, matrix metalloproteinase (MMP)-7 and c-Myc proteins was significantly down-regulated, and that of E-cadherin protein was significantly up-regulated (t=-1.000, P<0.01; t=-1.000, P<0.01; t=-1.000, P<0.01; t=-1.000, P<0.01; t=-1.000, P<0.01; t=-8.205, P<0.01). SKL2001 could reverse the inhibitory effect on EMT and invasion of SGC-7901 cells after PLAGL2-siRNA transfection, while XAV939 enhanced the above effects of PLAGL2-siRNA. Conclusion PLAGL2 is highly-expressed in gastric cancer. Inhibition of PLAGL2 gene by siRNA can inhibit the invasion of gastric cancer cells probably by down-regulating the Wnt/β-catenin signaling pathway. Key words: Gastric cancer; Invasion; Pleomorphic adenoma gene like-2; Epithelial-mesenchymal transition; Wnt/β-cateninn signaling pathway

Abstract 2491: Nuclear exporter protein XPO1 a novel prognostic and therapeutic target in gastric cancer

Abstract The high mortality rate associated with Gastric Cancer (GC) indicates the urgent need for actionable therapeutic targets. The nuclear exporter protein exportin 1 (XPO1/CRM1) is the exclusive exporter of many tumor suppressor proteins (TPSs) and growth regulators. XPO1 is often over-expressed in different malignancies leading to aberrant cytoplasmic localization of TSPs and subsequent inactivation. A detailed analysis on the correlation of XPO1 with inflammation-metaplasia-dysplasia-carcinoma sequence progression was performed using immunohistochemistry in 70 GC cases: (a) 10 cases from normal gastric mucosa, (b) 10 cases of stomach with intestinal metaplasia with and without inflammation, (c) 10 cases of mucosa with low-grade dysplasia (d) 10 cases of mucosa with high-grade dysplasia, (e) 10 cases of gastric adenocarcinoma and (f) 20 cases of metastatic gastric carcinoma. A correlation between XPO1 expression, the pathological and clinical features of the disease as well as survival were analyzed. Gastric cancer cell lines were exposed to the Selective Inhibitors of Nuclear Export (SINE) compounds (selinexor, KPT-8602, KPT-185, or the natural agent, leptomycin B (LMB), then analyzed using cytotoxicity and molecular assays. In addition, the activity of selinexor was evaluated in a sub-cutaneous xenograft of gastric cancer cell line NCI-N87. XPO1 served as a prognostic marker for poor outcome as positive staining of XPO1 in GC correlated with aggressive behavior of the disease. Targeting XPO1 using SINE compounds or LMB resulted in inhibition of GC cellular growth (IC50&amp;lt;200 nM), induction of apoptosis and suppression of colony formation (p&amp;lt;0.01). Molecular analysis revealed nuclear retention of several important TSPs, induction of pro-apoptotic proteins and suppression of pro-survival factor Bcl-2. Selinexor given orally at doses of 15 mg/kg twice a week for three weeks caused statistically significant reduction of NCI-N87 tumors in mice (p&amp;lt;0.05). Efficacy studies of SINE compounds in patient derived models of GC are ongoing. Our findings strongly demonstrate the potential of XPO1 to serve not only as a prognostic marker but also as a therapeutic marker in GC that warrants further clinical investigations. Citation Format: Irfana Muqbil, Zaid Mahdi, Rahman Choudhary, Erkan Baloglu, William Senapedis, Yosef Landesman, Sharon Shacham, Michael Kauffman, Steve Kim, Rafic Beydoun, Richard N. Berri, Anthony Shields, Ramzi M. Mohammad, Asfar S. Azmi. Nuclear exporter protein XPO1 a novel prognostic and therapeutic target in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2491.

Polyethylene glycol-conjugated HER2-targeted peptides as a nuclear imaging probe for HER2-overexpressed gastric cancer detection in vivo

BackgroundThe human epidermal growth factor receptor 2 (HER2) involved proliferation, angiogenesis, and reduced apoptosis in gastric cancer (GC), which is a common target for tumor therapy. HER2 is usually overexpressed in more than 15% GC patients, developing a reliable diagnostic tool for tumor HER2 detection is important. In this study, we attend to use polyethylene glycol (PEG) linked anti-HER2/neu peptide (AHNP-PEG) as a nuclear imaging agent probe for HER2 detection in GC xenograft animal model.MethodsThe HER2 expression of human sera and tissues were detected in GC patients and normal subjects. GC cell lines NCI-N87 (high HER2 levels) and MKN45 (low HER2 levels) were treated with AHNP-PEG to assess the cell viability and HER2 binding ability. The NCI-N87 was treated with AHNP-PEG to observe the level and phosphorylation of HER2. The MKN45 and NCI-N87-induced xenograft mice were intravenous injection with fluorescence labeled AHNP-PEG for detecting in vivo fluorescence imaging properties and biodistribution. The AHNP-PEG was conjugated with diethylenetriaminopentaacetic acid (DTPA) for indium-111 labeling (111In-DTPA-AHNP-PEG). The stability of was assessed in vitro. The imaging properties and biodistribution of 111In-DTPA-AHNP-PEG were observed in NCI-N87-induced xenograft mice.ResultsThe serum HER2 (sHER2) levels in GC patients were significantly higher than the normal subjects. The sHER2 levels were correlated with the tumor HER2 levels in different stages of GC patients. The AHNP-PEG inhibited the cell growth and down-regulated HER2 phosphorylation in HER2-overexpressed human GC cells (NCI-N87) via specific HER2 interaction of cell surface. In addition, the GC tumor tissues from HER2-postive xenograft mice presented higher HER2 fluorescence imaging as compared to HER2-negative group. The HER2 levels in the tumor tissues were also higher than other organs in NCI-N87-induced xenograft mice. Finally, we further observed that the 111In-DTPA-AHNP-PEG was significantly enhanced in tumor tissues of NCI-N87-induced xenograft mice compared to control.ConclusionsThese findings suggest that the sHER2 measurement may be as a potential tool for detecting HER2 expressions in GC patients. The radioisotope-labeled AHNP-PEG may be useful to apply in GC patients for HER2 nuclear medicine imaging.

Inhibitory effect of NVP-BEZ235 on gastric cancer in nude mice

Objective To investigate the effect of NVP-BEZ235 alone and combined with 5-fluorouracil (5-Fu) on NCI-N87 gastric cancer in nude mice. Methods human gastric cancer cell line NCI-N87 was used to prepare tumor bearing mice. Divided into control group (normal saline every 3 days 0.02 ml/g, every 3 days by intraperitoneal injection of saline 0.02 ml/g), group 5-Fu (normal saline every 3 days every 3 days by intraperitoneal injection of 0.02 ml/g, 5-Fu 0.025 mg/g), group NVP-BEZ235 (every 3 days by gavage once every 3 days NVP-BEZ235 0.04 mg/g, intraperitoneal injection of saline.0.02 ml/g), combination group (gavage once every 3 days NVP-BEZ235 0.04 mg/g, every 3 days by intraperitoneal injection of 5-Fu 0.025 mg/g). Recorded the volume and weight of tumor. Detection of apoptosis related proteins, phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway related proteins and invasion related proteins by Western blotting. Results Compared with 5-Fu group (6.84±0.98), the relative volume of tumor in nude mice with NCI-N87 gastric cancer xenograft model was (8.47±1.44), and the difference was statistically significant (t=2.303, P=0.044)). Tumor weight (g): the combination group (0.83±0.16) was less than the 5-Fu group (1.20±0.32), the difference was statistically significant (t=2.524, P=0.038). Akt, p-Akt, the relative expression of mTOR in group NVP-BEZ235 (0.56±0.05, 0.23±0.04, 0.50±0.03) was significantly lower than the control group (0.96±0.11, 0.68±0.03, 0.64±0.12; t=9.739, 21.823, 3.366, P=0.000, 0.000, 0.006)), combination group (0.53±0.06, 0.20±0.03, 0.51±0.07) was significantly lower than that of group 5-Fu (0.93±0.10, 0.51±0.05, 0.67±0.04; t=8.234, 12.489, 4.628, P=0.000, 0.000, 0.002), there was significant difference between the four groups (F=46.457, 263.318, 6.678, P=0.000, 0.000, 0.002). The activation of apoptosis related protein cysteinyl aspartate-specific protease (Caspase)-3, B cell lymphoma/leukemia-2 associated X protein (bax) and B cell lymphoma/leukemia-2 (bcl-2), NVP-BEZ235 (1.28±0.06, 1.39±0.08, 0.59±0.04) was significantly higher than the control group (0.94±0.10, 0.99±0.11, 1.07±0.07; t=8.660, 8.077, 17.456, P=0.000, 0.000, 0.000) combined. The medicine group (1.57±0.09, 1.70±0.10, 0.37±0.07) was significantly higher than that of group 5-Fu (1.42±0.09, 1.50±0.05, 0.48±0.07; t=2.737, 4.472, 2.861, P=0.021, 0.003, 0.017), compared with a significant difference between the four groups (F=71.181, 81.393, 199.848, P=0.000, 0.000, 0.000). The relative expression of matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF), NVP-BEZ235 (0.44±0.04, 0.30±0.05) was significantly lower than the control group (0.59±0.06, 0.41±0.03; t=5.335, 5.550, P=0.000, 0.001), combination group (0.37±0.04, 0.24±0.07) was significantly lower than that of 5-Fu group (0.58±0.12, 0.36±0.04; t=4.144, 3.691, P=0.006, 0.005), compared with a significant difference between the four groups (F=15.164, 20.205, P=0.000, 0.000). Conclusion NVP-BEZ235 can down regulate the activation level of PI3K/Akt/mTOR pathway, promote apoptosis, inhibit tumor invasion and metastasis, and play a synergistic role with 5-Fu. Key words: Gastric cancer; NVP-BEZ235; Phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin signaling pathway; Apoptosis; Invasion

A desirability-based multi objective approach for the virtual screening discovery of broad-spectrum anti-gastric cancer agents.

Gastric cancer is the third leading cause of cancer-related mortality worldwide and despite advances in prevention, diagnosis and therapy, it is still regarded as a global health concern. The efficacy of the therapies for gastric cancer is limited by a poor response to currently available therapeutic regimens. One of the reasons that may explain these poor clinical outcomes is the highly heterogeneous nature of this disease. In this sense, it is essential to discover new molecular agents capable of targeting various gastric cancer subtypes simultaneously. Here, we present a multi-objective approach for the ligand-based virtual screening discovery of chemical compounds simultaneously active against the gastric cancer cell lines AGS, NCI-N87 and SNU-1. The proposed approach relays in a novel methodology based on the development of ensemble models for the bioactivity prediction against each individual gastric cancer cell line. The methodology includes the aggregation of one ensemble per cell line using a desirability-based algorithm into virtual screening protocols. Our research leads to the proposal of a multi-targeted virtual screening protocol able to achieve high enrichment of known chemicals with anti-gastric cancer activity. Specifically, our results indicate that, using the proposed protocol, it is possible to retrieve almost 20 more times multi-targeted compounds in the first 1% of the ranked list than what is expected from a uniform distribution of the active ones in the virtual screening database. More importantly, the proposed protocol attains an outstanding initial enrichment of known multi-targeted anti-gastric cancer agents.