HGC-27 Cells Research Articles

Troponin C-1 Activated by E2F1 Accelerates Gastric Cancer Progression via Regulating TGF-β/Smad Signaling.

Troponin C-1 (TNNC1) has been previously characterized as an oncogenic gene. This study aimed to reveal the roles of TNNC1 in gastric cancer and the potential underlying mechanisms. TNNC1 siRNAs and TNNC1 overexpression plasmid were used to alter its expression in AGS, MKN45, and HGC-27 cells. CCK-8 assay, colony formation, EdU assay, flow cytometry, transwell assay, and scratch test were conducted to measure the phenotype changes. In vivo effects of TNNC1 silence were confirmed by using a xenograft mouse model. Bioinformatics analysis was conducted to screen out the transcription factor and downstream signaling of TNNC1. TNNC1 was highly expressed in gastric cancer tissues and cell lines, and its expression was associated with poor prognosis. TNNC1 silence suppressed the proliferation, migration, and invasion of AGS and MKN45 cells. However, TNNC1 silence induced apoptosis by mediating the cleavage of caspase-3 and caspase-9. Overexpression of TNNC1 in HGC-27 cells led to the contrary effects. The anti-tumor effects of TNNC1 silence were also confirmed in a xenograft animal model. E2F1 was validated as an upstream transcription factor of TNNC1. Effects of TNNC1 silence on AGS cell migration and invasion were attenuated by E2F1 overexpression. Besides, TGF-β/Smad was a downstream signaling pathway of TNNC1. The anti-tumor impacts of TNNC1 silence were weaken by SB431542 (a specific inhibitor of TGF-β signaling) while accelerated by TGF-β. TNNC1 activated by E2F1 functioned as an oncogenic gene through regulating TGF-β/Smad signaling. TNNC1 was suggested as a potential molecular drug target of gastric cancer.

Effect of KLF17 overexpression on epithelial–mesenchymal transition of gastric cancer cells

ObjectiveTo investigate Krüppel-like factor 17 (KLF17) expression in normal and gastric cancer tissues and cell lines.MethodsLevels of KLF17 mRNA and protein in GES-1 normal gastric mucosal cells, and NCI-N87, SGC-7901, BGC-823 and HGC-27 gastric cancer cells were analysed by quantitative polymerase chain reaction (qPCR) and western blot. Differences in KLF17 expression between gastric cancer and adjacent tissues were analysed by qPCR and immunohistochemistry. Invasion/migration effects of KLF17 overexpression in BGC-823 and HGC-27 cells were analysed by wound-healing and Transwell chamber assays. Changes in expression of KLF17 and epithelial–mesenchymal transition (EMT)-related genes (matrix metalloproteinase [MMP]-9, vimentin and E-cadherin) were analysed in BGC-823 and HGC-27 cells before and after transfection using qPCR and western blot. Transforming growth factor (TGF)-β1, Smad family member (Smad)2/3 and phosphorylated-Smad2/3 levels in BGC-823 and HGC-27 cells were assessed by qPCR and western blot.ResultsKLF17 expression was lower in gastric cancer versus adjacent tissues, and in gastric cancer cell lines versus GES-1 normal gastric mucosal cells, and was positively correlated with degree of cancer-cell differentiation. Wound-healing and Transwell assays showed decreased migration and invasion ability of BGC-823 and HGC-27 cells transfected to overexpress KLF17. KLF17 overexpression was associated with decreased MMP-9 and vimentin in BGC-823 and HGC-27 cancer cells, and increased KLF17 and E-cadherin. KLF17 overexpression also resulted in decreased levels of TGF-β1 and p-Smad2/3 in BGC-823 and HGC-27 cancer cells.ConclusionKLF17 is poorly expressed in gastric cancer tissues and cell lines. KLF17 overexpression might inhibit EMT via the TGF-β/Smad pathway, thereby reducing gastric cancer cell invasion and migration. Therefore, KLF17 may become a novel target for treating gastric cancer.

Tumor-Associated Macrophage-Derived Exosomes Promote the Progression of Gastric Cancer by Regulating the P38MAPK Signaling Pathway and the Immune Checkpoint PD-L1.

Objective: To investigate the effects of M2 macrophage-derived exosomes (M2-Exos) on proliferation, migration, and apoptosis of gastric cancer cells in the tumor microenvironment and to further explore their possible molecular mechanism. Materials and Methods: M2 macrophages were induced from THP-1 cells and identified by qRT-PCR. Exosomes were extracted by ultracentrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot analysis. Fluorescence labeling was used to detect the internalization of exosomes in receptors. The proliferation, migration, and invasion of AGS and HGC27 cells were determined by EdU and MTS, wound healing and Transwell assay, and flow cytometry, respectively. Proteins in the related pathway of M2-Exos affecting the progression of gastric cancer were detected by Western blot analysis. Results: In this study, M2 macrophages and M2-Exos were successfully obtained. The purified M2-Exos were observed as small round vesicles with diameters of 50-90 nm and positive expression of CD63, CD9, and TSG101. Besides, M2-Exos can be effectively taken up and internalized by AGS and HGC27 cells. Cell behavior studies showed that M2-Exos promoted proliferation and migration and inhibited the apoptosis of AGS and HGC27. Further research illustrated that M2-Exos promoted the phosphorylation of P38 and high expression of programmed death ligand 1 (PD-L1). Conclusions: This study demonstrated that M2-Exos promoted proliferation and migration and inhibited the apoptosis of gastric cancer cells. Mechanically, M2-Exos may promote gastric cancer progression through the P38MAPK signaling pathway and achieve immune escape through elevating the expression of PD-L1.

Novel evidence for m6A methylation regulators as prognostic biomarkers and FTO as a potential therapeutic target in gastric cancer.

While emerging evidence indicates that N6-methyladenosine (m6A) regulators play crucial roles in cancer progression, their clinical significance in gastric cancer (GC) has thus far not been elucidated. We investigated the expression of the m6A regulator genes and their prognostic potential in a large clinical cohort of 173 GC patients using qRT-PCR assays. In addition, we undertook a series of in-vitro and in-vivo functional studies to investigate the oncogenic role of FTO. GC patients with low expression of METTL3, METTL14, ALKBH5, WTAP and YTHDF1 demonstrated significantly poor OS, while patients with high FTO expression exhibited markedly worse OS. Furthermore, the cumulative risk-score derived from these gene panel also significantly associated with poor OS, with a corresponding hazard ratio of 5.47 (95% CI: 3.18-9.41, p < 0.0001). We observed that FTO expression was frequently upregulated in GC cell lines, with epithelial-mesenchymal-transition (EMT) features. FTO knockdown in HGC27 and AGS cells inhibited cell proliferation and migratory potential, while its overexpression in MKN28 cells resulted in enhanced proliferation and migration. Finally, confirming our in-vitro findings, FTO suppression led to significant tumour growth inhibition in a HGC27 xenograft model. We demonstrate that m6A regulators may serve as promising prognostic biomarkers in GC. Our functional studies reveal that FTO is an important oncogene and may be a promising therapeutic target associated with EMT-alterations in gastric cancer.

Direct interaction between Rab5a and Rab4a enhanced epidermal growth factor-stimulated proliferation of gastric cancer cells.

BACKGROUNDGastric cancer (GC) is one of the leading causes of cancer-related death worldwide. Although targeted therapies such as antibodies against human epidermal growth factor receptor 2 or vascular endothelial growth factor receptor 2 have been widely used in the treatment of metastatic cancer, the overall outcomes are poor. Therefore, elucidation of the mechanism underlying cancer progression is important to improve prognosis. Overexpression of the Rab5a gene has been confirmed to correlate with tumorigenesis of many cancers, but the mechanism underling, especially of GC, is still unclear.AIMTo investigate the effects of Rab5a overexpression on the tumorigenesis of GC.METHODSFirst, the expression levels of Rab5a and Rab4a in primary tumorous tissues of GC patients diagnosed between 2015 and 2018 were analyzed. Then we constructed HGC-27 cell lines overexpressing green fluorescent protein-Rab5a or red fluorescent protein-Rab4a and investigated the interaction between Rab5a or Rab4a using Western blotting, co-immunoprecipitation, confocal microscopy, and colocalization analysis. Finally, epidermal growth factor-stimulated proliferation of these cell lines was analyzed using cell counting kit-8 cell viability assay.RESULTSCompared with normal gastric tissues, the expression levels of Rab5a and Rab4a increased progressively both in paracancerous tissues and in advanced cancerous tissues. Epidermal growth factor could promote the proliferation of HGC-27 cells, especially Rab5a-overexpressing HGC-27 cells. Notably, Rab5a and Rab4a co-overexpression promoted the proliferation of HGC-27 cells to the greatest extent. Further analysis identified a direct interaction between Rab5a and Rab4a in HGC-27 cells.CONCLUSIONCo-overexpression of Rab5a and Rab4a in GC may promote the endosomal recycling of epidermal growth factor receptor, which in turn contributes to poor prognosis and tumor progression in GC patients. Inhibition of Rab5a or Rab4a expression might be a promising therapy for refractory GC.

KLK6 mediates stemness and metabolism of gastric carcinoma cells via the PI3K/AKT/mTOR signaling pathway.

Gastric cancer is a common tumor of the digestive system, which can occur in any part of the stomach. Kallikrein 6 (KLK6) is a trypsin-like serine protease and has been found to be involved in extracellular matrix remodeling, tumor invasion and nervous system plasticity. Our previous study reported that KLK6 suppressed HGC-27 gastric cancer cell growth by inhibiting epithelial-mesenchymal transition; however, the mechanism of action underlying the effect of KLK6 still remains unclear. The aim of the present study was to investigate the effect and the underlying mechanism of KLK6 on stem cell-like properties and metabolism in gastric carcinoma cells. The HGC-27 cell line was transfected with KLK6 overexpression (OV-KLK6) and interference (short hairpin-KLK6) vectors, then the transfection efficiency was confirmed using western blot analysis and reverse transcription-quantitative PCR. The percentage of CD133+ and CD44+ cells was detected using flow cytometry, while the protein expression levels of the stem-associated genes, Nanog, Oct-4, SOX2 and Notch1, the metabolic markers, hexokinase (HK)1, HK2, GLUT1, and the proteins within the PI3K signaling pathway, phosphorylated (p)-PI3K, p-AKT and p-mTOR, were determined using western blot analysis. Biochemical kits were used to measure ATP production, lactic acid content and glucose uptake. A tumorigenicity assay was performed with nude mice to detect gastric tumor volume, and the protein expression level of Oct-4, Nanog, HK1, HK2 and GLUT1, and the mRNA expression level of KLK6 was also determined in gastric tumor tissues of mice. Compared with that in the control group, KLK6 protein and mRNA expression levels were significantly decreased in the four sh-RNA groups (P<0.05). Among them, sh-RNA-3 induced the lowest KLK6 expression and was used to silence KLK6 in subsequent experiments. Compared with that in the control and negative control groups, the percentage of CD133+ and CD44+ cells, the protein expression level of Oct-4, Nanog, HK1, HK2, GLUT1, p-PI3K, p-AKT and p-mTOR, and ATP content, lactic acid production, glucose uptake and gastric tumor volume were significantly decreased by sh-KLK6 (P<0.05), whereas KLK6 overexpression induced the opposite effect (P<0.05). In conclusion, KLK6 modulated stemness properties and cell metabolic profile in gastric carcinoma cells and the mechanism may be associated with the PI3K/AKT/mTOR signaling pathway.

Design, synthesis and biological evaluation of novel o-aminobenzamide derivatives as potential anti-gastric cancer agents in vitro and in vivo

Although gastric cancer has become a major public health problem, oral agents applied in clinics for gastric cancer therapy are scarce. Therefore, to explore new oral chemical entities with high efficiency and low toxicity, 41 o-aminobenzamide derivatives based on the scaffolds of MS-275 and SAHA were designed, synthesized, and evaluated for their anti-gastric cancer abilities in vitro and in vivo. Structure-activity relationships were discussed, leading to the identification of compounds F8 (IC50 = 0.28 μM against HGC-27 cell) and T9 (IC50 = 1.84 μM against HGC-27 cell) with improved cytotoxicity, anti-gastric cancer proliferation potency, induction of cell apoptosis and cell cycle arrest ability, inhibition of cell migration and invasion. What is worth mentioning is that compound F8 was more efficient and less toxic than the positive drug capecitabine in vivo on the HGC-27-xenograft model. Meanwhile, compound F8 exhibited suitable pharmacokinetic properties and less acute toxicity (LD50 > 1000 mg/kg). Besides, western blotting analysis, IHC analysis, differentially expressed proteins analysis and ABPP experiment indicated that compound F8 could modulate molecular pathways involved in apoptosis and cell cycle progression. Consequently, compound F8 is a strong candidate for the development of human gastric cancer therapy.

MiR-511-5p Suppresses Cell Migration, Invasion and Epithelial-Mesenchymal Transition Through Targeting PAK2 in Gastric Cancer.

As a malignant tumor, gastric cancer (GC) is closely related with gastric mucosa and has a high mortality in the world. Since microRNA (miRNA) has become more and more important in tumor research, we intend to find out the functional role and mechanism of miR-511-5p in GC. Firstly, miR-511-5p level was examined in human GC cell lines and tissues, and its effect on cell migration and invasion of BGC-823 or HGC-27 cells was tested by migration assay and transwell assay. Then, we confirmed the association between miR-511-5p and p21 activated kinase 2 (PAK2) by the luciferase reporter assay, and further assessed their role in cell migration and invasion. Moreover, we verified the function of miR-511-5p and PAK2 in epithelial-mesenchymal transition (EMT). In our study, miR-511-5p was downregulated in GC cell lines and tissues, and inversely associated with PAK2. Luciferase reporter assay confirmed that miR-511-5p could bind to PAK2. MiR-511-5p mimics significantly upregulated E-cadherin and downregulated N-cadherin, Vimentin and Snail, and consequently inhibited cell migration and invasion. However, reintroduction of PAK2 reversed the inhibitory function of miR-511-5p on BGC-823 and HGC-27 cells. Our research suggested that tumor-suppressive function of miR-511-5p in GC was inhibited by PAK2, and miR-511-5p/PAK2 axis may serve as a new strategy in GC management.

Mechanism of piperine in affecting apoptosis and proliferation of gastric cancer cells via ROS-mitochondria-associated signalling pathway.

Piperine (PIP), the main active ingredient in pepper, belongs to the cinnamamide alkaloid. PIP has been found to have functions, including anti‐oxidation, immune regulation, anti‐tumour and promotion of drug metabolism. The present study was mainly designed to reveal the anti‐tumour effect of PIP against gastric cancer and the relevant mechanism. In brief, the undifferentiated human gastric cancer cell HGC‐27 was used, which was treated with different concentrations of PIP. As a result, PIP could inhibit proliferation and induce apoptosis of HGC‐27 cells in a dose‐dependent manner. The mechanism of PIP was associated with ROS increase and mitochondrial damage, simultaneously, the expression of key proteins of apoptosis was affected, including Bcl‐2, Bax, Cyt‐c, Caspase‐9 and Caspase‐3. Pre‐treatment of ROS scavenger NAC HGC‐27 cells could significantly reduce PIP‐induced apoptosis and inhibit the activation of apoptotic signals. Consistently, PIP could induce ROS to increase and activate apoptotic signals in the animal model. Therefore, the present study showed that PIP can induce the generation of ROS, thereby promoting the activation of mitochondrial apoptotic pathway and exerting anti‐tumour effects.

Apatinib combined with Keytruda treatment induces apoptosis of gastric carcinoma cells through CES4/miR-616-5p/DUSP2 axis.

Gastric carcinoma (GC) is a highly malignant and heterogeneous tumour. Long non-coding RNA CES4 is down-regulated in GC. However, whether CES4 can participate in GC remains unclear; we have carried out research on this topic. GC cells (HGC-27 and MKN-7) were treated with anti-tumour drugs: apatinib combined with Keytruda. Cell viability and apoptosis were detected by CCK-8 assay and flow cytometry. Gene and protein expression were examined by quantitative real-time PCR and western blot. Luciferase reporter assay was performed to verify the relationship among CES4, miR-616-5p and dual-specificity phosphatase-2 (DUSP2). CES4 was highly expressed in the apatinib combined with Keytruda-treated HGC-27 and MKN-7 cells. Apatinib combined with Keytruda treatment repressed cell viability and promoted apoptosis of HGC-27 and MKN-7 cells, which was abrogated by CES4 knockdown. Furthermore, CES4 promoted DUSP2 expression by sponging miR-616-5p in HGC-27 and MKN-7 cells. CES4 knockdown promoted cell viability and inhibited apoptosis of drug-treated HGC-27 and MKN-7 cells by regulating miR-616-5p/DUSP2 axis. In conclusion, these data demonstrate that apatinib combined with Keytruda treatment induces apoptosis of GC cells through CES4/miR-616-5p/DUSP2 axis. Thus, this work provides the experimental basis for the combination of apatinib and Keytruda as a treatment for GC.

Gentiopicroside inhibits proliferation, migration, and invasion of gastric cancer HGC-27 cells by regulating miR-34c-5p/XBP1 axis

Gentiopicroside inhibits proliferation, migration, and invasion of gastric cancer HGC-27 cells by regulating miR-34c-5p/XBP1 axis

Knockdown of hsa_circ_0091994 constrains gastric cancer progression by suppressing the miR-324-5p/HMGA1 axis.

CircRNAs have emerged as potential therapeutic targets for diseases such as gastric cancer (GC). We identified highly dysregulated circRNAs in GC tissue and further explored their potential mechanisms in the progression of GC. Hsa_circ_0091994 (cicrRNA_105040) was identified as a highly upregulated circRNA in GC tissues, whose host gene is negatively associated with the overall survival of patients. Using cell counting kit-8 and Annexin V assays, we observed that hsa_circ_0091994 knockdown inhibited the viability of AGS and HGC-27 cells by inducing apoptosis. Scratch wound healing assays showed that hsa_circ_0091994 knockdown also inhibited GC cell healing. Bioinformatics analysis and a luciferase assays revealed that hsa_circ_0091994 knockdown inhibits GC progression by suppressing miR-324-5p and HMGA1 expression. The antitumor effect of hsa_circ_0091994 knockdown was confirmed in vivo using a mouse xenograft model. Hsa_circ_0091994 knockdown inhibited the progression of GC by inhibiting the miR-324-5p/HMGA1 axis.

The m6A Methyltransferase METTL14-Mediated N6-Methyladenosine Modification of PTEN mRNA Inhibits Tumor Growth and Metastasis in Stomach Adenocarcinoma.

BackgroundStomach adenocarcinoma (STAD) is a common reason for tumor-related fatalities globally, as it results in distant metastasis. Methyltransferase-like 14 (METTL14), a notable RNA N6-adenosine methyltransferase (m6A), plays a significant role in the growth of tumor through controlling the RNA working. This study aims to highlight METTL14 in STAD’s biological function and molecular mechanism.MethodsBioinformatics and immunohistochemical (IHC) assays have been utilized for the detection of METTL14 expression in the STAD. METTL14’s biological function has been shown while making use of HGC-27 and AGS cells in vitro experiments. MeRIP-qPCR and luciferase reporter assays were employed for the exploration of METTL14’s mechanism modifying the target of phosphatase and tensin homologue (PTEN). Subcutaneous xeno transplantation model and STAD liver metastasis orthotopic tumor model were used to study METTL14 in STAD in vivo.ResultsMETTL14 expression was substantially downregulated in STAD reflecting contribution to major tumors, progressed TNM stage as well as poor overall survival (OS) in STAD. Moreover, METTL14’s inhibition of STAD cells proliferation, migration and invasion has been verified in vitro assays. Furthermore, an identification of PTEN being METTL14-mediated m6A modification’s substrate has been made. METTL14’s overexpression highly enhanced PTEN mRNA m6A variation, stabilized PTEN mRNA and increased protein expression. Further, it has been found out that METTL14-mediated STAD cells inhibition of proliferation and invasion dependent on PTEN. At last, we demonstrated that METTL14 inhibit STAD growth and metastasis in vivo models.ConclusionsMETTL14 inhibits tumor growth and metastasis of STAD via stabilization of PTEN mRNA expression. Therefore, METTL14 is a potential biomarker of prognosis and therapeutic targets for STAD.

ELK4 promotes the development of gastric cancer by inducing M2 polarization of macrophages through regulation of the KDM5A-PJA2-KSR1 axis

BackgroundWe tried to elaborate the molecular mechanism of ETS-like transcription factor 4 (ELK4) affecting gastric cancer (GC) progression through M2 polarization of macrophages mediated by lysine-specific demethylase 5A (KDM5A)-Praja2 (PJA2)-kinase suppressor of ras 1 (KSR1) axis.MethodsGC expression dataset was obtained from GEO database, and the downstream regulatory mechanism of ELK4 was predicted. Tumor-associated macrophages (TAMs) were isolated from GC tissues. The interaction among ELK4, KDM5A, PJA2 and KSR1 was analyzed by dual luciferase reporter gene, ChIP and Co-IP assays. The stability of KSR1 protein was detected by cycloheximide (CHX) treatment. After TAMs were co-cultured with HGC-27 cells, HGC-27 cell biological processes were assessed through gain- and loss-of function assays. Tumorigenicity was detected by tumorigenicity test in nude mice.ResultsIn GC and TAMs, ELK4, KDM5A and KSR1 were highly expressed, while PJA2 was lowly expressed. M2 polarization of macrophages promoted the development of GC. ELK4 activated KDM5A by transcription and promoted macrophage M2 polarization. KDM5A inhibited the expression of PJA2 by removing H3K4me3 of PJA2 promoter, which promoted M2 polarization of macrophages. PJA2 reduced KSR1 by ubiquitination. ELK4 promoted the proliferative, migrative and invasive potentials of GC cells as well as the growth of GC xenografts by regulating KSR1.ConclusionELK4 may reduce the PJA2-dependent inhibition of KSR1 by transcriptional activation of KDM5A to promote M2 polarization of macrophages, thus promoting the development of GC.

MYBL2 in synergy with CDC20 promotes the proliferation and inhibits apoptosis of gastric cancer cells.

Gastric cancer (GC) is a common malignant tumor with a high morbidity and mortality worldwide. It has been reported that V-Myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2) could be a promising prognostic biomarker for GC. However, the specific role of MYBL2 in GC progression remains unclear. To examine the role of MYBL2 in GC progression and investigate the underlying mechanisms. The mRNA levels of target genes were detected using quantitative real-time polymerase chain reaction (RT-qPCR) and protein expression was measured with western blot analysis. Cell Counting Kit-8 (CCK-8) and colony formation assays were employed to inspect HGC-27 cell proliferation, and cellular apoptosis was determined with TUNEL staining. Finally, the interaction of MYBL2 and cell division cycle 20 (CDC20) was verified with immunoprecipitation. MYBL2 was confirmed to be overexpressed in GC cells. MYBL2 knockdown inhibited HGC-27 cell proliferation and promoted cellular apoptosis, and these effects were reversed by CDC20 overexpression. Interestingly, MYBL2 interacted with CDC20 and regulated its expression. MYBL2 knockdown also inhibited activation of the Wnt/β-catenin signaling pathway, while CDC20 overexpression showed the opposite effect. In summary, the synergy between MYBL2 and CDC20 induced the proliferation of GC cells and inhibited cell apoptosis; these effects may have involved the Wnt/β-catenin signaling pathway. Thus, MYBL2 may be a promising target for GC treatment.

Pannexin 1 Mediates Gastric Cancer Cell Epithelial-Mesenchymal Transition via Aquaporin 5.

Pannexin 1 (PANX1) has been implicated in cancer emergence and progression. However, its roles in gastric cancer remain unclear. In the present study, the function and molecular mechanisms of PANX1 in gastric cancer were investigated in vitro. Two gastric cancer cell lines exhibiting low and high PANX1 expression (SNU-16 and HCG-27, respectively) were transfected using a PANX1-containing plasmid or PANX1 transcript-targeting short hairpin (sh)RNA. In addition, HCG-27 cells and PANX1-overexpressing SNU-16 cells were subjected to short interfering (si)RNA-mediated aquaporin 5 (AQP5) knockdown. In vitro cell migration (scratch) and transwell invasion assays were performed to evaluate the cell migratory and invasive abilities. Real-time fluorescence quantitative PCR was used to detect transcripts encoding epithelial-mesenchymal transition markers. Immunofluorescence and Western blotting were conducted to quantify corresponding proteins. In SNU-16 cells, PANX1 overexpression induced conversion from round (cobblestone-like) to elongated (spindle-like) morphologies and enhanced the cell migratory and invasive abilities. PANX1 knockdown had the opposite effect in HGC-27 cells. In PANX1-overexpressing SNU-16 cells, expression of SLUG, vimentin, and AQP5 was significantly upregulated, whereas expression of E-cadherin was downregulated. In HGC-27 cells, PANX1 knockdown showed the opposite effect. In both PANX1-overexpressing SNU-16 cells and untransfected HGC-27 cells, silencing of AQP5 expression significantly inhibited PANX1-induced upregulation of SLUG and vimentin expression, as well as downregulation of E-cadherin expression and enhanced migratory and invasive abilities. In summary, elevated PANX1 expression induces gastric cancer cell epithelial-mesenchymal transition and the associated promotion of migratory and invasive abilities by inducing expression of AQP5, which facilitates SLUG-mediated regulation of vimentin and E-cadherin expression.

Cytotoxic tetracyclic triterpenes from the endophytic fungus Aspergillus fumigatus of Cleidion brevipetiolatum

• Two new tetracyclic triterpenes were isolated from Aspergillus fumigatus . • Their structures were elucidated by HRESIMS, 1D and 2D NMR analysis. • Asperfumins A ( 1 ) and B ( 2 ) exhibited cytotoxic activities against A549, HGC-27 and H1975 cells. In our search for cytotoxic natural products from endophytic fungi of traditional Chinese medicine (TCM), two previously unreported tetracyclic triterpenes, namely asperfumins A ( 1 ) and B ( 2 ), were isolated from Aspergillus fumigatus , an endophytic fungus derived from Cleidion brevipetiolatum . Their structures were unequivocally elucidated by extensive spectroscopic analysis including HRESIMS, 1D and 2D NMR. Asperfumins A ( 1 ) and B ( 2 ) exhibited cytotoxic activities on three human cancer cell lines (A549, HGC-27, and H1975) with the IC 50 values of 4.2–26.5 μM.

Dihydroartemisinin suppresses proliferation, migration, the Wnt/β-catenin pathway and EMT via TNKS in gastric cancer.

Gastric cancer is a common malignancy worldwide. However, the molecular mechanisms underlying this malignancy remain unclear and there are a lack of effective drugs. The present study aimed to investigate the antitumor effect of Dihydroartemisinin (DHA) or inhibition of Tankyrases (TNKS), and determine the underlying molecular mechanisms of gastric cancer. Immunohistochemistry and immunofluorescence analyses were performed to detect the expression levels of TNKS, epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin pathway-related proteins in gastric cancer tissues and adjacent normal tissues. The Cell Counting Kit-8 assay was performed to assess the viability of HGC-27 and AGS cells following treatment with different concentrations of HLY78 (a Wnt activator) or DHA. Following treatment with HLY78, DHA or small interfering (si)-TNKS1/si-TNKS2, colony formation and migratory abilities were assessed via the colony formation, wound healing and Transwell assays. Furthermore, western blot and immunofluorescence analyses were performed to detect the expression levels of TNKS, EMT- and Wnt/β-catenin-related proteins. The results demonstrated that the expression levels of TNKS, AXI2, β-catenin, N-cadherin and Vimentin were upregulated, whereas E-cadherin expression was downregulated in gastric cancer tissues compared with normal tissues. Furthermore, HLY78 and DHA suppressed the viability of HGC-27 and AGS cells, in a concentration-independent manner. Notably, TNKS knockdown or treatment with DHA suppressed colony formation, migration, TNKS expression, EMT and the Wnt/β-catenin pathway. Opposing effects were observed following treatment with HLY78, which were ameliorated following co-treatment with DHA. Taken together, these results suggest that DHA or inhibition of TNKS can suppress the proliferation and migration of gastric cancer cells, which is partly associated with inactivation of the Wnt/β-catenin pathway and EMT process.

TRAIL produced by SAM-1-activated CD4+ and CD8+ subgroup T cells induces apoptosis in human tumor cells through upregulation of death receptors

Bacterial superantigens potently activate conventional T-cells to induce massive cytokine production and mediate tumor cell death. To engineer superantigens for immunotherapy against tumors in clinic, we previously generated SAM-1, a staphylococcal enterotoxins C2 (SEC2) mutant, that exhibited significantly reduced toxicity but maintained the superantigen activity in animal models. This present study aimed to investigate whether SAM-1 activates T cells and induces apoptosis in human tumor cells. We found that SAM-1 induced the maturation of dendritic cells (DCs) with upregulating expression of the surface markers CD80, CD86 and HLA-DR, which secreted high levels of IL-12p70 by activating TLR2-NF-κB signaling pathways. SAM-1 could activate human CD4+ subgroup T cells and CD8+ subgroup T cells in the presence of mature dendritic cells (DCs), leading to the productions of cytokines TRAIL, IL-2, IFN-γ and TNF-α. We observed that TRAIL mediated the apoptosis and S-phase and G2/M-phase arrest in HGC-27 tumor cells via binding to upregulated death receptors DR4 and DR5. Using shRNA knockdown in HGC-27 cells or constitutive overexpression in ES2 cells for DR4 and DR5, we demonstrated the vital requirement of DR4 and DR5 in apoptosis of tumor cells in response to TRAIL secreted from SAM-1-activated T cells. Collectively, our results will facilitate better understanding of SAM-1-based immunotherapies for cancer.

Distinctive Prognostic Value and Cellular Functions of Osteopontin Splice Variants in Human Gastric Cancer.

Background: Osteopontin (OPN) splice variants are identified as predictors of tumour progression and therapeutic resistance in certain types of solid tumours. However, their roles in gastric cancer (GC) remain poorly characterized. The current study sought to assess the prognostic value of the three OPN splice variants (namely OPN-a, OPN-b, and OPN-c) in gastric cancer and their potential functions within gastric cancer cells. Methods: RNA extraction and reverse transcription were performed using our clinical cohort of gastric carcinomas and matched normal tissues (n = 324 matched pairs). Transcript levels were determined using real-time quantitative PCR. Three OPN splice variants overexpressed cell lines were created from the gastric cancer cell line HGC-27. Subsequently, biological functions, including cell growth, adhesion, migration, and invasion, were studied. The potential effects of OPN isoforms on cisplatin and 5-Fu were evaluated by detecting cellular reactive oxygen species (ROS) levels in the HGC-27-derived cell lines. Results: Compared with normal tissues, the expression levels of three splice variants were all elevated in gastric cancer tissues in an order of OPN-a > OPN-b > OPN-c. The OPN-a level significantly increased with increasing TNM staging and worse clinical outcome. There appeared to be a downregulation for OPN-c in increasing lymph node status (p < 0.05), increasing TNM staging, and poor differentiation. High levels of OPN-a and OPN-b were correlated with short overall survival and disease-free survival of gastric cancer patients. However, the low expression of OPN-c was significantly associated with a poor prognosis. Functional analyses further showed that ectopic expression of OPN-c suppressed in vitro proliferation, adhesiveness, migration, and invasion properties of HGC-27 cells, while the opposite role was seen for OPN-a. Cellular ROS detection indicated that OPN-a and OPN-c significantly promoted ROS production after treatment with 5-Fu comparing to OPN-vector, while only OPN-a markedly induced ROS production after treatment with cisplatin. Conclusion: Our results suggest that OPN splice variants have distinguished potential to predict the prognosis of gastric cancer. Three OPN variants exert distinctive functions in gastric cancer cells. Focusing on specific OPN isoforms could be a novel direction for developing diagnostic and therapeutic approaches in gastric cancer.