SNU-1 Cells Research Articles

Identification and Characterization of Cell Lines HepG2, Hep3B217 and SNU387 as Models for Porcine Epidemic Diarrhea Coronavirus Infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies.

Decitabine enhances the tumoricidal potential of TRAIL via the epigenetic regulation of death receptor 4 in gastric cancer

Deoxyribonucleic acid (DNA) methyltransferase inhibitors, such as decitabine, have made great advances in cancer therapy as combinational drugs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has an obvious anti-tumor effect; however, some gastric cancer (GC) cells are resistant to TRAIL-induced cell death. This study sought to explore the synergistic anti-tumor effect of TRAIL and decitabine, and the potential synergetic mechanism. The cell growth inhibition effect was monitored by the IncuCyte ZOOM Live-Cell Analysis System, and cell viability was determined by Cell Counting Kit-8 assays. Apoptosis was detected by Annexin V/Propidium Iodide double staining. Death receptor 4 (DR4) was knocked down by ribonucleic acid (RNA) interference, and the effect of DR4 deletion on TRAIL sensitivity was analyzed. Methylation-specific polymerase chain reaction (PCR) was applied to determine the methylation status of DR4. The messenger RNA (mRNA) and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The expression of the DRs on the cell membrane surfaces was analyzed by flow cytometry. The combined use of decitabine and TRAIL synergistically inhibited cell growth in 2 TRAIL-resistant cell lines. Further, decitabine augmented TRAIL-induced apoptosis in a caspase-dependent manner. The co-application of decitabine and TRAIL facilitated the activation of caspase-7, -8, -9, and poly ADP-ribose polymerase (PARP). Notably, decitabine increased the expression of DR4 at the transcriptional and post-transcriptional levels. DR4 expression on the cell membrane surfaces was also upregulated after decitabine exposure. The depletion of DR4 by specific inhibitors attenuated TRAIL-induced apoptosis and weakened the synergistic effects of decitabine and TRAIL. In addition, DR4 gene presented methylation status in SNU-1 cells. The low mRNA and protein expression of DR4 were also detected in SNU-1 cells. Decitabine enhances the effect of TRAIL by inhibiting the growth and inducing the apoptosis of GC cells. This is achieved by the epigenetic modification of decitabine, which upregulates DR4. Decitabine may act as a sensitizing agent of TRAIL. The combined use of decitabine and TRAIL may provide a novel idea for GC treatment.

Dehydrocostus lactone ameliorates lipid accumulation, insulin resistance, and endoplasmic reticulum stress in palmitate-treated hepatocytes

Fatty liver disease is caused by lipid accumulation in the liver, insulin resistance (IR), reactive oxygen species (ROS), and endoplasmic reticulum (ER) stress. Dehydrocostus lactone (DHE) has anticancer, anti-inflammatory, and anti-ulcer effects. However, its effects on hepatic steatosis and IR remain unclear. In this study, we investigated whether DHE has antisteatotic effect on fatty liver in vitro. Hepatocytes HepG2 and SNU-449 cells were exposed to 0.25 mM palmitate (PA), and then antisteatotic effect was evaluated by treatment with 10 μM DHE. DHE treatment reduced lipid accumulation and lipogenesis factor protein levels, compared with PA-treated hepatocytes. DHE treatment also decreased gluconeogenesis marker expression and recovered IR in PA-treated hepatocytes, and promoted glucose uptake in PA-treated HepG2 cells. Additionally, the levels of ROS and ER stress factors in PA-treated HepG2 cells were reduced by DHE treatment, compared with PA-treated HepG2 cells. Overall, DHE decreased lipid accumulation and lipogenesis factors as well as recovered IR, gluconeogenesis, and glucose uptake by reducing ER stress and ROS levels in PA-treated hepatocytes. Thus, DHE is a potential antisteatotic agent.

Increased Expression of NXPH4 Correlates with Immune Cell Infiltration and Unfavorable Prognosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading malignant carcinomas. Despite the advancement in the treatment for HCC, such as precise hepatectomy, radiotherapy, transarterial therapies, chemotherapy, targeted treatments, and immunotherapy, the 5-year overall survival rate of HCC is extremely low. Hence, novel biomarkers are urgently needed for advancing the therapy and prognosis of HCC. Neurexophilin 4 (NXPH4) is a neuropeptide-like glycoprotein. The study is designed to investigate the function of NXPH4 in HCC through a comprehensive bioinformatics analysis. NXPH4 expression status and prognostic values were analyzed via multiple datasets, such as TCGA, GEO, and ICGC. The association between NXPH4 and immune cell infiltration was estimated by TIMER, TISIDB, and CIBERSORT. In vitro, we explored the biological function of NXPH4 in JHH7 and SNU182 cells through knocking down the expression of NXPH4 via siRNA. In general, NXPH4 was predominantly upregulated in HCC tumors, and increased NXPH4 expression predicted unfavorable prognosis. The gene enrichment analysis displayed that NXPH4 was related with metabolic pathways. NXPH4 expression was correlated with immune cell infiltration. NXPH4 knockdown significantly suppressed proliferation, migration, and invasion of JHH7 and SNU182 cells. This study suggested that the upregulation of NXPH4 is associated with adverse prognosis and immune cell infiltration in HCC. NXPH4 could be a novel biomarker of unfavorable prognosis and an underlying target for immunotherapy in HCC.

Overexpression of hsa_circ_0006470 inhibits the malignant behavior of gastric cancer cells via regulation of miR-1234/TP53I11 axis.

Gastric cancer (GC) is a subtype of a common malignant tumor found in the digestive system. Hsa_circ_0006470 is known to be closely associated with the development of GC. Nevertheless, the mechanism by which hsa_circ_0006470 regulates the tumorigenesis of GC has not been fully elucidated. To investigate the role of hsa_circ_0006470 in GC, its expression levels were assessed in GES-1, AGS, MKN45, and SNU5 cells by reverse transcription-quantitative PCR. Fluorescence in situ hybridization was used to evaluate the localization of hsa_circ_0006470 in AGS and MKN45 cells. In addition, cell counting kit-8 and 5-ethynyl- 2’-deoxyuridine assays were performed to evaluate the viability and proliferation of GC cells, respectively. The dual-luciferase reporter assay was used to explore the interaction among hsa_circ_0006470, microRNA (miR)- 1234, and TP53I11. The expression levels of TP53I11, Akt, p-Akt, forkhead box O1, and cyclin dependent kinase 2 in AGS cells were analyzed by Western blotting. The data indicated that hsa_circ_0006470 expression was downregulated in AGS cells. In addition, overexpression (OE) of hsa_circ_0006470 could inhibit the viability and proliferation of GC cells. Moreover, OE of hsa_circ_0006470 inhibited the migration of GC cells and induced G1 cell cycle phase arrest. Moreover, miR-1234 was bound to hsa_circ_0006470 and TP53I11 was targeted by miR-1234. Furthermore, OE of hsa_circ_0006470 inhibited the tumorigenesis of GC via the regulation of the miR-1234/TP53I11 axis. In summary, the present study demonstrated that OE of hsa_circ_0006470 notably inhibited the tumorigenesis of GC by regulating the miR-1234/TP53I11 axis. Therefore, the present study may provide a theoretical basis for exploring novel therapeutic strategies for the treatment of GC.

FAM83D promotes the proliferation and migration of hepatocellular carcinoma cells by inhibiting the FBXW7/MCL1 pathway.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors and has a poor 5-year survival rate. Family with sequence similarity 83, member D (FAM83D) is characterized as an oncogenic gene related to cell proliferation in many tumors, but the role and underlying mechanism of FAM83D in the development of HCC are still unclear. FAM83D expression profiles and clinicopathological data were obtained from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC). Additionally, 2 data sets from the Gene Expression Omnibus (GEO) database were used to further validate the FAM83D profile in HCC. We then downregulated the expression of FAM83D in HCC cells transfected with FAM83D small-interfering ribonucleic acid (siRNA) and upregulating its expression by FAM83D-overexpression transfection for further in vitro studies. TCGA and the GEO databases showed that FAM83D was significantly more upregulated in tumor tissues than non-tumor tissues. The high expression of FAM83D in HCC is associated with poor prognostic clinical factors. The knockdown of FAM83D in SNU449 and HUH7 cells in vitro impaired cell proliferation and migration, and promoted apoptosis, while the overexpression of FAM83D in BEL7402 cells had the opposite effect. Further, combined transfection with FBXW7 siRNA or MCL1-overexpression reversed the role of FAM83D knockdown on cell proliferation, migration, and apoptosis in vitro, while FBXW7 expression was negatively correlated with both the FAM83D and MCL1 levels in TCGA-LIHC patients. FAM83D played a significant role in HCC progression by enhancing cell proliferation and migration and inhibiting apoptosis, which may have been caused by the inhibition of the FBXW7/MCL1 signaling pathway. Thus, FAM83D may be a promising therapeutic target for HCC.

MiR-711 regulates gastric cancer progression by targeting CD44.

MicroRNAs (miRNAs) have been reported to play an important role in tumor progression by regulating the expression of target genes. This study attempted to verify the role of miR-711 in gastric cancer (GC) progression by in vitro and in vivo assays. The expression of miR-711 in tumor tissues and cells was detected by real-time quantitative PCR (qRT-PCR). Expression of MiR-711 in NCI-N87 and SNU-1 cells was detected by FISH. We transfected GC cells with miR-711 mimics or inhibitors. The effects of miR-711 on the proliferation and metastasis of GC cells were detected by CCK-8, wound healing and transwell assays. Dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-711 and CD44. Xenograft assays was used to verify the regulatory effect of miR-711 on tumor growth. In GC tissues and cell lines, the expression of miR-711 was down-regulated when compare with adjacent tissues or normal epithelial cells. The results indicated that overexpressing of miR-711 could suppress the GC cell proliferation, migration, and invasion through targeting CD44. The knockdown of CD44 showed similar effects as miR-711 overexpression in GC cells. Moreover, we confirmed these effects in the in vivo assays. Furthermore, we found that miR-711 could play a role by influencing tumor cell stemness. MiR-711 plays vital roles as a tumor-suppressor by targeting CD44 and may be a therapeutic target for GC treatment.

Circular RNA hsa_circ_0062682 Binds to YBX1 and Promotes Oncogenesis in Hepatocellular Carcinoma.

Simple SummaryCircular RNA (circRNA) have a role in carcinogenesis in different cancers, also in hepatocellular carcinoma (HCC). The transcriptome analyses of HCC tumours identified an upregulated circRNA hsa_circ_0062682. We show that this circRNA affects several aspects of oncogenesis, which are cell proliferation, migration, and invasion. Using transcriptome analyses we identified modulated signalling pathways and transcription factors, confirming the observed phenotype in cells. We identified Y-box-binding protein 1 (YBX1), a known oncogene and RNA-binding protein, as a binding partner, which was in line with transcriptome analyses. We also identified a cell-specific response to sorafenib after circRNA modulation, which is in line with a heterogeneous molecular pathology of HCC subtypes.Circular RNAs (circRNAs) have been shown to play an important role in the pathogenesis of hepatocellular carcinoma (HCC). By implementing available transcriptomic analyses of HCC patients, we identified an upregulated circRNA hsa_circ_0062682. Stable perturbations of hsa_circ_0062682 in Huh-7 and SNU-449 cell lines influenced colony formation, migration, cell proliferation, sorafenib sensitivity, and additionally induced morphological changes in cell lines, indicating an important role of hsa_circ_0062682 in oncogenesis. Pathway enrichment analysis and gene set enrichment analysis of the transcriptome data from hsa_circ_0062682 knockdown explained the observed phenotypes and exposed transcription factors E2F1, Sp1, HIF-1α, and NFκB1 as potential downstream targets. Biotinylated oligonucleotide pulldown combined with proteomic analyses identified protein interaction partners of which YBX1, a known oncogene, was confirmed by RNA immunoprecipitation. Furthermore, we discovered a complex cell-type-specific phenotype in response to the oncogenic potential of hsa_circ_0062682. This finding is in line with different classes of HCC tumours, and more studies are needed to shed a light on the molecular complexity of liver cancer.

Abstract PO005: Obesity-associated Visfatin, and Silibinin in an In Vitro and In Vivo Model of Liver Cancer

Abstract Introduction: Obesity, specifically body fatness, is a risk factor for the development of liver cancer. The link between obesity and liver cancer is likely to be mediated in part by a state of chronic inflammation. However, it has yet to be determined if visfatin inhibition, a pro-inflammatory adipocytokine upregulated by obesity, is sufficient to prevent or delay the progression of liver cancer. Obesity-induced inflammatory factors may promote cancer progression by increasing cell proliferation, motility, invasion, and metastasis.The use of dietary compounds may break the pro-inflammatory cycle induced by the obese state, thus attenuating the cancer process. Silibinin, a polyphenol in milk thistle seed, has been shown to have anti-inflammatory properties. Studies have yet to determine whether silibinin can be used to break the obesity-cancer link to delay progression of liver cancer. The objective of this cell culture and mouse study is to determine the efficacy of silibinin in the prevention of obesity-associated liver cancer progression. Methods: SNU-449 and HepG2 liver cancer cells were exposed to the following experimental conditions; control, FBS, visfatin (80 ng/ml), visfatin + silibinin 40 μM, silibinin (40 μM). Cell proliferation, lipogenesis, lactate dehydrogenase secretion, and ROS production were measured. Immunoblot was used to measure phospho-Akt, phospho-Erk, NF-kB, Bcl-xL, and actin. Eight-week-old C57BL/6J male mice were randomized to a high-fat DIO (n=6) or control diet (n=6). At 14 weeks, mice will be injected subcutaneously with syngeneic Hepa1-6 murine liver cancer cells. At 15-weeks, mice will receive peritoneal injections of silibinin 3 times per week for 2 weeks. Tumors will be measured and assessed for visfatin, Akt, Erk, and NF-kB protein expression. Results from the animal study are on-going and will be presented at time of the conference. Results: SNU-449 and HepG2 cells exposed to visfatin significantly increased cell proliferation and lipogenesis when compared to control. Further, the addition of silibinin inhibited the visfatin-induced proliferation and lipogenesis in both cell lines. Visfatin increased the phosphorylation of Akt and Erk, and NF-kB expression in both liver cancer cell lines. The addition of silibinin differently suppressed phosphorylation of Akt and Erk in SNU-449 and HepG2 cells. Conclusion: Silibinin differentially inhibits visfatin-induced cell growth, lipogenesis, and promotes cytotoxicity which may lead to increased cancer cell death. Citation Format: Elisa Pedone, Kelly N Green, Kelly R Thornton, Ramona S. Price. Obesity-associated Visfatin, and Silibinin in an In Vitro and In Vivo Model of Liver Cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO005.

Abstract PO006: The Role of Obesity-associated Allograft Inflammatory Factor-1 in Liver Cancer Cell Growth

Abstract Introduction: Obesity is a risk factor in the development of liver cancer. The obesogenic tumor microenvironment is of particular importance in liver tumorigenesis due in part to lipid accumulation and monocyte recruitment which differentiate into pro-inflammatory macrophages. A novel macrophage-associated protein, whose role in obesity-associated liver tumorigenesis has yet to be explored, is allograft inflammatory-factor 1 (AIF-1). AIF-1 is a calcium binding inflammation response scaffold protein. Although, proliferation and migration have been studied, https://scholar.google.com/citations?view_op=view_citation&hl=en&user=AAeKZnoAAAAJ&sortby=pubdate&citation_for_view=AAeKZnoAAAAJ:8dzOF9BpDQo Cactivation of kinase pathways, cell survival, cell death, ROS production and lipogenesis have yet to be explored. AIF-1 is upregulated in both liver cancer and obese individuals. Our research objective is to determine the functional role of AIF-1 in promoting physiological processes important for liver tumorigenesis including proliferation, survival, ROS, and lipogenesis, and 2) determine the contribution of Akt and Erk in promoting this in-vitro phenotype. Methods: HepG2 and SNU-449 liver cancer cells were exposed to AIF-1 (10uM) with or without 10 μM ly294002 (PI3K inhibitor), 10 μM PD98059 (MAPK inhibitor), or 5μM NPS-2143 (calcium sensing receptor inhibitor). Cells were assessed for proliferation, ROS production, lipogenesis, cytotoxicity, and cell survival. Immunoblot analysis was used to measure phosphorylated Akt, Erk, IL-6, and NF-kB protein levels. Results: AIF-1 increased proliferation in HepG2 and SNU-449 liver cancer cells. Inhibiting Akt, and Erk signaling decreased AIF-1-induced proliferation while NPS-2143 CaSR inhibitor decreased proliferation only in HepG2 cells. AIF-1 promoted ROS production and the addition of Akt inhibitor blocked AIF-1 ROS production in both cell lines. Significant differences in lipogenesis were not observed among various treatments in HepG2 and SNU-449 cells. AIF-1 increased cell survival, cytotoxicity and phosphorylation of Akt and Erk, when compared to control. Conclusions: Obesity-associated AIF-1 increased proliferation, ROS, and survival while differential effects were observed with Akt or Erk inhibition. Citation Format: Jessie Waltenbaugh, Curissa Groll, Kelsie Raign, Ramona Price. The Role of Obesity-associated Allograft Inflammatory Factor-1 in Liver Cancer Cell Growth [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO006.

Abstract PO018: The development of chemoresistant liver cancer cells to explore visfatin and the IGF-1/IR/Akt axis

Abstract Background: Obesity is a risk factor for liver cancer a lethal cancer that has increased in incidence and mortality. Understanding how obesity increases the risk for liver cancer progression and chemoresistance is important because of the high prevalence of obesity. In late-stage liver cancer, sorafenib, a multi-kinase inhibitor, is an effective first-line of chemotherapy until resistance emerges. Considering the potential for obesity-related promotion of hepatocellular carcinoma (HCC), there is also a potential link between obesity and HCC Sorafenib resistance. In addition to higher levels of insulin and IGF-1, obese individuals also have higher circulating amounts of adipocytokines such as visfatin, which may have a role in resistance to treatment. The purpose of this study is to develop an in vitro model of chemoresistance to identify strategies to break the obesity-cancer link.Methods: HepG2, Huh7, and SNU-449 liver cancer cells were used to develop sorafenib resistant cells. Cells continuously exposed to sorafenib are referred to as HepG2R, Huh7R, and SNU-449R. Cells were exposed to Sorafenib concentration beginning at 5 µM to 10 µM gradually, over approximately four months. To characterize resistance, cell viability, survival, and protein signaling were analyzed every four weeks. MTT, colony formation, and immunoblot assays were used to determine viability survival and protein signaling. Resistant cells were exposed to the following conditions; control, 80 ng/mL of visfatin, visfatin + 100 µM of HNMPA, visfatin + 500 nM of PPP, visfatin + 10 µM of LY294002, visfatin + 100 µM of HNMPA + 500 nM of PPP.Results: For the first two time points HepG2, SNU-449 and Huh7 cell lines did not demonstrate resistance. Huh7R and HepG2R cells did not differ in proliferation compared to cells not exposed to sorafenib. SNU-449R cells when treated with Sorafenib were more proliferative and increased survival compared to SNU-449 non-resistant cells. HepG2R cells had increased survival and BCL-xL protein expression compared to non-resistant cells. At time point 4, SNU-449R and HepG2R cells demonstrated resistance to sorafenib with increased anti-apoptotic signaling, survival, and cell viability. HUH7 non-resistant and HUH7R cells had no significant differences when treated with Sorafenib or the control. Visfatin increased proliferation compared to cells exposed to visfatin plus inhibitors. Visfatin plus iNAMPT or IR inhibitor decreased proliferation.Conclusion: HepG2 and SNU-449 liver cancer cells that were continuously exposed to Sorafenib resulted in increased proliferation, survival, and anti-apoptotic signaling. Visfatin promoted proliferation and inhibited IR, and iNAMPT inhibition suppressed these effects. Using these cell lines, future studies can identify strategies to sensitize chemoresistance cells to chemotherapy in the context of obesity by inhibiting visfatin ligand or enzyme activity. Citation Format: Curissa Groll, Ramona Salcedo Price. The development of chemoresistant liver cancer cells to explore visfatin and the IGF-1/IR/Akt axis [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO018.

HER3 PET Imaging Predicts Response to Pan Receptor Tyrosine Kinase Inhibition Therapy in Gastric Cancer.

New generation of receptor tyrosine kinase inhibitors (RTKIs) have shown to improve survival in many solid tumors. However, an imaging biomarker is needed for patient selection and prediction of treatment response. This study evaluates the use of quantitative changes of HER3 on 68Ga-NOTA-HER3P1 PET/MRI for prediction of early response to pan-RTKIs in gastric cancer (GCa). GCa cell lines were evaluated for expression of RTKs, and downstream signaling pathways (AKT and MAPK). Cell viability was assessed following 24-72h of treatment with 0.01-1µmol/L of afatinib, a pan-RTKI. HER3-expressing afatinib-sensitive (NCI-N87) and resistant cells (SNU16) were selected for evaluation of changes in RTKs expression and downstream pathways, with 24-72h of 0.1µmol/L afatinib treatment. 68Ga-NOTA-HER3P1 PET/MRI was performed in subcutaneous NCI-N87 and SNU16 xenografts (nu:nu, n = 12/group) at baseline and 4days after afatinib treatment (10mg/kg, PO, daily). Temporal changes in PET measures were correlated to HER3 expression in tumors, tumor growth rate, and treatment response. With afatinib therapy, NCI-N87 cells showed increased total HER3 expression, and reduction of other RTKs and downstream nodes within 72h, while SNU16 cells showed no significant change in total HER3 and downstream nodes. 68Ga-HER3P1 PET/MRI showed increased uptake in NCI-N87 and no significant change in SNU16 tumors (day 4 vs. baseline SUVmean: 3.8 ± 0.7 vs. 1.6 ± 0.6, p < 0.05 in NCI-N87, and 1.5 ± 0.7 vs. 1.7 ± 0.7, p > 0.05 in SNU16). These findings were in concordance with HER3 expression in histopathological analyses and tumor growth over 3weeks of treatment (mean tumor volume in treated vs. control: 11 ± 17 mm3 vs. 293 ± 79 mm3, p < 0.001 in NCI-N87, and 238 ± 91 mm3 vs. 282 ± 35 mm3, p > 0.05 in SNU16). Quantitative changes in HER3 PET could be used to predict response to pan-RTKI within few days after initiation of treatment and can help with personalizing GCa management.

Biochemical characterization and detection of antitumor activity of l-asparaginase from thermophilic Geobacillus kaustophilus DSM 7263T

L-asparaginases, which are oncolytic enzymes, have been used in clinical applications for many years. These enzymes are also important in food processing industry due to their potential in acrylamide-mitigation. In this study, the gene for l-asparaginase (GkASN) from a thermophilic bacterium, Geobacillus kaustophilus, was cloned and expressed in E. coli Rosetta™2 (DE3) cells utilizing the pET-22b(+) vector. The 6xHis-tag attached enzyme was purified and analyzed both biochemically and structurally. The molecular mass of GkASN was determined as ∼36 kDa by SDS-PAGE, Western Blotting, and MALDI-TOF MS analyses. Optimum temperature and pH for the enzyme was determined as 55 °C and 8.5, respectively. The enzyme retained 89% of its thermal stability at 37 °C and 75% at 55 °C after 6 h of incubation. The enzyme activity was inhibited in the presence of Cu2+, Fe3+, Zn2+, and EDTA, while the activity was enhanced in the presence of Mn2+, Mg2+, and thiol group protective agents such as 2-mercaptoethanol and DTT. The structural modeling analysis demonstrated that the catalytic residues of the enzyme were partially similar to other asparaginases. The therapeutic potential of GkASN was tested on hepatocellular carcinoma cells, a solid cancer type with high mortality rate and rapidly increasing incidence in recent years. We showed that the GkASN-induced asparagine deficiency effectively reduced the metastatic synergy in HCC SNU387 cells on a xCELLigence system with differentiated epithelial Hep3B and poorly differentiated metastatic mesenchymal HCC SNU387 cells.

Design, synthesis, and biological evaluation of dinuclear bismuth(III) complexes with Isoniazid-derived Schiff bases

Four dinuclear bismuth(III) Schiff-base complexes bearing Schiff-base ligands have been synthesized and structurally characterized by single-crystal X-ray diffraction, elemental analysis, and spectral techniques (FT-IR, NMR and MS). The analytical data reveal the bismuth(III) complexes possess 1:1 metal–ligand ratios. In vitro biological studies have revealed that bismuth(III) complexes displayed much higher antibacterial and antitumor activities than their parent ligands, which involves two gram-negative (S. aureus, B. subtili) and two gram-positive (E. coli, P. aeruginosa) bacteria, and human gastric cancer SNU-16 cells. The power–time curves of S. pombe exposed to tested compounds were detected by bio-microcalorimetry. Some thermokinetic parameters (k, Pmax,tG and Qtotal) were derived based on the metabolic power–time curves, and their quantitative relationships with the concentrations (c) were further discussed.

Policosanol suppresses tumor progression in a gastric cancer xenograft model.

Gastric cancer (GC) is the most common cancer worldwide and the third leading cause of cancer death, with the fifth highest incidence. The development of effective chemotherapeutic agents is needed to decrease GC mortality. Policosanol (PC) extracted from Cuban sugar cane wax is a healthy functional food ingredient that helps improve blood cholesterol levels and blood pressure. Its various physiological activities, such as antioxidant, anti-inflammatory, and anticancer activities, have been reported recently. Nevertheless, the therapeutic efficacy of PC in gastric xenograft models is unclear. We aimed to investigate the anticancer effect of PC on human GC SNU-16 cells and a xenograft mouse model. PC significantly inhibited GC cell viability and delayed tumor growth without toxicity in the SNU-16-derived xenograft model. Therefore, we investigated protein expression levels in tumor tissues; the expression levels of Ki-67, a proliferation marker, and cdc2 were decreased. In addition, we performed proteomic analysis and found thirteen differentially expressed proteins. Our results suggested that PC inhibited GC progression via cdc2 suppression and extracellular matrix protein regulation. Notably, our findings might contribute to the development of novel and effective therapeutic strategies for GC.

Circ_0061395 functions as an oncogenic gene in hepatocellular carcinoma by acting as a miR-1182 sponge

ABSTRACT Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in liver cancer, with a high rate of metastasis and recurrence. Circular RNA_0061395 (circ_0061395) has been shown to be involved in the advance of HCC. However, the interaction between circ_0061395 and microRNA (miRNA) in HCC has not been studied. Quantitative real-time polymerase-chain reaction (qRT-PCR) was used to detect the expression of related genes in liver cancer tissues and cells. The stability of circ_0061395 was verified by RNase R digestion. Through detection of cell malignant behavior and apoptosis, the capping experiment was carried out to verify the regulatory relationship between miR-1182 and circ_0061395 or SPARC/osteonectin, CWCV and Kazal-like domains proteoglycan 1 (SPOCK1). The expression of related proteins was detected by western blot. The interaction of miR-1182 with circ_0061395 or SPOCK1 has been notarized by Dual-luciferase reporter analysis and RNA immunoprecipitation (RIP) assay. Xenotransplantation experiments using BALB/C nude mice were used to confirm the function of circ_0061395 in vivo. Circ_0061395 and SPOCK1 were significantly expressed in liver cancer tissues and cells. Silencing circ_0061395 reduced the proliferation, migration, invasion, tube formation and tumor spheroid formation rate of Huh-7 and SNU-387 cells. MiR-1182 was a target of circ_0061395. Silencing circ_0061395 inhibited the malignant behavior of HCC cells by releasing miR-1182. In addition, SPOCK1 was the target of miR-1182. Overexpression of SPOCK1 partially restored the inhibitory effect of miR-1182 on cell proliferation. Animal experiments confirmed the anti-tumor effect of silence circ_0061395. Circ_0061395 induced the changes of the expression of SPOCK1 by regulating miR-1182, thereby mediating the process of HCC, and at least partially promoting the development of HCC cells, providing a novel targeted therapy for HCC.

Synaptotagmin 11 scaffolds MKK7–JNK signaling process to promote stem-like molecular subtype gastric cancer oncogenesis

BackgroundIdentifying biomarkers related to the diagnosis and treatment of gastric cancer (GC) has not made significant progress due to the heterogeneity of tumors. Genes involved in histological classification and genetic correlation studies are essential to develop an appropriate treatment for GC.MethodsIn vitro and in vivo lentiviral shRNA library screening was performed. The expression of Synaptotagmin (SYT11) in the tumor tissues of patients with GC was confirmed by performing Immunohistochemistry, and the correlation between the expression level and the patient’s survival rate was analyzed. Phospho-kinase array was performed to detect Jun N-terminal kinase (JNK) phosphorylation. SYT11, JNK, and MKK7 complex formation was confirmed by western blot and immunoprecipitation assays. We studied the effects of SYT11 on GC proliferation and metastasis, real-time cell image analysis, adhesion assay, invasion assay, spheroid formation, mouse xenograft assay, and liver metastasis.ResultsSYT11 is highly expressed in the stem-like molecular subtype of GC in transcriptome analysis of 527 patients with GC. Moreover, SYT11 is a potential prognostic biomarker for histologically classified diffuse-type GC. SYT11 functions as a scaffold protein, binding both MKK7 and JNK1 signaling molecules that play a role in JNK1 phosphorylation. In turn, JNK activation leads to a signaling cascade resulting in cJun activation and expression of downstream genes angiopoietin-like 2 (ANGPTL2), thrombospondin 4 (THBS4), Vimentin, and junctional adhesion molecule 3 (JAM3), which play a role in epithelial-mesenchymal transition (EMT). SNU484 cells infected with SYT11 shRNA (shSYT11) exhibited reduced spheroid formation, mouse tumor formation, and liver metastasis, suggesting a pro-oncogenic role of SYT11. Furthermore, SYT11-antisense oligonucleotide (ASO) displayed antitumor activity in our mouse xenograft model and was conferred an anti-proliferative effect in SNU484 and MKN1 cells.ConclusionSYT11 could be a potential therapeutic target as well as a prognostic biomarker in patients with diffuse-type GC, and SYT11-ASO could be used in therapeutic agent development for stem-like molecular subtype diffuse GC.

PSMB2 knockdown suppressed proteasome activity and cell proliferation, promoted apoptosis, and blocked NRF1 activation in gastric cancer cells.

Proteasome 20S Subunit Beta 2 (PSMB2) has been suggested to play several roles in cancer. However, the role of PSMB2 and its underlying mechanisms in gastric cancer have not been studied. In this study, qRT-PCR was employed to detect the expression of genes that encode for 26s proteasome subunit proteins. PSMB2 expression and its prognostic ability were assessed by collecting patient tissue samples and reviewing the TCGA and Kaplan-Meier Plotter databases. Immunofluorescence and western blotting experiments were performed to evaluate the expression of PSMB2 in human gastric cancer cells and normal gastric epithelial cells. Subsequently, PSMB2 was knocked down in HGC-27 and SNU-1 cells and overexpressed in N-87 and AGS cells. Proteasome activity assays, 5-Ethynyl-2'-deoxyuridine staining, and TUNEL assays were used to assess proteasome activity, cell proliferation, and apoptosis. Tumor xenograft assays were conducted to evaluate PSMB2 function in vivo. Our results showed that a total of 8 genes encoding for the 26s proteasome subunit protein were highly expressed in a variety of gastric cancer cells. Next, PSMB2 was selected as the focus of subsequent studies which showed that PSMB2 was highly expressed in samples of gastric cancer tissue. Furthermore, a review of the TCGA database revealed that a high level of PSMB2 expression was associated with a poor clinical prognosis. Our results indicated that PSMB2 overexpression promoted proteasome activity, cell proliferation, and suppressed the apoptosis of gastric cancer cells, while those effects were reversed by treatment with a proteasome inhibitor (MG132). In contrast, PSMB2 knockdown produced the opposite effects and also blocked NRF1 activation. Moreover, PSMB2 knockdown inhibited tumor growth in vivo, decreased PSMB2 expression and cell proliferation, and promoted apoptosis in tumor tissues. Our findings revealed the role played by PSMB2 in gastric cancer and suggest PSMB2 as a new target molecule for use in diagnosing and treating gastric cancer.

Glaucocalyxin A Inhibits the Malignancies of Gastric Cancer Cells by Downregulating MDM2 and RNF6 via MiR-3658 and the SMG1-UPF mRNA Decay Pathway.

Gastric cancer (GC) ranks as the most common gastrointestinal cancer and is among the leading causes of cancer death worldwide. Glaucocalyxin A (GLA), an entkauranoid diterpene isolated from Rab-dosia japonica var., possesses various bioactivities. To date, the data on the effect of GLA on GC are still minimal, and the molecular mechanisms remain largely unknown. Herein, we found that GLA could significantly inhibit the proliferation, cell adhesion, and invasion of HGT-1, SNU-1, SNU-6, and NCI-N87 GC cells in a dose-dependent manner. GLA enhanced the apoptosis of the GC cells as evidenced by the increased caspase-3 activity and the elevated levels of cleaved caspase-3 and cleaved PARP in GC cells in the presence of GLA. We then showed that the downregulation of Murine Double Minute Clone 2 (MDM2) and Ring Finger Protein 6 (RNF6) by GLA was implicated in the GLA-induced inhibition of the GC cells. Furthermore, MDM2 and RNF6 were identified as the targets of miR-3658 that was downregulated in the GC cells and upregulated by GLA. Moreover, it was shown that miR-3658 was hypermethylated in the GC cells, and GLA could rescue the expression of miR-3658 via demethylation by abrogating EZH2-mediated epigenetic silencing. In addition to the miR-3658-MDM2/RNF6 regulatory axis, activation of the SMG1-UPF mRNA decay pathway contributed to the downregulation of MDM2 and RNF6 by GLA in the GC cells. The inhibitory effect of GLA on gastric cancer and the expression of MDM2 and RNF6 was also validated in in vivo study. Our findings suggest that has the therapeutic potential for GC by downregulating oncogenes via posttranscriptional regulation.

Volatilomic Signatures of AGS and SNU-1 Gastric Cancer Cell Lines

In vitro studies can help reveal the biochemical pathways underlying the origin of volatile indicators of numerous diseases. The key objective of this study is to identify the potential biomarkers of gastric cancer. For this purpose, the volatilomic signatures of two human gastric cancer cell lines, AGS (human gastric adenocarcinoma) and SNU-1 (human gastric carcinoma), and one normal gastric mucosa cell line (GES-1) were investigated. More specifically, gas chromatography mass spectrometry has been applied to pinpoint changes in cell metabolism triggered by cancer. In total, ten volatiles were found to be metabolized, and thirty-five were produced by cells under study. The volatiles consumed were mainly six aldehydes and two heterocyclics, whereas the volatiles released embraced twelve ketones, eight alcohols, six hydrocarbons, three esters, three ethers, and three aromatic compounds. The SNU-1 cell line was found to have significantly altered metabolism in comparison to normal GES-1 cells. This was manifested by the decreased production of alcohols and ketones and the upregulated emission of esters. The AGS cells exhibited the increased production of methyl ketones containing an odd number of carbons, namely 2-tridecanone, 2-pentadecanone, and 2-heptadecanone. This study provides evidence that the cancer state modifies the volatilome of human cells.