MGC-803 Cells Research Articles

Isoliquiritigenin inhibits gastric cancer growth through suppressing GLUT4 mediated glucose uptake and inducing PDHK1/PGC-1α mediated energy metabolic collapse

BackgroundIsoliquiritigenin (ISL), a natural flavonoid, has anti-tumor activity. But, the understanding of the impact and molecular mechanism of ISL on the growth of gastric cancer (GC) remains limited. PurposeThe study was to explore the tumor suppressive effect of ISL on GC growth both in vitro and in vivo, meanwhile, clarify its molecular mechanisms. MethodsCell viability was detected by cell counting kit-8 (CCK-8) assay. Apoptotic cells in vitro were monitored by Hoechst 33,342 solution. Protein expression was assessed by Western blot. Reactive oxygen species (ROS) level was evaluated by utilizing 2′,7′- dichlorofluorescin diacetate (DCFH-DA). Lactic acid level was detected with L-lactate assay kit. Glucose uptake was monitored with fluorescently tagged glucose 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). Glycolytic proton efflux rate (GlycoPER) was evaluated by glycolytic rate assay kit. Oxygen consumption rate (OCR) was conducted by mito stress test kit. A nude mouse model of gastric cancer cell xenograft was established by subcutaneous injection with MGC803 cells. Pathological changes were evaluated by using H&E staining. Cell apoptosis in vivo was evaluated by terminal deoxy-nucleotide transferase mediated dUTP nick end labeling (TUNEL) assay. ResultsISL remarkably suppressed GC growth and increased cell apoptosis. It regulated apoptosis-related and metabolism-related protein expression both in vitro and in vivo. ISL blocked glucose uptake and suppressed production and secretion of lactic acid, which was accompanied with suppressed mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis but increased ROS accumulation. Overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), cellular-myelocytomatosis viral oncogene (c-Myc), hypoxia inducible factor-1α (HIF-1α), glucose transporter 4 (GLUT4) or pyruvate dehydrogenase kinase 1 (PDHK1), could abolish ISL-induced inhibition of cell viability in GC cells. ConclusionThese findings implicated that ISL inhibits GC growth by decreasing GLUT4 mediated glucose uptake and inducing PDHK1/PGC-1α-mediated energy metabolic collapse through depressing protein expression of c-Myc and HIF-1α in GC, suggesting its potential application for GC treatment.

CircRNA_15430 reduced by Helicobacter pylori infection and suppressed gastric cancer progression via miR-382-5p/ZCCHC14 axis

BackgroundHelicobacter pylori (H.pylori, HP) is one of the main causes of gastric cancer (GC). CircRNAs have been reported to play a crucial role in developing many types of cancer. However, the role of circRNAs in the development and progression of HP infected-GC has not been studied.MethodsThe location of circRNA_15430 in GC cells were detected by nuclear and cytoplasmic RNA fractionation and RNA fluorescence in situ hybridization analysis (FISH) assays, and circRNA_15430, miR-382-5p and ZCCHC14 expression in GC cell lines and tissues were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The function of circRNA_15430 in GC cells were examined by using colony formation, cell counting kit-8 (CCK-8) and Transwell assays, flow cytometry and laser scanning confocal microscopy. The protein levels were detected by Western blotting. Whether circRNA_15430 sponges miR-382-5p was monitored with a dual-luciferase reporter assay. Furthermore, circRNA_15430 was analyzed in vivo in tumor growth with nude mice.ResultsCircRNA_15430 is primarily localized in the cytoplasm of GC cells, and downregulated in the GC cell lines and tissues, and is negatively correlated with the tumor size. Downregulation of circRNA_15430 promotes proliferation, migration and suppresses cell apoptosis and autophagy in GC cells. Mechanically, circRNA_15430 acts as a miR-382-5p sponge, alleviating the inhibitory effect of miR-382-5p on its target ZCCHC14. Knockdown circRNA_15430 enhances tumor growth in vivo. In addition, circRNA_15430 was reduced in HP + gastritis tissues and HP-infected MGC-803 cells, reversing the pro-HP effect on autophagy. Additionally, miR-382-5p was increased in HP + gastritis tissue and HP-infected MGC-803 cells while ZCCHC14 decreased in HP-infected MGC-803 cells. MiR-382-5p reverses the effect of si-ZCCHC14 on autophagosome numbers in MGC-803 cells.ConclusionsTherefore, circRNA_15430 plays an inhibitory role in GC and regulates the progression of HP infection-related GC, providing a novel molecular marker for GC therapy.

A novel long non-coding RNA XLOC_004787, is associated with migration and promotes cancer cell proliferation by downregulating mir-203a-3p in gastric cancer

BackgroundLong noncoding RNAs (lncRNAs) have been identified as important regulatory factors implicated in a wide array of diseases, including various forms of cancer. However, the roles of most lncRNAs in the progression of gastric cancer (GC) remain largely unexplored. This study investigates the biological function and underlying mechanism of a novel lncRNA, XLOC_004787 in GC.MethodsThe location of XLOC_004787 in GES-1 cells and HGC-27 cells were detected by fluorescence in situ hybridization (FISH) assay. The expression levels of XLOC_004787 were assessed using quantitative real-time fluorescence PCR (qRT-PCR) in various cell lines, including GES-1, MGC-803, MKN-45, BGC-823, SGC-7901, and HGC-27 cells. Functional assays such as Transwell migration, cell counting kit-8 (CCK-8), and colony formation experiments were employed to analyze the effects of XLOC_004787 and miR-203a-3p on cell migration and proliferation. Protein levels associated with GC in these cell lines were examined by Western blotting. The intracellular localization of β-catenin and P-Smad2/3 was assessed using immunofluorescence (IF) assay. Additionally, the interaction between XLOC_004787 and miR-203a-3p was investigated using a dual luciferase assay.ResultsXLOC_004787 was localized at both the cytoplasm and nucleus of GES-1 cells and HGC-27 cells. Compared to normal tissues and GES-1 cells, XLOC_004787 expression was significantly upregulated in GC tissues and cells, with the highest and lowest expression observed in SGC-7901 and HGC-27 cells, respectively. Furthermore, a reduced expression of XLOC_004787 was seen to inhibit migration and proliferation in SGC-7901 cells. Western blotting analysis revealed that a decrease in XLOC_004787 expression correspondingly decreased the expression of N-cadherin, mmp2, mmp9, Snail, Vimentin, β-catenin, C-myc, Cyclin D1, and TGF-β, while concurrently increasing E-cadherin expression. This was also associated with diminished expression of P-Smad2/3 in relation to Smad2/3, and reduced P-Gsk3β expression in comparison to Gsk3β. Additionally, the nuclear entry of P-Smad2/3 and β-catenin was reduced by lower XLOC_004787 expression. Amplifying XLOC_004787 expression via pcDNA_XLOC_004787 suggested a potential for cancer promotion. Notably, XLOC_004787 was found to negatively regulate mir-203a-3p expression, with potential binding sites identified between the two. Higher mir-203a-3p expression was observed to decrease migration and proliferation, and enhance E-cadherin expression. Conversely, suppression of mir-203a-3p expression suggested a potential promotion of proliferation and migration in GC cells.ConclusionsThese results suggest that XLOC_004787, found to be upregulated in GC tissues, potentially promotes proliferation and migration in GC cells. This occurs through the activation of TGF-β and Wnt/β-catenin signaling pathways and the expression of EMT-related proteins. Additionally, XLOC_004787 may influence cell migration and proliferation by modulating the signaling pathway via the adsorption and inhibition of mir-203a-3p.

Multiple approaches revealed MGc80-3 as a somatic hybrid with HeLa cells rather than a gastric cancer cell line.

The short-tandem-repeats (STR) profiles of MGc80-3 and HeLa partially overlap, raising suspicion of contamination in the MGc80-3 cell line. However, there has not been any relevant study demonstrating whether MGc80-3 was fully replaced by HeLa cells, just mixed with HeLa cells (co-existing), or was a somatic hybrid with HeLa cells. In addition to STR profiling, various approaches, including single nucleotide polymorphisms genotyping, polymerase chain reaction, screening for human papillomaviruses type 18 (HPV-18) fragment, chromosome karyotyping, pathological examination of xenografts, tissue-specific-90-gene expression signature and high-throughput RNA sequencing were used to determine the nature of MGc80-3. Our study found that the abnormal STR profile, partially overlapping with that of HeLa cells (64.62% to 71.64%), could not verify MGc80-3 as a HeLa cell line. However, the STR 13.3 repeat allele in the D13S317 locus that seemed to be unique to HeLa cells was detected in MGc80-3. Almost all the MGc80-3 cells exhibited HPV-18 fragments in the genome as well as certain HeLa marker chromosomes, such as M7 and M12. The molecular assay of the 90-gene expression signature still considered MGc80-3 as a stomach cancer using an algorithmic analysis. The expression pattern of multiple genes in MGc80-3 was quite different from that in HeLa cells, which showed that certain characteristics belonged to gastric cancer cell lines. High throughput RNA sequencing showed the distinct patterns of gene expression in MGc80-3. In conclusion, MGc80-3 cell line is a somatic hybrid with HeLa cells rather than a pure gastric cancer cell line.

Construction of gigantic highly-porous microcarriers as a three-dimensional cell culture platform

3D cell carriers with porous structures have the potential to promote cellular interactions and extracellular matrix remolding, making it a promising strategy to investigate the complex mechanisms behind drug treatments for tumors. Nevertheless, the applicability of currently reported porous microcarriers is still limited by the relatively small size and poor pore interconnectivity. Herein, a well-defined gigantic highly-porous poly(lactic-co-glycolic acid) (PLGA) microsphere (named LPMs) fabrication platform is reported. Utilizing this “LPMs” platform, arrays of well-uniform particles with sizes up to approximately 1000 µm are prepared in an orderly and controlled manner. The preparation matrix and crucial parameters affecting the particle size and pore size were established through extensive single-factor screening. It was also found that the loading of human gastric cancer cells MGC-803 to the LPMs cell carriers was facilitated not only by the increased pore size, but the particle size should also be enlarged along with the increased pore size. Through surface modification of gelatin on LPMs, cell loading could be dramatically enhanced, and microtissue-like structures were formed through cross-linking LPMs under extended 3D culture time. Under the guidance of these findings, we successfully constructed massive tumor cell carriers with 1000 µm LPMs, a preliminary prototype of a massive tumor tissue mimic. Together, the fabrication of these gigantic highly-porous microcarriers possibly allows for the high-efficiency 3D cell culture and represents the attractive potential of the construction of artificial tumor tissue models with massive and multiple-type cells.

High expression of death-associated protein 5 promotes glucose metabolism in gastric cancer cells and correlates with poor survival outcomes

To investigate the prognostic value of death-associated protein 5 (DAP5) in gastric cancer (GC) and its regulatory effect on aerobic glycolysis in GC cells. We analyzed DAP5 expression levels in GC and adjacent tissues and its association with survival outcomes of GC patients using public databases. We collected paired samples of GC and adjacent tissues from 102 patients undergoing radical resection of GC in our hospital from June, 2012 to July, 2017, and analyzed the correlation of DAP5 expression level detected immunohistochemically with the clinicopathological parameters of the patients. Cox regression analysis, Kaplan-Meier analysis, and ROC curves were used to explore the independent risk factors and the predictive value of DAP5 expression for 5-year survival of the patients. In the cell experiments, we observed the changes in aerobic glycolysis in MGC-803 cells following lentivirus-mediated DAP5 knockdown or overexpression by measuring glucose uptake and cellular lactate level and using qRT-PCR and Western blotting. Analysis using the public databases showed that DAP5 was highly expressed in GC and correlated with tumor progression and poor survival outcomes of the patients (P < 0.05). In the clinical samples, DAP5 expression was significantly higher in GC than in the adjacent tissues (3.19±0.60 vs 1.00±0.12; t=36.863, P < 0.01), and a high expression of DAP5 was associated with a reduced 5-year survival rate of the patients (17.6% vs 72.5%; χ2=29.921, P < 0.05). A high DAP5 expression, T3-4, N2-3, and CEA≥5 ng/mL were identified as independent risk factors affecting 5-year survival outcomes of GC (P < 0.05), for which DAP5 expression showed a prediction sensitivity, specificity and accuracy of 73.2%, 80.4% and 79.0%, respectively. In MGC-803 cells, DAP5 knockdown significantly reduced glucose uptake, lactate level and the expressions of GLUT1, HK2 and LDHA, and DAP5 overexpression produced the opposite effects (P < 0.05). A high expression of DAP5 in GC, which enhances cellular aerobic glycolysis to promote cancer progression, is correlated with a poor survival outcome and may serve as a biomarker for evaluating long-term prognosis of GC patients.

Silencing Rac1 and Prex1 Inhibit Epithelial-Mesenchymal Transition in Human Gastric Cancer Cells Induced by Transforming Growth Factor-β1.

Transforming growth factor-beta can influence tumor cells, causing epithelial-mesenchymal transition and enhancing their invasion and metastasis ability. Rac1 protein could be used as an independent tumor diagnostic marker and survival predictor. Prex1 is closely related to cell metastasis. In this study, the impact of silencing Rac1 and Prex1 on transforming growth factor-beta 1-induced epithelial-mesenchymal transition and apoptosis of human gastric cancer cells MGC-803 and MKN45 was investigated. MGC-803 and MKN45 cells received recombinant transforming growth factor-beta 1 (rTGF-β1) treatments at various concentrations. Cell Counting Kit-8 kit was used to determine cell viability. Rac1 and Prex1 interference vectors were transfected into the rTGF-β1-treated MGC-803 and MKN45 cells. Cell apoptosis and migration were detected by flow cytometry and scratch test, respectively. Western blot was used to detect the epithelial-mesenchymal transition-related markers E-cadherin, N-cadherin, vimentin, and PDLIM2 expression levels. The rTGF-β1 (10 ng/mL) could promote MGC-803 and MKN45 cell viability. Silencing Rac1 and Prex1 could increase E-cadherin and PDLIM2 expression, decrease N-cadherin and vimentin expression, inhibit cell viability and migration, and promote apoptosis in rTGF-β1-treated MGC-803 and MKN45 cells. Silencing Rac1 and Prex1 could inhibit epithelial-mesenchymal transition, reduce cell viability and migration, and promote apoptosis in human gastric cancer cells.

A network pharmacology approach to investigate dehydrocostus lactone inhibits the proliferation and epithelial-mesenchymal transition of human gastric cancer cells via regulating the PI3K/Akt and extracellular signal-regulated kinases/mitogen-activated protein kinase signalling pathways.

Dehydrocostus lactone (DHE), a sesquiterpene lactone, has been proven the significant inhibition of multiple cancer cells. However, there are limited reports on the activity of DHE in gastric cancer (GC). In this research, Network pharmacology predicted the anti-GC mechanism of DHE, and the prediction was verified by in-vitro experiments. Network pharmacology confirmed the major effect signalling pathway of DHE in treating GC. Cell viability assay, colony formation assay, wound healing assay, cell migration and invasion assay, apoptosis assay, western blot and real-time quantitative polymerase chain reaction verified the mechanism of DHE in GC cell lines. The results showed that DHE inhibited the growth and metastasis of MGC803 and AGS GC cells. Mechanistically, the analysis results indicated that DHE significantly induced the apoptosis process by suppressing the PI3K/protein kinase B (Akt) signalling pathway, and inhibited epithelial-mesenchymal transition by suppressing the extracellular signal-regulated kinases (ERK)/MAPK signalling pathway. The Akt activator (SC79) inhibited DHE induced apoptosis, and DHE had similar effects with the ERK inhibitor (FR180204). All results suggested that DHE was a potential natural chemotherapeutic drug in GC treatment.

Genome-wide characterization of extrachromosomal circular DNA in gastric cancer and its potential role in carcinogenesis and cancer progression

Extrachromosomal circular DNAs (eccDNAs) carrying random genomic segments are broadly found across different cancer types, but their molecular functions and impact in gastric cancer (GC) are rarely known. In this study, we aimed to investigate the potential role of eccDNA in GC. Using the Circle-seq strategy, we observed the eccDNA abundance in gastric cancer tissues (GCT) was aberrantly higher than that of normal adjacent tissues (NAT). The high abundance of eccDNAs carrying oncogene-segments in GCT may represent the DNA damage products of amplified oncogenes. Analysis of GCT over-represented eccDNA carrying enhancer (eccEnhancer) based on data from FANTOM5 project combined with TCGA database suggested the GC over-represented eccEnhancers may contribute to development of GC. GC over-represented eccDNAs carrying pre-miRNA (eccMIR) were enriched to multiple cancer-relevant signal pathways by KEGG analysis. We then synthesized the top six GC over-represented eccMIRs and found four of them enabled high expression of miRNAs and down-regulation of miRNA-target genes in MGC803 cells. Furthermore, we observed the inheritance of GC over-represented eccMIRs benefited host cell proliferation and promoted the aggressive features of host cells. Altogether, this study revealed the GC over-represented eccDNAs carrying functional genomic segments were related to the carcinogenesis of GC and presented the capability to facilitate cancer progression, suggesting the cancerous eccDNAs may serve as a dynamic reservoir for genome plasticity and rapid adaptive evolution of cancer. Therefore, blocking the pathways for eccDNAs generation may provide a novel therapeutic strategy for the treatment of gastric cancer.

Design, synthesis and biological evaluation of nitric oxide-releasing 5-cyano-6-phenyl-2, 4-disubstituted pyrimidine derivatives

In this study, a series of nitric oxide (NO) -releasing 5-cyano-6-phenyl-2, 4-disubstituted pyrimidine derivatives were designed and synthesized. In the in vitro biological evaluation, compound 24l exhibited optimal antiproliferative activity against MGC-803 cells with the IC50 value of 0.95 µM, significantly better than that of the positive control 5-FU. In addition, preliminary mechanistic studies indicated that 24l inhibited colony formation and blocked MGC-803 cells in the G0/G1 phase. DAPI staining, reactive oxygen species and apoptosis assays demonstrated that 24l induced apoptosis of MGC-803 cells. Particularly, the most potent compound 24l produced the highest level of NO, and the antiproliferative activity was significantly reduced after preincubation with NO scavengers. In conclusion, compound 24l may be considered as a potential candidate antitumor agent.

Anthracycline chemicals with anthracyclinone structure exert antitumor effects by inhibiting angiogenesis and lymphangiogenesis in a xenografted gastric tumor model.

It is vital to screen or develop alternative therapeutic drugs with higher curative characteristics and fewer side effects for the clinical treatment of gastric cancer. Gastric cancer cells were exposed to different auramycin G doses while determining the impact on cell viability, migration, and invasion. Then the antitumor effects of auramycin G, 5-fluorouracil (5-Fu) and their combination were evaluated. Furthermore, the molecular mechanisms of angiogenesis and lymphangiogenesis regulated by auramycin G and its analogs were investigated. Auramycin G inhibited cell viability in a dose-dependent manner, with a 50% inhibitory concentration of 23.72 ± 6.36mg/L and 32.54 ± 5.91mg/L for AGS and MGC803 cells, respectively. The migration and invasion of gastric cancer cells were significantly inhibited by 10mg/L auramycin G, which was consistent with the down-regulation of the VEGFR2-VEGFA-pPI3K-pAkt-pErk1 and VEGFR3-VEGFC-pPI3K-pAkt-pmTOR proteins. Notably, the average tumor weights were significantly reduced in both the auramycin G (2.21 ± 0.45g) of 50mg/kg body weight and auramycin G + 5-fluorouracil (5-Fu) groups (1.33 ± 0.28g), compared with the control (3.73 ± 0.56g). Considering that auramycin G decreased the growth of blood and lymphatic vessels while reducing the degree of tumor malignancy, it effectively suppressed tumors by regulating the angiogenic and lymphangiogenic pathways. The present study confirmed that auramycin G displayed a prominent antitumor activity in gastric tumor models, both in vitro and in vivo. Moreover, it was confirmed that auramycin G played a specific role in certain gastric cancer cell types, while the mechanism was validated to be associated with angiogenesis- and lymphangiogenesis-related pathway suppression.

Comparison of two kinds of Agrocybe cylindracea polysaccharides: Structural characteristic and antitumor activity

Agrocybe cylindracea is one of the common sources of functional polysaccharides. But the structure and function of Agrocybe cylindracea polysaccharides obtained by different preparation methods are very different. A comprehensive and detailed study of its fine structure and systematic activity is helpful for its development and utilization. In this study, a cold water-souble polysaccharide (ACP) and an alcohol-soluble polysaccharide (ACAP) from Agrocybe cylindracea were extracted. Their physicochemical property, structural feature and antitumor activity in vitro were compared and investigated. The results showed that ACP (2840.9 kDa) was mainly composed of 1,6-linked-Glcp, 1,3,6-linked-Glcp, 1,6-linked-Galp and T-Glcp and inhibited MGC-803 cells proliferation via inducing tumor cells apoptosis by the cytochrome c/caspases pathway. However, ACAP (1823.9 Da) just contained glucose. By comparison, ACAP exhibited no inhibitory effect on MGC-803 cells in vitro. This work would provide an additional explanation for the structure-activity relationship of polysaccharides from Agrocybe cylindracea and provide a theoretical support for its application in biomedicine industries.

Targeting Snail1 by CRISPR/Cas9 System Inhibits the Proliferation and Migration of Human Gastric Cancer Cells

The zinc-finger transcriptional repressor Snail1 affects cancer progression by controlling the epithelial cell-mesenchymal transition. The RNA-guided clustered regularly interspaced short palindromic (CRISPR) with a CRISPR-associated nuclease 9 (Cas9) nuclease system has been extensively used for gene editing. Here, we used two distinct sgRNAs to successfully target Snail1 in the gastric cancer cell line MGC803 with the CRISPR/Cas9 system. Furthermore, we discovered that Snail1 knockout reduced the proliferation and migration of MGC803 cells.

MiRNA-219a-1-3p inhibits the malignant progression of gastric cancer and is regulated by DNA methylation

Abstract Objective Worldwide, gastric cancer (GC) is one of primary reasons for cancer-related deaths. However, the pathogenic mechanism underlying GC remains to be fully understood. MicroRNAs are momentous regulators of diverse biological progression in cancer. Even though the ability of miR-219a-1-3p to inhibit malignant progression in pancreatic cancer have been previously reported, its role in GC remains to be elucidated. Methods Quantitative real-time PCR (qRT-PCR) was performed to measure miR-219a-1-3p expression levels in collected GC samples (n=98) and paired nearby non-tumor tissues. Cell proliferation, migration, and invasion assays were then conducted to explain the biological influences of miR-219a-1-3p in vitro. In vivo effects were confirmed by subcutaneously injecting miR-219a-1-3p overexpressing MGC-803 cells into nude mice. Methylation-specific PCR was employed to evaluate the CpG island upstream methylation condition of miR-219a-1-3p in collected clinical tissues (n=22), GC cell lines and GES-1 cells. GC cells were supplemented with 5-aza-2′-deoxycytidine to identify the miR-219a-1-3p expression changes using qRT-PCR. Results The miR-219a-1-3p expression was obviously suppressed in GC tissues relation to nearby non-tumor tissues, along with in GC cell lines in comparison to GES-1. Moreover, in vivo and in vitro functional evaluations indicated the function of miR-219a-1-3p in inhibiting the malignant characteristics of GC cells. Mechanistically, MiR-219a-1-3p expression was partly regulated utilizing DNA hypermethylation in GCs. In addition, overexpression of miR-219a-1-3p inhibited PI3K/AKT signaling. Conclusions MiR-219a-1-3p might function as a tumor suppressor in GC, and our investigation creates a foundation to diagnose of GC.

Aloin inhibits gastric cancer cell proliferation and migration by suppressing the STAT3/HMGB1 signaling pathway

To investigate the molecular mechanism underlying the inhibitory effect of aloin on the proliferation and migration of gastric cancer cells.Human gastric cancer MGC-803 cells treated with 100, 200 and 300 μg/mL aloin were examined for changes in cell viability, proliferation and migration abilities using CCK-8, EdU and Transwell assays. HMGB1 mRNA level in the cells was detected with RT-qPCR, and the protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 were determined using Western blotting. JASPAR database was used to predict the binding of STAT3 to HMGB1 promoter. In a BALB/c-Nu mouse model bearing subcutaneous MGC-803 cell xenograft, the effect of intraperitoneal injection of aloin (50 mg/kg) on tumor growth was observed. The protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 in the tumor tissue was examined using Western blotting, and tumor metastasis in the liver and lung tissues was detected using HE staining.Treatment with aloin concentration-dependently inhibited the viability of MGC-803 cells (P < 0.05), significantly reduced the number of EdU-positive cells (P < 0.01), and attenuated the migration ability of the cells (P < 0.01). Aloin treatment dose-dependently down-regulated HMGB1 mRNA expression (P < 0.01), lowered the protein expressions of HMGB1, cyclin B1, cyclin E1, MMP-2, MMP-9 and p-STAT3, and up-regulated E-cadherin expression in MGC-803 cells. Prediction based on JASPAR database suggested that STAT3 could bind to the promoter region of HMGB1. In the tumor-bearing mice, aloin treatment significantly reduced the tumor size and weight (P < 0.01), lowered the protein expressions of cyclin B1, cyclin E1, MMP-2, MMP-9, HMGB1 and p-STAT3 and increased the expression of E-cadherin in the tumor tissue (P < 0.01).Aloin attenuates the proliferation and migration of gastric cancer cells by inhibiting the STAT3/HMGB1 signaling pathway.

Discovery of Novel 1,3-Diphenylpyrazine Derivatives as Potent S-Phase Kinase-Associated Protein 2 (Skp2) Inhibitors for the Treatment of Cancer.

F-box protein S-phase kinase-associated protein 2 (Skp2) is a component of cullin-RING ligases, which is responsible for recruiting and ubiquitinating substrates and subsequently plays its proteolytic and non-proteolytic role. High expression of Skp2 is frequently observed in multiple aggressive tumor tissues and associated with poor prognosis. Several of the Skp2 inhibitors have been reported in the last decades; however, few of them have shown detailed structure-activity relationship (SAR) and potent bioactivity. Herein, based on the hit compound 11a from our in-house library, we optimize and synthesize a series of new 2,3-diphenylpyrazine-based inhibitors targeting the Skp2-Cks1 interaction and further systematically study the SAR. Among them, compound 14i shows potent activity against the Skp2-Cks1 interaction with an IC50 value of 2.8 μM and against PC-3 and MGC-803 cells with IC50 values of 4.8 and 7.0 μM, respectively. Most importantly, compound 14i exhibited effectively anticancer effects on PC-3 and MGC-803 xenograft mice models without obvious toxicity.

Acacetin exerts antitumor effects on gastric cancer by targeting EGFR.

Background: Gastric cancer (GC) is a common malignant tumor with a poor prognosis. Combination treatments may prolong the survival of patients with GC. Acacetin, which is a flavonoid, exerts potent inhibitory effects on several types of cancer cells; however, the mechanisms of action remain poorly understood. Methods: Network pharmacology and RNA sequencing were used to predict the targets of acacetin, which were then verified by drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA) and molecular docking. The biological functions of acacetin in MKN45 and MGC803 cells were investigated using TUNEL assays, crystal staining and colony formation assays. The pathways affected by acacetin were verified through reverse experiments. The in vivo antitumor efficacy of acacetin was assessed in a subcutaneous xenotransplanted tumor model. Results: In this study, we identified EGFR from more than a dozen predicted targets as a protein that directly binds to acacetin. Moreover, acacetin affected the level of phosphorylated EGFR. In vitro, acacetin promoted the apoptosis of GCcells. Importantly, EGFR agonists reversed the inhibitory effects of acacetin on the STAT3 and ERK pathways. In vivo, acacetin decreased the protein levels of pEGFR in tumors, resulting in increased GC xenograft tumor regression without obvious toxicity. Conclusion: Our findings highlight EGFR as one of the direct targets of acacetin in GCcells. Acacetin inhibited the phosphatase activity of EGFR in vitro and in vivo, which played a role in the antitumor effects of acacetin. These studies provide new evidence for the use of acacetin as a potential reagent for the treatment of GC.

Discovery of a novel Coumarin-Dihydroquinoxalone derivative MY-673 as a tubulin polymerization inhibitor capable of inhibiting the ERK pathway with potent anti-gastric cancer activities

As a class of microtubule targeting agents, colchicine binding site inhibitors (CBSIs) are considered as promising drug candidates for cancer therapy. However, due to adverse reactions, there are currently no CBSIs approved by FDA for cancer treatment. Therefore, extensive efforts are still encouraged to find novel CBSIs with different chemical structures and better anticancer efficacies. In this work, we designed and synthesized a new coumarin-dihydroquinoxalone derivative, MY-673, and evaluated its anticancer potency in vitro and in vivo. We confirmed that MY-673 was a potent CBSI that it not only inhibited tubulin polymerization, but also exhibited significant inhibitory potency on the growth of 13 cancer cells with IC50 values from 11.7 nM to 395.9 nM. Based on the results of kinase panel screening, MY-673 could inhibit ERK (extracellular regulated protein kinases) pathways-related kinases. We further confirmed that MY-673 could inhibit ERK signaling pathway in MGC-803 and HGC-27 cells, and then affected the expression level of SMAD4 protein in TGF-β (transforming growth factor β) /SMAD (small mother against decapentaplegic) signaling pathway using the western blotting assay. In addition, compound MY-673 could effectively inhibit cell proliferation, migration and induce cell apoptosis. We also further confirmed the in vivo efficacy of MY-673 in inhibiting tumor growth using the MGC-803 xenograft tumor model. At 20 mg/kg, the TGI rate was 85.9%, and it did not cause obvious toxicity to the main organs of mice. Together, the results we report here indicated that MY-673 was a promising CBSI for cancer treatment, which was capable of inhibiting the ERK pathway with potent antiproliferative activities in vitro and in vivo.

Bioactive compounds and mechanism of Xianglian pill in the treatment of gastric cancer: Network pharmacology analysis and experimental validation

Ethnopharmacological relevanceGastric cancer (GC) affects people's quality of life because of its high incidence rate and mortality. The Xianglian Pill (XLP) is a traditional Chinese medicine (TCM) prescription used to treat gastrointestinal (GI） diseases. Its anti-tumor effect has been found in recent years, but it's bioactive compounds and mechanism of action in treating GC are remain unknown. Aim of the studyThis study reveals the bioactive compounds and mechanisms of XLP in the treatment of GC through network pharmacology analysis and experimental verification. Materials and methodsThe main compounds in XLP were searched and the active compounds with anti-GC activity were selected. Compounds targets and GC- related targets were predicted, and common targets were obtained. Subsequently, a protein-protein interaction (PPI) network of common targets is constructed, while GO and KEGG enrichment analyses were performed on common targets. Finally, the anti-GC effects of active compounds in XLP were verified in GC cell lines MGC-803 and HGC-27 by wound healing assay, cell cycle assay, cell apoptosis assay and western blotting (WB) assay. ResultsA total of 33 active compounds of XLP were obtained. MTT assay showed that dehydrocostus lactone (DHL) and berberrubine (BRB) had lower IC50 value in GC cells HGC-27 and MGC-803, and has a less inhibitory effect on normal gastric epithelial cells. Further, 73 common targets were obtained after the total target of DHL and BRB intersected with GC. Among them, CASP3, AKT1, SRC, STAT3,and CASP9 were the most associated genes in the PPI network. GO and KEGG enrichment analyses indicated that apoptosis played a major role in the biological processes and signaling pathways involved. Moreover, the in vitro experiment revealed that DHL and BRB inhibited GC cell viability via inducing cell cycle arrest at G2/M phase, and promoting cell apoptosis by up-regulating the caspase3 expression and down-regulating the expression of Bcl2/Bax. ConclusionsDHL and BRB are the two main anti-GC active compounds in XLP, and their mechanism is mainly to inhibit cell cycle and promote cell apoptosis.

Discovery of novel tranylcypromine-based derivatives as LSD1 inhibitors for gastric cancer treatment.

As an important epigenetic regulator, histone lysine specific demethylase 1 (LSD1) has become an attractive target for the discovery of anticancer agents. In this work, a series of tranylcypromine-based derivatives were designed and synthesized. Among them, compound 12u exhibited the most potent inhibitory potency on LSD1 (IC50=25.3nM), and also displayed good antiproliferative effects on MGC-803, KYSE450 and HCT-116cells with IC50 values of 14.3, 22.8 and 16.3μM, respectively. Further studies revealed that compound 12u could directly act on LSD1 and inhibit LSD1 in MGC-803cells, thereby significantly increasing the expression levels of mono-/bi-methylation of H3K4 and H3K9. In addition, compound 12u could induce apoptosis and differentiation, inhibit migration and cell stemness in MGC-803cells. All these findings suggested that compound 12u was an active tranylcypromine-based derivative as a LSD1 inhibitor that inhibited gastric cancer.