SNU-1 Cells Research Articles

Identification of alternative lengthening of telomeres-related genes prognosis model in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide, characterized by high mortality. This study aimed to explore the prognostic value and function of alternative lengthening of telomeres (ALT)-related genes in HCC. Differentially expressed genes (DEGs) were identified based on The Cancer Genome Atlas (TCGA) and then intersected with ALT-related genes to obtain ALTDEGs. Risk score model was constructed using the least absolute shrinkage and selection operator (LASSO) algorithm and Cox regression and validated with Gene Expression Omnibus (GEO) datasets. The predictive efficacy of the risk score and ALTs-score was evaluated by Kaplan-Meier curves, time-ROC curves, and the nomogram analyses. The impacts of SMG5 silencing on the HCC cell behaviors were assessed by CCK-8, wound healing, and Transwell assays. A total of 500 ALTDEGs were screened and 13 genes (CDCA8, SMG5, RAD54B, FOXD2, NOL10, RRP12, CCT5, CCT4, HDAC1, DDX1, HRG, HDAC2, and PPP1CB) were identified for constructing a prognostic model. The overall survival (OS) curves, time-ROC curves, and nomograms based on the risk score or ALTs-score were developed to optimally predict the survival of HCC patients. ALTs-score was correlated with immune infiltration and confirmed its value in predicting immunotherapy outcomes. Furthermore, RT-qPCR demonstrated that eight risk signature genes were up-regulated in HCC cells. SMG5 silencing suppressed the proliferation, migration, and invasion of HCC cells. It was also found that SMG5 silencing reduced C-circle level in SNU-387 cells. We identified new ALT-related prognostic biomarkers for HCC. SMG5 knockdown inhibited the HCC progression, which might be a promising target for HCC therapy.

The role of neopterin in cross-talk between tumor and tumor microenvironment in hepatocellular carcinoma

Abstract Neopterin is a marker of activated immune response, but its role in hepatocarcinogenesis is unknown. The present study aims to evaluate the effects of neopterin on prooncogenic/proinflammatory, apoptotic pathways, and other molecular mechanisms in HCC. We used SNU449, Huh-7, SK-Hep-1, and HepG2 cell lines. A cell viability assay was performed with different concentrations of neopterin. RT-PCR, Western blotting, transwell migration, scratch assay, and reactive oxygen species (ROS) production assays were performed at inhibition concentration 50 of neopterin, which was 40 µM for SNU449 and 80 µM for other cell lines. There were significant changes in mTOR, STAT3, PI3K, and interleukin-6 gene expressions, which were also supported by the protein expressions. Neopterin did not affect apoptosis in SNU449, while apoptosis increased by all doses of neopterin in SK-Hep-1 and HepG2. ROS production was increased in all cell lines in response to neopterin. Cell migration was reduced in SK-Hep1 and HepG2 but did not change in SNU449 and Huh-7. Our study showed that neopterin is not just a byproduct. The results suggest that neopterin may be a paracrine factor that modulates pro-inflammatory and pro-oncogenic pathways responsible for the biological behavior of HCC in a chronic inflammatory tumor microenvironment.

CXC chemokine receptor 4 - mediated immune modulation and tumor microenvironment heterogeneity in gastric cancer: Utilizing multi-omics approaches to identify potential therapeutic targets.

G-protein-coupled receptors (GPRs) are critical regulators of various biological behaviors, and their role in gastric cancer (GC) progression is gaining increasing attention. Among them, the immune regulatory mechanisms mediated by chemokine receptor 4 (CXCR4) remain insufficiently understood. This study aims to explore the immune regulatory functions of CXCR4 and the heterogeneity of the tumor microenvironment (TME) by examining GPR-related gene expression in GC. Through multi-omics approaches, including spatial transcriptomics and single-cell RNA sequencing, we investigated the oncogenic mechanisms of CXCR4, particularly its role in T cell immune exhaustion. In vitro experiments, including ELISA, PCR, CCK8 assays, cell scratch assays, and colony formation assays, were used to validate the role of CXCR4 in the migration and invasion of AGS and SNU-1 cell lines. CXCR4 silencing using siRNA further demonstrated its regulatory effects on these cellular processes. Our results revealed a strong correlation between elevated CXCR4 expression and increased exhaustion of regulatory T cells (Tregs) in the TME. Furthermore, heightened CXCR4 expression was linked to increased TME heterogeneity, driven by oxidative stress and activation of the NF-κB pathway, promoting immune evasion and tumor progression. Silencing CXCR4 significantly inhibited the invasive and proliferative abilities of AGS and SNU-1 cells, while also reducing the expression of pro-inflammatory cytokines IL-1β and interleukin-6, thus alleviating chronic inflammation and improving TME conditions. In conclusion, our comprehensive investigation highlights CXCR4 as a key mediator of TME dynamics and immune modulation in GC. Targeting CXCR4 presents a promising therapeutic strategy to slow tumor progression by reducing Tregs-mediated immune exhaustion and TME heterogeneity, positioning it as a novel therapeutic target in GC treatment.

YBX1 promotes epithelial-mesenchymal transition in hepatocellular carcinoma via transcriptional regulation of PLRG1.

Hepatocellular carcinoma (HCC) ranks as the sixth most prevalent cancer worldwide. The epithelial-mesenchymal transition (EMT) is a critical process in cancer progression, contributing to increased malignancy. While Pleiotropic Regulator 1 (PLRG1) is upregulated in HCC and is associated with enhanced cell proliferation, its oncogenic role in EMT remains unclear. In this study, we demonstrate that PLRG1 promotes EMT in HCC cells. Knockdown of PLRG1 in Huh7 cells resulted in decreased expression of the EMT markers N-cadherin and Snail, and impaired cell migration and invasion. Chromatin immunoprecipitation (ChIP) and luciferase assays identified Y-box binding protein 1 (YBX1) as a direct regulator of PLRG1 transcription, binding to its promoter region. Overexpression of YBX1 in SNU-449 cells led to increased PLRG1 expression and subsequent EMT activation, as well as enhanced migration, and invasion. These effects were attenuated by PLRG1 knockdown. Our findings indicate that YBX1 drives EMT in HCC by upregulating PLRG1, offering novel insights into the molecular mechanisms underlying HCC progression.

ANGPTL4 plays a paradoxical role in gastric cancer through the LGALS7 and Hedgehog pathways.

Gastric cancer (GC) is a malignant disease worldwide. Angiopoietin-like protein 4 (ANGPTL4) plays a role in pathophysiological processes, including metabolic reprogramming, angiogenesis, proliferation, and metastasis. Current evidence shows conflicting findings regarding the role of ANGPTL4 in the progression of GC. ANGPTL4 in GC was confirmed through bioinformatic analysis and immunofluorescence staining. The impact of ANGPTL4 was subsequently validated in GC cell lines using various assays, including 5-ethynyl-2-deoxyuridine (EdU), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Flow Cytometry (FCM), wound healing, transwell, tube formation, chorioallantoic membrane model, and nude mouse model assays. RNA-seq analysis, polymerase chain reaction (PCR), western blotting (WB), immunofluorescence (IF) and coimmunoprecipitation (co-IP) were conducted to determine the potential downstream mechanism of ANGPTL4. In SNU5 and MKN7 cells, ANGPTL4 was found to augment proliferation, migration, invasion, evasion of apoptosis, and angiogenesis. Conversely, in the AGS cell line, ANGPTL4 was observed to suppress these processes. Notably, the overexpression of ANGPTL4 in AGS cells led to the upregulation of LGALS7, which has emerged as a pivotal factor contributing to the manifestation of an anticancer phenotype induced by ANGPTL4. LGALS7, which is involved in the regulation of the hedgehog pathway and subsequent promotion of GC progression through various processes, such as proliferation, migration, apoptosis evasion, angiogenesis, and lymphangiogenesis, was found to contribute to the contradictory effects of ANGPTL4.

High KHSRP expression promotes gastric adenocarcinoma metastasis: the mediating role of the JAK1/STAT3 signaling axis

To investigate the regulatory effect of KHSRP on progression of gastric adenocarcinoma and the role of the JAK1/STAT3 signaling axis in mediating its effect. KHSRP mRNA expression level was detected using qRT-PCR in 120 pairs of gastric adenocarcinoma and adjacent tissues, 4 gastric adenocarcinoma cell lines (MKN-28, HGC-27, CRL-5822, and SNU-1) and normal human gastric mucosal GES-1 cells. In HGC-27 cells with KHSRP knockdown and SNU-1 cells with KHSRP overexpression, cell proliferation, migration, invasion and expression levels of JAK/STAT were evaluated using CCK-8 assay, Transwell migration and invasion assays, and Western blotting. In BALB/c-nude mice, HGC-27 cells with KHSRP knockdown and SNU-1 cells overexpressing KHSRP were injected either subcutaneous or via the tail vein to observe subcutaneous xenograft growth and lung metastasis of the tumor cells. Gastric adenocarcinoma tissues and cell lines all showed significantly increased KHSRP expression as compared with the adjacent tissues and GES-1 cells. In HGC-27 cells, KHSRP knockdown significantly inhibited cell proliferation, migration and invasion, while KHSRP overexpression enhanced the malignant behaviors of SNU-1 cells. In nude mice, inoculation of HGC-27 cells with KHSRP knockdown resulted in smaller tumor volume and weight, slower cell proliferation rate and fewer lung metastatic foci, and KHSRP-overexpressing SNU-1 cells produced the opposite results. KHSRP knockdown in HGC-27 cells significantly down-regulated the expression levels of JAK1 and STAT3, which were obviously increased in KHSRP-overexpressing SNU-1 cells. High expressions of KHSRP promote progression and metastasis of gastric adenocarcinoma possibly by regulating the JAK1/STAT3 signaling axis.

Investigation of Folate-Functionalized Magnetic-Gold Nanoparticles Based Targeted Drug Delivery for Liver: In Vitro, In Vivo and Docking Studies.

Magnetic nanoparticles used for targeted drug administration present a promising approach in cancer treatment owing to its notable advantages, such as targeted and enhanced encapsulation ability and improved bio protection compared with conventional drug delivery methods. Au shell-iron core nanoparticles (Fe3O4@Au) were manufactured by a chemical process, coated with dextran to encapsulate curcumin, and functionalized for precision drug delivery using folic acid to combat liver cancer. Dynamic light scattering, scanning electron microscopy, transmission electron microscopy, vibrational spectroscopy, and magnetometry were applied to assess the synthesis of the Fe3O4@Au-DEX-CU-FA compound. The mean size, zeta potential, and polydispersity of Fe3O4@Au-DEX-CU-FA were 63.3 ± 2.33 nm, -68.3 ± 1.78 mV, and 0.041 ± 0.008, respectively. Molecular docking models were created to examine the relationship between Fe3O4@Au-CU and BCL-XL, BAK, and to identify potential binding sites. The loading efficiency and release profile tests examined the medication delivery system's ability. MTT assay was subsequently utilized to determine the optimal dosage and therapeutic efficacy of Fe3O4@Au-DEX-CU-FA on cancer SNU-449 and healthy THLE-2 cell lines. Flow cytometry demonstrated that Fe3O4@Au-DEX-CU-FA effectively induced cancer cell death. Fe3O4@Au-DEX-FA showed a regulated release profile of free curcumin at 37 °C and pH values of 7.4 and 5.4. Real-time PCR revealed increased BAK expression and decreased BCL-XL expression. Nude tumor-bearing mice were used for in vivo experiments. Fe3O4@Au-DEX-CU-FA treatment dramatically reduced the swelling size compared with free CU and control treatments. It also resulted in a longer lifespan, expanded splenocyte proliferation, increased IFN-γ levels, and decreased IL-4 levels. The regular cells showed no cytotoxic effect compared with the cancer type, confirming that Fe3O4@Au-DEX-CU-FA maintained its potent anticancer actions. The data suggests that Fe3O4@Au-DEX-CU-FA possesses a promising potential as a therapeutic agent for combating tumors.

Mito-LND and (E)-Akt inhibitor-IV: novel compounds inducing endoplasmic reticulum stress and ROS accumulation against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality. Although multi-kinase inhibitors can prolong the overall survival of late-stage HCC patients, the emergence of drug resistance diminishes these benefits, ultimately resulting in treatment failure. Therefore, there is an urgent need for novel and effective drugs to impede the progression of liver cancer. This study employed a concentration gradient increment method to establish acquired sorafenib or regorafenib-resistant SNU-449 cells. Cell viability was assessed using the cell counting kit-8 assay. A library of 793 bioactive small molecules related to metabolism screened compounds targeting both parental and drug-resistant cells. The screened compounds will be added to both the HCC parental cells and the drug-resistant cells, followed by a comprehensive assessment. Intracellular adenosine triphosphate (ATP) levels were quantified using kits. Flow cytometry was applied to assess cell apoptosis and reactive oxygen species (ROS). Real-time quantitative PCR studied relative gene expression, and western blot analysis assessed protein expression changes in HCC parental and drug-resistant cells. A xenograft model in vivo evaluated Mito-LND and (E)-Akt inhibitor-IV effects on liver tumors, with hematoxylin and eosin staining for tissue structure and immunohistochemistry staining for endoplasmic reticulum stress protein expression. From the compound library, we screened out two novel compounds, Mito-LND and (E)-Akt inhibitor-IV, which could potently kill both parental cells and drug-resistant cells. Mito-LND could significantly suppress proliferation and induce apoptosis in HCC parental and drug-resistant cells by upregulating glycolytic intermediates and downregulating those of the tricarboxylic acid (TCA) cycle, thereby decreasing ATP production and increasing ROS. (E)-Akt inhibitor-IV achieved comparable results by reducing glycolytic intermediates, increasing TCA cycle intermediates, and decreasing ATP synthesis and ROS levels. Both compounds trigger apoptosis in HCC cells through the interplay of the AMPK/MAPK pathway and the endoplasmic reticulum stress response. In vivo assays also showed that these two compounds could significantly inhibit the growth of HCC cells and induce endoplasmic reticulum stress. Through high throughput screening, we identified that Mito-LND and (E)-Akt inhibitor-IV are two novel compounds against both parental and drug-resistant HCC cells, which could offer new strategies for HCC patients.

Raddeanin A promotes the apoptosis of gastric cancer in conjunction with autophagy inhibitor Hydroxychloroquine via MAPK signaling pathway

PurposeGastric cancer (GC) is among the malignant cancers with the highest incidence and mortality worldwide. As GC is not very sensitive to current chemotherapy drugs, there is an urgent need to develop new effective drugs. Raddeanin A (RA) is extracted from the traditional Chinese medicine Anemone raddeana Regel, which has an anti-cancer effect. The purpose of this study was to explore the effects of RA on GC in vitro and in vivo. MethodsWe explored the targets of RA in GC through network pharmacology. MTT assay, flow cytometry, Western blotting, and other methods were used to detect the effects of RA on the proliferation, apoptosis, and autophagy of GC cells. After preconditioning with hydroxychloroquine (HCQ) and rapamycin, we observed the effects of RA-induced autophagy on apoptosis. We further verified the antitumor effect and safety of RA in vivo. Using SNU-1 xenograft tumor model in nude mice, tumor volume was observed and liver toxicity was observed by immunohistochemistry. ResultsMany cancer-related signaling pathways were visualized using Cytoscape software. Among them, the MAPK signaling pathway was one of the highest-ranked pathways. The MTT assay results suggested that RA could inhibit the proliferation of HGC-27 and SNU-1 cells effectively. Flow cytometry and Western blotting confirmed that RA could significantly induce apoptosis of HGC-27 and SNU-1 cells. Electron microscopy and Western blotting demonstrated that RA could induce autophagy of HGC-27 and SNU-1 cells. Further experiments suggested that HCQ, an autophagy inhibitor, could enhance the capacity of RA to induce apoptosis. In animal studies, we found that intraperitoneal injection of RA could effectively and safely inhibit gastric tumors. ConclusionsRA significantly inhibited the proliferation and induced autophagy and apoptosis of GC cells. In combination with HCQ, RA-induced apoptosis increased in vitro. The combined application of RA and autophagy inhibitors may serve as an added approach to the treatment of GC, but the underlying mechanism needs further exploration. In vivo, it was observed that RA has a good antitumor effect without increasing liver toxicity.

The 3,3'-dimethoxy-4,4'-dihydroxy-stilbene Triazole (STT) Inhibits Liver Cancer Cell Growth by Targeting Akt/mTOR Pathway.

The present study was aimed to investigate the proliferation inhibitory ability of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene triazole (STT) on SNU449 and Huh7 cells. Moreover, the mechanism associated with the suppression of liver cancer cell proliferation by STT was also studied. The results revealed that STT suppresses proliferation of SNU449 and Huh7 cells to 28 and 21%, respectively treatment with 20 µM. The clonogenic survival of SNU449 and Huh7 cells was also significantly reduced after incubation with STT compared to the control cultures. In comparison to the control, STT treatment significantly decreased the invasive potential of SNU449 cells. Treatment with STT led to a prominent suppression in p62 and increase in LC3B protein expression in SNU449 cells compared to the control cells. The STT treatment dramatically decreased p-Akt and p-mTOR protein expression in SNU449 cells. Docking study revealed that STT interacts via traditional hydrogen bonding with the glutamine, phenylalanine, leucine, serine, arginine, aspartic acid, and lysine residues of Akt protein. In summary, the current study demonstrates that STT effectively suppresses the viability of SNU449 and Huh7 liver cancer cells. Moreover, STT treatment of the liver cancer cells also significantly reduces the clonogenic survival and invasive potential of SNU449 cells. Treatment of liver cancer cells with STT increases the expression of autophagic, targets anti-autophagic protein expression and down-regulates Akt/mTOR pathway to inhibit cancer growth and proliferation. Thus, STT exhibits prominent anticancer effect and needs to be investigated further as a potential candidate for the treatment of liver cancer.

Differential effects of choline on TLR2/4 mediated signaling through possible regulation of Toll-interacting protein in hepatocellular carcinoma cell lines

Abstract Objectives Toll-like receptor (TLR) mediated inflammatory status plays an important role in development and progression of hepatocellular carcinoma (HCC). Toll-interacting protein (TOLLIP) has an inhibitory effect on TLR-mediated inflammatory signalling and expression profile of TOLLIP varies between malignancies including HCC. Cholinergic anti-inflammatory pathway (CAP) is an endogenous mechanism that controls inflammatory status via α7nicotinic acetylcholine receptors (α7nAChR). This study aims to investigate the effect of CAP-acting agent choline on TOLLIP and its related TLR-mediated inflammatory response in HCC cells with distinct differentiation stages. Methods The expression patterns of α7nAChR, TLR2/4, TOLLIP, IL6, NFkB genes were evaluated by RT-PCR and ELISA in the presence of choline, along with the real-time cell proliferation and migration in HEP3B and SNU449 HCC cell lines. The interaction between choline and TOLLIP assessed by using in-silico analyses. Results Choline downregulated TOLLIP in Hep3B and SNU449 cells. However, the expressions of α7nAChR, NF-κB, IL-6, TLR2 and TLR4 showed a decreased pattern in well differentiated HEP3B cells, while an increased pattern in poorly differentiated SNU449 cells. Conclusions Choline might exert differential effects in TLR2/4-dependent signalling based on the differentiation stages of the HCC cells, suggesting its potential therapeutic effects in earlier stages of HCC which might be result of its partial modulation of TOLLIP.

Characterization of MET Alterations in 37 Gastroesophageal Cancer Cell Lines for MET-Targeted Therapy.

Capmatinib and savolitinib, selective MET inhibitors, are widely used to treat various MET-positive cancers. In this study, we aimed to determine the effects of these inhibitors on MET-amplified gastric cancer (GC) cells. Methods: After screening 37 GC cell lines, the following cell lines were found to be MET-positive with copy number variation >10: SNU-620, ESO51, MKN-45, SNU-5, and OE33 cell lines. Next, we assessed the cytotoxic response of these cell lines to capmatinib or savolitinib alone using cell counting kit-8 and clonogenic cell survival assays. Western blotting was performed to assess the effects of capmatinib and savolitinib on the MET signaling pathway. Xenograft studies were performed to evaluate the in vivo therapeutic efficacy of savolitinib in MKN-45 cells. Savolitinib and capmatinib exerted anti-proliferative effects on MET-amplified GC cell lines in a dose-dependent manner. Savolitinib inhibited the phosphorylation of MET and downstream signaling pathways, such as the protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) pathways, in MET-amplified GC cells. Additionally, savolitinib significantly decreased the number of colonies formed on the soft agar and exerted dose-dependent anti-tumor effects in an MKN-45 GC cell xenograft model. Furthermore, a combination of trastuzumab and capmatinib exhibited enhanced inhibition of AKT and ERK activation in human epidermal growth factor receptor-2 (HER2)- and MET-positive OE33 cells. Targeting MET with savolitinib and capmatinib efficiently suppressed the growth of MET-amplified GC cells. Moreover, these MET inhibitors exerted synergistic effects with trastuzumab on HER2- and MET-amplified GC cells.

Chalcomoracin promotes apoptosis and endoplasmic reticulum stress in hepatocellular carcinoma cells.

Chalcomoracin (CMR), a Diels-Alder adduct obtained from mulberry leaves, demonstrated wide-spectrum anti-cancer activity. Herein, we aimed to explore the function of CMR and how it works in hepatocellular carcinoma (HCC). Human HCC cell lines Hep3B and SNU-387 were cultured and treated with various concentrations of CMR (1.5, 3, and 6 µM). Subsequently, the effects of CMR on cell viability, colony formation, apoptosis, migration, and invasion abilities were studied in vitro. Furthermore, the levels of endoplasmic reticulum (ER) stress-related proteins and mitogen-activated protein kinase (MAPK) pathway-related proteins in cells under CMR exposure were detected using western blot. Experiments in vivo were conducted to examine the effects of CMR on tumor growth in HCC. CMR administration inhibited the viability and clonogenic, migration, and invasion abilities, as well as promoted cell apoptosis and ER stress in Hep3B and SNU-387 cells. In addition, CMR treatment reduced the phosphorylation levels of ERK, P38, and JNK in the MAPK pathway. Moreover, an in vivo study showed that CMR administration could inhibit tumorigenesis and MAPK pathway activity in HCC. Our data indicate that CMR has the potential to inhibit the development of HCC, potentially through the inhibition of the MAPK pathway. These findings suggest that CMR may have promising applications as an anticancer agent in future therapeutics for HCC.

Synthesis and cytotoxicity of novel 6,8,9-trisubstituted purine analogs against liver cancer cells

A series of novel 6-(substituted phenyl piperazine)-8-(4-substituted phenyl)-9-cyclopentyl purines, 10–51, were synthesized by a four-step synthesis, achieving an overall yield of about 43 %. The reaction conditions were effectively optimized, and the final products were obtained with high purity and yield in all synthesis steps. The synthesized nucleobases were evaluated for their in vitro cytotoxic activities on selected human cancer cell lines (HUH7 (liver), HCT116 (colon), and MCF7 (breast)) using the Sulforhodamine B (SRB) assay. Among these analogs, compounds bearing 4-trifluoromethyl phenyl (19, 20 and 21), 4-methoxy phenyl (27) and 4-fluoro phenyl (34) substitutions at C-8 of purine were the most potent, and they were also analyzed in drug-resistance and drug-sensitive hepatocellular cancer cell (HCC) panels. Compound 19 displayed remarkable anticancer activities (IC50 = 2.9–9.3 μM) against Huh7, FOCUS, SNU475, SNU182, HepG2, and Hep3B cells compared to the positive control, Fludarabine. Additionally, the pharmacological properties and toxicity profiles of the molecules were investigated computationally by the Swiss-ADME and Pro-Tox II online tools, respectively. Results showed that our compounds have favorable physicochemical characteristics for oral bioavailability and do not reveal any toxicity endpoints such as carcinogenicity, immunotoxicity, mutagenicity, or cytotoxicity.

SMARCA4 (BRG1) activates ABCC3 transcription to promote hepatocellular carcinogenesis

AimsHepatocellular carcinoma (HCC) is a lead cause of cancer-related deaths. In the present study we investigated the role of Brahma-related gene 1 (BRG1), a chromatin remodeling protein, in HCC the pathogenesis focusing on identifying novel transcription targets. Methods and materialsHepatocellular carcinogenesis was modeled in mice by diethylnitrosamine (DEN). Cellular transcriptome was evaluated by RNA-seq. ResultsHepatocellular carcinoma was appreciably retarded in BRG1 knockout mice compared to wild type littermates. Transcriptomic analysis identified ATP Binding Cassette Subfamily C Member 3 (ABCC3) as a novel target of BRG1. BRG1 over-expression in BRG1low HCC cells (HEP1) up-regulated whereas BRG1 depletion in BRG1high HCC cells (SNU387) down-regulated ABCC3 expression. Importantly, BRG1 was detected to directly bind to the ABCC3 promoter to activate ABCC3 transcription. BRG1 over-expression in HEP1 cells promoted proliferation and migration, both of which were abrogated by ABCC3 silencing. On the contrary, BRG1 depletion in SNU387 cells decelerated proliferation and migration, both of which were rescued by ABCC3 over-expression. Importantly, high BRG1/ABCC3 expression predicted poor prognosis in HCC patients. Mechanistically, ABCC3 regulated hepatocellular carcinogenesis possibly by influencing lysosomal homeostasis. SignificanceIn conclusion, our data suggest that targeting BRG1 and its downstream target ABCC3 can be considered as a reasonable approach for the intervention of hepatocellular carcinoma.

LncRNA BACE1-AS Accelerates the Progression of Gastric Cancer Through Regulating as a ceRNA of miR-422a to Positively Control BRD4 Expression

Gastric cancer (GC) is a leading global cause of cancer-related mortality, necessitating urgent research on its pathogenesis, prevention, and treatment. In this study, we investigated the expressions of LncRNA BACE1-AS, mRNA BRD4, and miR-422a in GES-1 and GC cells under various treatments using RT-PCR. Western Blots confirmed protein expressions in HGC-27 and SNU-1 cells. EDU and MTT assays assessed cell proliferation, while Transwell tests determined invasion capacity, and flow cytometry analyzed apoptosis. BACE1-AS and BRD4 were significantly elevated in cancerous tissues compared to paired non-cancerous tissues. BACE1-AS knockdown inhibited invasion and proliferation, promoting apoptosis. miR-422a mimics suppressed proliferation and invasion while enhancing apoptosis, and miR-422a mimics with BRD4 overexpression had the opposite effect. Moreover, BAX protein increased in the si-BACE1-AS group but decreased in the si-BACE1-AS+miR-422a inhibitor group. Si-BACE1-AS and miR-422a mimics reduced the expression of C-Myc, CyclinD1, Survivin, CDK4, and Bcl-2, while the si-BACE1-AS+miR-422a inhibitor and miR-422a mimics+BRD4-OV groups showed the opposite trend. Our findings suggest that LncRNA BACE1-AS positively regulates gastric cancer progression by modulating BRD4 as a competitive endogenous RNA for miR-422a. This LncRNA BACE1-AS/BRD4/miR-422a signaling axis presents potential targets for developing therapeutic strategies against gastric cancer.

Abstract 5939: Enhancing nab-paclitaxel chemotherapy response in gastric cancer preclinical models through Inhibition of the HGF/c-Met pathway with merestinib

Abstract Background: Current treatments for primary metastatic or recurrent gastric adenocarcinoma (GAC) primarily involve combination chemotherapy, but the median survival time remains under a year. Nab-paclitaxel has displayed significant antitumor activity in preclinical GAC studies. GAC often exhibits overexpression of various growth factors and their receptors, which play a critical role in its pathophysiology. Aberrant activation of the HGF/c-Met pathway has been reported in up to 50% of GAC patients. We investigated the therapeutic potential of merestinib, a novel inhibitor targeting c-Met, along with Axl and DDR1/2 pathways, in combination with nab-paclitaxel in GAC preclinical models. Methods: Animal survival studies were conducted using peritoneal dissemination xenografts in NOD/SCID mice implanted with MKN-45 cells. Tumor growth was assessed in subcutaneous xenografts using MKN-45 and SNU-1 cells in NOD/SCID mice. IHC analyses were performed to evaluate tumor cell proliferation and microvessel density. In vitro cell proliferation and protein expression were assessed using the WST-1 assay and Western blot analysis, respectively. Results: Animal survival significantly improved with nab-paclitaxel (118%) and merestinib (41%) individually. The combination of merestinib with nab-paclitaxel (153%) resulted in further increased animal survival. In MKN-45 xenografts with high c-Met expression, a substantial reduction in tumor growth was observed with nab-paclitaxel and merestinib, showing a synergistic response. The net change in tumor size in the control, nab-paclitaxel, merestinib, and combination groups was 503 mm3, 115 mm3, 91 mm3, and -9.7 mm3 (tumor regression). In low c-Met expressing SNU-1 xenografts, the effect of merestinib and nab-paclitaxel was less pronounced compared to MKN-45 xenografts. The net change in tumor size in the control, nab-paclitaxel, merestinib, and combination groups was 219 mm3, 105 mm3, 131 mm3, and 57 mm3. Tumor cell proliferation and microvessel density corroborated the tumor growth study results. Nab-paclitaxel and merestinib reduced in vitro cell proliferation, with an additive effect in combination. The reduction in cell proliferation by nab-paclitaxel (10 nM), merestinib (100 nM), and their combination was 87%, 82%, and 94% in MKN-45, 59%, 50%, and 82% in SNU-1, and 53%, 19%, and 66% in gastric fibroblasts. In MKN-45 cells, merestinib increased the expression of pro-apoptotic proteins and decreased the expression of phospho-c-Met, phospho-EGFR, phospho-IGF-1R, phospho-ERK, and phospho-AKT. Conclusion:Merestinib has strong antitumor activity in GAC, especially when administered with nab-paclitaxel, demonstrating a synergistic effect. These results provide compelling evidence for the potential clinical relevance of this combination therapy in improving the survival of GAC patients. Citation Format: Quinn Kaurich, Jennifer Huang, Sazzad Hassan, Urs von Holzen, Niranjan Awasthi. Enhancing nab-paclitaxel chemotherapy response in gastric cancer preclinical models through Inhibition of the HGF/c-Met pathway with merestinib [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 5939.

Synergistic toxicity with copper contributes to NAT2-associated isoniazid toxicity

Anti-tuberculosis (AT) medications, including isoniazid (INH), can cause drug-induced liver injury (DILI), but the underlying mechanism remains unclear. In this study, we aimed to identify genetic factors that may increase the susceptibility of individuals to AT-DILI and to examine genetic interactions that may lead to isoniazid (INH)-induced hepatotoxicity. We performed a targeted sequencing analysis of 380 pharmacogenes in a discovery cohort of 112 patients (35 AT-DILI patients and 77 controls) receiving AT treatment for active tuberculosis. Pharmacogenome-wide association analysis was also conducted using 1048 population controls (Korea1K). NAT2 and ATP7B genotypes were analyzed in a replication cohort of 165 patients (37 AT-DILI patients and 128 controls) to validate the effects of both risk genotypes. NAT2 ultraslow acetylators (UAs) were found to have a greater risk of AT-DILI than other genotypes (odds ratio [OR] 5.6 [95% confidence interval; 2.5–13.2], P = 7.2 × 10−6). The presence of ATP7B gene 832R/R homozygosity (rs1061472) was found to co-occur with NAT2 UA in AT-DILI patients (P = 0.017) and to amplify the risk in NAT2 UA (OR 32.5 [4.5–1423], P = 7.5 × 10−6). In vitro experiments using human liver-derived cell lines (HepG2 and SNU387 cells) revealed toxic synergism between INH and Cu, which were strongly augmented in cells with defective NAT2 and ATP7B activity, leading to increased mitochondrial reactive oxygen species generation, mitochondrial dysfunction, DNA damage, and apoptosis. These findings link the co-occurrence of ATP7B and NAT2 genotypes to the risk of INH-induced hepatotoxicity, providing novel mechanistic insight into individual AT-DILI susceptibility.Yoon et al. showed that individuals who carry NAT2 UAs and ATP7B 832R/R genotypes are at increased risk of developing isoniazid hepatotoxicity, primarily due to the increased synergistic toxicity between isoniazid and copper, which exacerbates mitochondrial dysfunction-related apoptosis.

Morusin suppresses the stemness characteristics of gastric cancer cells induced by hypoxic microenvironment through inhibition of HIF-1α accumulation

Gastric cancer (GC) is a common, life-threatening malignancy that contributes to the global burden of cancer-related mortality, as conventional therapeutic modalities show limited effects on GC. Hence, it is critical to develop novel agents for GC therapy. Morusin, a typical prenylated flavonoid, possesses antitumor effects against various cancers. The present study aimed to demonstrate the inhibitory effect and mechanism of morusin on the stemness characteristics of human GC in vitro under hypoxia and to explore the potential molecular mechanisms. The effects of morusin on cell proliferation and cancer stem cell-like properties of the human GC cell lines SNU-1 and AGS were assessed by MTT assay, colony formation test, qRT-PCR, flow cytometry analysis, and sphere formation test under hypoxia or normoxia condition through in vitro assays. The potential molecular mechanisms underlying the effects of morusin on the stem-cell-like properties of human GC cells in vitro were investigated by qRT-PCR, western blotting assay, and immunofluorescence assay by evaluating the nuclear translocation and expression level of hypoxia-inducible factor-1α (HIF-1α). The results showed that morusin exerted growth inhibitory effects on SNU-1 and AGS cells under hypoxia in vitro. Moreover, the proportions of CD44+/CD24– cells and the sphere formation ability of SNU-1 and AGS reduced in a dose-dependent manner following morusin treatment. The expression levels of stem cell-related genes, namely Nanog, OCT4, SOX2, and HIF-1α, gradually decreased, and the nuclear translocation of the HIF-1α protein was apparently attenuated. HIF-1α overexpression partially reversed the abovementioned effects of morusin. Taken together, morusin could restrain stemness characteristics of GC cells by inhibiting HIF-1α accumulation and nuclear translocation and could serve as a promising compound for GC treatment.

Predictive role of homologous recombination deficiency (HRD) for irinotecan in combination with venadaparib, a novel PARP1/2 inhibitor, as third- or fourth-line treatment in patients with advanced gastric cancer.

380 Background: Tumors with homologous recombination deficiency (HRD), including BRCA and ATM mutations, have shown to predict response to poly (adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibitors. The associations between treatment efficacy of PARP inhibitor-based regimen and mutations in BRCA (BRCAm) or ATM gene (ATMm) are not well known in previously treated advanced gastric cancer (GC). Combining PARP inhibitor and irinotecan is known to synergize cytotoxicity. The aim of this analysis was to evaluate the association of HRD and efficacy of irinotecan and venadaparib combined, in patients with metastatic GC who had failed at least 2 lines of therapy. Methods: MTT assays were performed in vitro to verify and characterize synergism between venadaparib and SN-38. Data were analyzed using the GraphPad Prism (version 9.5.1) and CompuSyn (version 1.0) software. Patients with at least two prior palliative treatments were enrolled in a multi-national phase Ib trial (NCT04725994). Tumor response was evaluated according to RECIST 1.1. Exploratory genomic analysis was included to assess the correlation between efficacy and HRD. Genomic analysis was conducted using ctDNA (GuardantOMNI Gene Panel version 1.0, Redwood City, CA) on Day 1 pre-dose. Results: In MTT assays, the combination of SN-38 and venadaparib exerted synergistic effect with combination indices (CI) ranging from 0.013 to 0.991 in SNU-638 (BRCA2m), SNU-668 (BRCA1m), AGS (low ATM) and SNU-484 (HR proficient) cells. We observe the synergistic effect between SN-38 and venadaparib remained consistent even at low concentrations of venadaparib (SN-38 IC50; without venadaparib → with 10 nM of venadaparib; SNU-638(1.72→0.28), SNU-668(10.7→1.5), AGS (0.62→0.049), SNU-484(2.25->0.38)). In the dose-finding part of the clinical study, 26 patients were enrolled, 13 had received two prior treatments and 13 had received three prior treatments. At data cut-off, median duration of follow-up (range) was 4.4 (1.3 – 23.7) months. Of the 26 patients enrolled, 5 (1 BRCA2m and 4 ATMm) had HRD (19.2%) and were treated with varying doses of irinotecan and venadaparib. In the five patients with HRD, an objective response rate (ORR) was 60% and median progression-free survival (mPFS) (95% CI) was not reached (1.2–21.5). Of the 26 patients enrolled, 11 received varying doses of venadaparib plus 100 mg/m2 of irinotecan, with an ORR of 36.4% and mPFS (95% CI) of 5.6 (3.5–not reached) months. Conclusions: Venadaparib in combination with irinotecan showed synergistic effect in vitro assays and promising clinical efficacy in the systemic treatment of refractory GC, particularly in patients with HRD. The dose expansion phase of the study is ongoing to investigate the optimal biological dose and patient selection strategies, in a randomized design. Clinical trial information: NCT04725994 .