| BackgroundAs a common malignant tumor of the digestive system,the incidence of gastric cancer was relatively high among different cancers.Resina Garciniae,an ancient Chinese medicine contains a variety of bioactive xanthones.Among them,morellic acid（MA）has been considered to be a promising anti-gastric cancer drug candidate.Its anti-gastric cancer effects and related molecular mechanisms deserve further investigation.Long non-coding RNAs（lncRNAs）,as important regulatory factors in cells,are closely related to various diseases including tumors.Previous studies have shown that lncRNAs can act as caspase upstream signaling molecules to influence the programmed cell death process.Therefore,in this study,we innovatively attempted to explore the mechanism of anti-tumor effects of MA from the perspective of lncRNAs regulating caspase-mediated apoptosis and pyroptosis.ObjectiveThe purpose of this study was to observe the efficacy of MA on gastric cancer in vitro and in vivo and to investigate the molecular mechanism of the anti-gastric cancer effect of MA by induce caspase-3-dependent apoptosis and pyroptosis of gastric cancer cells through regulating lncRNA GAS5/miR-23a/Apaf-1 signaling pathway.Methods（1）MA extraction,isolation,purification and quantitative determinationThe isolation of MA from Resina Garciniae was performed by ultrasonic extraction with ethanol,semi-preparative and preparative chromatography;the content and stability of MA in different solvent（Me OH、DMSO、Et OH）were determined by high performance liquid chromatography.（2）Studies on anti-gastric cancer activity of MA and its induction of caspase-3-dependent apoptosis and pyroptosis in gastric cancer cells in vitro.MTT assay was used to observe the in vitro inhibitory effects of graded concentrations of MA treatment on different gastric cancer cells.The migration of gastric cancer cells was observed using scratch test.The apoptosis rate of gastric cancer cells was estimated by Annexin V-PI double-staining assay.The expression levels of the apoptosis-associated proteins:cleaved caspase-9,cleaved caspase-3,Bax and Bcl-2 in gastric cancer cells were determined by western blot.Lactate dehydrogenase（LDH）kit was used to detect the of LDH release rate of gastric cancer cells;The expression levels of GSDME N-terminal peptide（GSDME-NT）and IL-18 in gastric cancer cells were determined by western blot.（3）Studies on anti-gastric cancer activity of MA and its induction of caspase-3-dependent apoptosis and pyroptosis in gastric cancer cells in vivo.A mouse subcutaneous transplantation tumor model of gastric cancer was prepared using BLAB/c mouse and MFC cells,and the animals were divided into:Model group（Model）,MA low dose group（MA-L）,MA medium dose group（MA-M）,MA high dose group（MA-H）and Positive drug 5-fluorouracil group（5-FU）,Normal control group（Normal）was also set.The tumor volume,tumor weight,tumor inhibition rate and animal body weight change were selected as indexes to observe the effect of MA on the growth of subcutaneous transplanted tumors in mice;HE staining was used to detect the effect of MA on the pathological morphology of tumors in mice;Immunohistochemistry was used to test the Ki67 expression levels;The expression levels of the apoptosis associated proteins:cleaved caspase-9,cleaved caspase-3,Bcl-2 and Bax and the pyroptosis associated proteins:GSDME-NT and IL-18in the gastric cancer tissue were measured.（4）Studies on molecular mechanism MA promoting caspase-3-dependent apoptosis and pyroptosis in gastric cancer cells.The levels of lncRNA GAS5 in MA treated MKN-45 and MFC cells and in gastric cancer tissues of each group were measured by RT-qRCR.The siRNA was used to construct lncRNA GAS5 low expression cells,and the cells were divided into:non-target sequence siRNA control group（NC-siRNA group）,non-target sequence siRNA administration group（NC-siRNA+MA group）,lncRNA GAS5 low expression control group（GAS5-siRNA group）,and lncRNA GAS5 low expression administration group（GAS5-siRNA+MA group）.The apoptosis rate,pyroptosis morphology,and LDH release of lncRNA GAS5 low-expressing gastric cancer cells were detected after administration;The levels of lncRNA GAS5,miR-23a and Apaf-1 mRNA in lncRNA GAS5 and the expression of cleaved caspase-9,cleaved caspase-3,GSDME-NT,Apaf-1 and IL-18 in lncRNA GAS5 low-expressing gastric cancer cells were detected.The shRNA lentiviral packaging was used to construct a stable lncRNA GAS5 low-expressing cell line,and the obtained cell line was used to prepare a lncRNA GAS5 low-expressing gastric cancer animal model.The animals were divided into:non-target sequence shRNA control group（NC-shRNA group）,non-target sequence shRNA administration group（NC-shRNA+MA group）,lncRNA GAS5 low expression control group（GAS5-shRNA group）,lncRNA GAS5 low expression administration group（GAS5-shRNA+MA group）.The effect of MA on tumor growth in lncRNA GAS5 low-expressing gastric cancer animals were observed.The levels of lncRNA GAS5,miR-23a and Apaf-1 mRNA in lncRNA GAS5 and the expression of cleaved caspase-9,cleaved caspase-3,GSDME-NT,Apaf-1 and IL-18 in lncRNA GAS5 low expression gastric cancer tissues were detected.Results（1）Isolation and stability of MAThe MA was successfully separated and the purity was 98.3%by HPLC peak area normalization method;the stability of MA in DMSO solution within 45 d was satisfactory.（2）MA had strong anti-gastric cancer activity and induced caspase-3-dependent apoptosis and pyroptosis in MKN-45 and MFC cells in vitro.The IC50 values of MA on gastric cancer BGC-823,SGC-7901,HGC-27 MKN-45 and MFC cells were 6.216±0.23μM,3.859±0.11μM,4.518±0.17μM,2.964±0.25μM and1.81±0.16μM,respectively.Among them,the strongest activity was observed for MKN-45 and MFC cells.Meanwhile,MA（1μM,2μM and 4μM）was able to significantly inhibit the migration of MKN-45 and MFC cells（P<0.05 or P<0.01）.The apoptosis rates of gastric cancer cells in the groups treated with MA（1μM,2μM and4μM）were significantly promoted and the levels of cleaved caspase-9,cleaved caspase-3 and Bax were significantly increased,while the expression of Bcl-2 was remarkablely suppressed in gastric cancer cells by MA（1μM,2μM and 4μM）（P<0.05 or P<0.01）.The two kinds of gastric cancer cells in the MA-treated（1μM,2μM and 4μM）group showed a significant pyroptosis morphology.LDH release and expression of GSDME-NT as well as IL-18 was significantly upregulated in gastric cancer cells by MA（1μM,2μM and 4μM）（P<0.05 or P<0.01）.（3）MA had anti-gastric cancer activity and induced caspase-3-dependent apoptosis and pyroptosis in MKN-45 and MFC cells in vivo.In a subcutaneous transplantation tumor model of gastric cancer in mice,both MA（1,2and 4mg/kg）and 5-FU（100mg/kg）showed significant inhibition of tumor growth in mice.Day 15 of the treatment process,the inhibition rates of tumor growth of animals in MA（1,2 and 4mg/kg）and 5-FU（100mg/kg）group were 79.32±7.69%,92.36±4.21%,92.18±2.99%and 87.64±6.30%,respectively.In the study of the effect of MA on the pathological morphology of tumor tissue in gastric cancer model mice,it was found that the nuclei of tumor cells were intact and closely arranged in the gastric cancer tissues of the model group,while apoptotic and necrotic cells were scattered in the gastric cancer tissues of the MA（1,2 and 4mg/kg）and 5-FU（100mg/kg）group,with gaps between the cells and nuclear consolidation and nuclear fragmentation in the nuclei of the cells.The expression of Ki67 protein in the tumor tissues was significantly downregulated and the expression of cleaved caspase-9,cleaved caspase-3,Bax GSDME-NT and IL-18 were significantly upregulated,while the expression of Bcl-2 was significantly downregulated by MA（1,2 and 4mg/kg）and 5-FU（100mg/kg）（P<0.05 or P<0.01）.（4）MA promoted caspase-3-dependent apoptosis and pyroptosis in gastric cancer cells via lncRNA GAS5/miR-23a/Apaf-1 pathway.In vitro,the levels of lncRNA GAS5 were significantly increased in MKN-45 cells and MFC cells treated MA（2μM）（P<0.05 or P<0.01）.In the mouse subcutaneous transplantation tumor model,lncRNA GAS5 levels were significantly increased in the tumor tissues of both MA（2μM）and positive drug（P<0.05 or P<0.01）.Gastric cancer cells in MA（2μM）treatment group showed significantly higher apoptosis rate,significant pyroptosis morphology and increased release of LDH.After knocking down the level of lncRNA GAS5,the effect of MA（2μM）induced apoptosis,pyroptosis and LDH release in gastric cancer cells was partially reversed（P<0.05 or P<0.01）.The levels of lncRNA GAS5 and Apaf-1 mRNA were significantly upregulated and miR-23a levels were significantly downregulated in gastric cancer cells in the MA（2μM）treatment group.The upregulation of lncRNA GAS5 and Apaf-1 mRNA and downregulation of miR-23a by MA（2μM）was partially reversed after knocking down the level of lncRNA GAS5（P<0.05 or P<0.01）.The expression of cleaved caspase-3,cleaved caspase-9,GSDME-NT,Apaf-1 and IL-18was significantly upregulated in MA（2μM）treated gastric cancer cells group,and the upregulation of these proteins by MA（2μM）was partially reversed by knocking down the level of lncRNA GAS5（P<0.05 or P<0.01）.In the lncRNA GAS5 low expression gastric cancer animal model,the tumor volume and weight of MA（2mg/kg）treated animals were significantly reduced,and the inhibitory effect of MA（2mg/kg）on tumor growth was partially reversed after knocking down the lncRNA GAS5 level（P<0.05 or P<0.01）.The levels of lncRNA GAS5 and Apaf-1 mRNA were significantly upregulated and miR-23a levels were significantly downregulated in gastric cancer tissues of MA（2mg/kg）treatment group.The upregulation of lncRNA GAS5 and Apaf-1 mRNA and downregulation of miR-23a by MA（2mg/kg）was partially reversed after knocking down the level of lncRNA GAS5（P<0.05 or P<0.01）.The expression of cleaved caspase-3,cleaved caspase-9,GSDME-NT,Apaf-1 and IL-18was significantly upregulated in gastric cancer tissues of the MA（2mg/kg）treatment group,and the upregulation of these proteins by MA（2mg/kg）was partially reversed after knocking down the level of lncRNA GAS5（P<0.05 or P<0.01）.ConclusionMA has anti-gastric cancer effects in vitro and in vivo,and its mechanism of action may be related to its upregulation of lncRNA GAS5 levels in gastric cancer MKN-45 and MFC cells,enhancing targeted adsorption of miR-23a,antagonizing the inhibitory effect of miR-23a on Apaf-1,which in turn activates caspase-9 and caspase-3,and promoting apoptosis and pyroptosis in gastric cancer MKN-45 and MFC cells. |
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