Study On The Effect And Mechanism Of Total Extract Of Celastrus Orbiculatus On The Invasion And Metastasis Of Gastric Cancer By Regulating Cytoskeleton Remodeling

IntroductionGrastric cancer is one of the most common digestive tract cancers in China.Its incidence rate and mortality rate are second.In some rural and underdeveloped areas,gastric cancer has even surpassed lung cancer,becoming the number one cancer that threatens people’s health.In some area with low prevalence of early screening for gastric cancer,it is not easy to be diagnosed early because of the lack of obvious symptoms of gastric cancer.Most of the patients were in the late stage when they came to the hospital,and the five-year survival period after treatment was not high.The recurrence and metastasis of gastric cancer greatly restrict the survival of patients with gastric cancer.The invasion and metastasis of gastric cancer cells need to undergo a series of biological characteristics changes,including epithelial stromal transformation,pseudopodia formation,invasion and metastasis related factors and enzyme secretion.However,tumor cytoskeleton remodeling plays an important role in the process of inducing tumor cells to acquire the ability of invasion and metastasis.The reconstruction of tumor cytoskeleton is that in the process of tumor cell movement,the fibrous microfilaments and spherical microfilaments in the cytoskeleton are disassembled and assembled again to form a new cytoskeleton structure,and its morphology changes(including pseudopodia formation),making tumor cells easy to migrate.Tumor cells transform from epithelial cells to stromal cells by remolding their skeletons.At the same time,they remodel the skeletons on the edge of cells,forming the structures of lamellar pseudopods and filiform pseudopods with polarity.Under the action of cell contraction force,the protrusion is repeatedly extended forward,and the cell body is continuously circulated forward to complete cell invasion and metastasis.It can be seen that the cytoskeleton remodeling of gastric cancer is involved in many links of invasion and metastasis of gastric cancer.The rate of cytoskeleton remodeling of gastric cancer directly affects the migration ability of tumor cells,which is the structural basis and precondition of invasion and metastasis of gastric cancer.Celastrus orbiculatus is a plant belonging to Celastrus orbiculatus Thunb.It has a good medicinal property.Its roots,stems,leaves,fruits and seeds can be used as medicine.Many components isolated from the root skin,stem,leaf and seed of Celastrus orbiculatus have antitumor,anti-inflammatory and antiviral activities.Our group has long been engaged in the pharmacodynamics research of ethyl acetate extract of Celastrus orbiculatus(Celastrus orbiculatus extract,COE)Ethyl acetate extract of Celastrus orbiculatus on anti-tumor(Patent No.:200710025343.3).Previous studies have found that the drug can inhibit the invasion and metastasis of tumor cells and EMT process,but the specific mechanism of cytoskeleton remodeling in COE mediated inhibition of gastric cancer invasion and metastasis is still unclear.Therefore,based on the previous studies,the mechanism of COE inhibiting the invasion and metastasis of gastric cancer through CFL1 was studied at the molecular level.At the same time,it is very important to establish the orthotopic transplanted tumor animal model which is highly suitable for the clinical gastric cancer environment,and to further explore the effect and mechanism of COE on the invasion and metastasis of gastric cancer in vivoThis study includes the following three parts.PART I Role of CFL1 in cytoskeleton remodeling of human gastric cancer cells and its regulatory mechanismObjective:To study the biological structural basis of invasion and metastasis of gastric cancer cells-the regulatory mechanism of cytoskeleton remodeling;to explore the key regulatory role of CFL1 in the cytoskeleton remodeling of gastric cancer cells;to clarify the key regulatory link of CFL1 in the cytoskeleton remodeling of gastric cancer cells,and to clarify the regulatory mechanism of CFL1 involved in the cytoskeleton remodeling of gastric cancer cells,so as to find a biological target for the development of drugs against invasion and metastasis of gastric cancer.Methods:Gastric cancer cells BGC-823 and AGS were cultured.TGF-β 15ng/mL was given to induce the phenotype changes of gastric cancer cells,and the changes of cytoskeleton were observed.At the same time,we constructed CFL1 low expression and over expression lentivirus vectors and transfected them into BGC-823 and AGS cell lines.Western blot and PCR were used to verify the expression and interference of lentivirus.Gastric cancer cells were divided into wild type group,empty vectors control group and low expression CFL1 group。The effects of CFL1 on the proliferation and cell viability of gastric cancer cells were detected by MTT method.The expression of CFL1 and the relationship between CFL1 and the activity of cytoskeleton remodeling were observed by cytoskeleton staining combined with immunofluorescence staining.After inducing gastric cancer cells with TGF-β 15ng/mL for 24 hours,the cell skeleton was stained with TRITC-Phalloidin.The changes of microtubules and microfilaments in the cytoskeleton with different degrees of CFL1 expression were observed by laser confocal microscopy,and the depolymerization and aggregation of actin in the cells were observed.The F-actin and G-actin proteins were extracted from the cytoskeleton of gastric cancer cells with different degrees of CFL1 expression and detected by Western blot.Transmission electron microscopy was used to detect and observe the ultrastructural changes of different cytoskeletons expressed in CFL1,such as the distribution and changes of F-actin and G-actin.Field emission scanning electron microscopy(FESEM)was used to detect and observe the ultrastructural changes of different CFL1 expression cells,including the changes of lamellar pseudopodia and filiform pseudopodia.Results:TGF-β can induce the depolymerization and rearrangement of microfilaments and microtubules in gastric cancer cells.The morphological changes of gastric cancer cells induced by TGF-β were significant.The cells grew from aggregation to dispersion,and the shape of cells changed from irregular quadrilateral to fusiform.There are long antennae around the cell.The formation of filamentous and lamellar pseudopods proves that the cytoskeleton has been remolded after TGF-β induction.The expression of CFL1 was significantly inhibited and enhanced in lentiviral Lv-siRNA-CFL1 and Lv-CFL1 cells,both at protein level and mRNA level.MTT results showed that there was no significant difference in the proliferation of gastric cancer cells whether the expression of CFL1 was decreased or increased.Combined immunofluorescence and cytoskeleton staining can clearly see that the expression of CFL1 in the cytosol of cytoskeleton remodeling induced by TGF-β is significantly enhanced,and the increase of CFL1 expression level is accompanied by the formation of lamellar pseudopodia and filiform pseudopodia.It was found that TGF-βcould significantly promote the activity of actin in the cells.In the negative control group,the microfilaments were evenly distributed,orderly arranged,and the lamellar pseudopods were relatively complete.In Lv-siRNA-CFL1 group,actin distribution became extremely uneven,the color in the inclusion was relatively light,and the color in the cell membrane was relatively deep,which indicated that actin in the cytoskeleton was enriched from the cytoplasm to the cell membrane.Western blot showed that after TGF-β induction,the expression of F-actin and G-actin in Lv-NC group increased,but the expression of F-actin in Lv-siRNA-CFL1 group was significantly higher than that in Lv-NC group,and the expression of G-actin was significantly reduced.The results of transmission electron microscopy showed that the number of microfilaments in cytoplasm and membrane of TGF-β treated group was more than that of Lv-NC treated group.Under the same induction conditions,the number of microfilaments in Lv-siRNA-CFL1 group also increased,but most of the microfilaments were concentrated on the cell membrane,and F-actin was polymerized into F-actin through G-actin,which increased significantly.The results of field emission scanning electron microscopy also showed that the filamentous pseudopodia of CFL1 decreased and the area of lamellar pseudopodia increased,but the number decreased.Conclusion:TGF-β can induce the cytoskeleton remodeling of gastric cancer cells;low and over expression of CFL1 has no significant effect on the proliferation of gastric cancer cells;CFL1 participates in the cytoskeleton remodeling of gastric cancer cells;low expression of CFL1 can inhibit the remodeling rate of gastric cancer cells and reduce the formation of filiform pseudopods of gastric cancer cells;CFL1 is one of the key regulatory factors of cytoskeleton remodeling.PART Ⅱ The role and mechanism of cytoskeleton remodeling regulated by CFL1 in the EMT,invasion and metastasis of gastric cancer cellsObjective:To study the role of CFL1 mediated cytoskeleton remodeling in EMT,invasion,metastasis and adhesion of gastric cancer cells.To elucidate the molecular mechanism of cytoskeleton remodeling involved in EMT and invasion and metastasis of gastric cancer cells.To explore the mechanism of how cytoskeleton remodeling affects cell adhesion,to clarify the mechanism of cytoskeleton remodeling involved in cell movement structure,and to lay the experimental foundation for the search and development of anti gastric cancer metastasis drugs based on CFL1.Methods:different gastric cancer cell lines and human normal gastric mucosa cells were cultured.Total protein and total RNA were extracted from each group.Western blot and PCR were used to detect the protein content and mRNA expression of CFL1 in different gastric cancer cell lines.The protein and RNA of normal gastric mucosa and gastric cancer were extracted and the expression of CFL1 was detected by western blot.CFL1 mRNA was detected by RT-PCR.BGC-823 and AGS cells were cultured and TGF-β 15ng/ml was given to induce EMT of gastric cancer cells,then the changes of cytomorphology and cytoskeleton were observed.Western blot was used to detect the expression of EMT related markers and CFL1 in this EMT process.Cytoskeleton staining combined with immunofluorescence staining was used to observe the expression of CFL1 in the cytoskeleton remodeling cells induced by TGF-β,and the correlation between CFL1 and cytoskeleton remodeling activity.Western blot was used to detect the expression of EMT related proteins in Lv-siRNA-CFLl cells and Lv-NC cells under the same induction conditions.Transwell cell experiment was used to detect the invasion and metastasis ability of Lv-siRNA-CFL1 cells and Lv-NC cells The peritoneal xenograft model of nude mice was used to detect the metastasis ability of lv-sirna-cfll cells and lv-nc cells in the abdominal cavity of nude mice.Mesenteric anatomy was used to visually observe the metastasis of different groups of cells in nude mice.The cell orthotopic tumor model was used to detect the true implantation and growth of Lv-siRNA-CFL1 cells and Lv-NC cells in the gastric wall of nude miceResults:Western blot and PCR results showed that compared with the GES group of normal gastric mucosa,the expression of CFL1 protein and mRNA in the gastric cancer group were significantly increased.At the same time,we detected 66 pairs of clinical samples of gastric cancer and found that 51 pairs of CFL1 protein in 66 pairs were significantly highly expressed in gastric cancer tissues(77.27%),and 52 pairs of CFL1 mRNA were highly expressed in gastric cancer tissues(78.78%).After being induced by TGF-β 15ng/mL for 24 hours,the growth morphology of gastric cancer cells changed significantly.The cells changed from irregular quadrilateral to spindle shape,the tentacles were extended at the cell edge,and the EMT-related proteins of the cells changed significantly.The expression of E-cadherin decreased(P<0.001),and the expression of N-cadherin,Vimentin and CFL1 increased(P<0.001).Combined immunofluorescence and cytoskeleton staining showed that the expression of CFL1 in the cytoplasm of the cells was significantly enhanced after TGF-βinduction.The microtubule structure of EMT cells was observed by laser confocal microscopy,and it was found that lamellar pseudopodia and filiform pseudopodia were extended around EMT cells.Western blot results showed that the expression of EMT-related proteins in Lv-siRNA-CFL1 cells and Lv-NC cells significantly changed under the same induction condition.The expression of E-cadherin in Lv-siRNA-CFLl group increased(P<0.01),while the expression of N-cadherin,Vimentin and Snail protein decreased(P<0.01).Transwell experiment showed that compared with Lv-NC group,the number of cells penetrating the membrane in Lv-siRNA-CFLl group was significantly reduced.The animal living imaging showed that the fluorescence number of cells in Lv-siRNA-CFL1 group was significantly smaller than that in Lv-NC group In vzivo.The number of tumor formation in peritoneum of Lv-siRNA-CFL1 group was significantly less than that of Lv-NC group.In situ cell transplantation experiment,the tumor volume of Lv-siRNA-CFL1 group was much smaller than that of Lv-NC groupConclusion:The protein and mRNA of CFL1 are highly expressed in gastric cancer cell lines,and most of CFL1 are highly expressed in clinical samples of gastric cancer.The high expression rate is 77.27%,and the high expression rate of mRNA is 78.78%.After TGF-βinduced EMT in gastric cancer,CFL1 was highly expressed.CFL1 was involved in the EMT process of gastric cancer.During the EMT process,CFL1 was highly expressed and the cytoskeleton changed dramatically.Knocking down CFL1 can inhibit the EMT process of gastric cancer cells and inhibit the invasion and metastasis of gastric cancer cells.In vivo experiments showed that knockdown of CFL1 could inhibit the peritoneal metastasis of gastric cancer cells and the growth of orthotopic transplanted tumor.PART Ⅲ The role and mechanism of CFL1 mediated cytoskeleton remodeling in the inhibition of human gastric cancer invasion and metastasis by COE.Objective:To explore the molecular mechanism of COE in inhibiting the cytoskeleton remodeling of gastric cancer through CFL1,and to clarify the molecular mechanism of COE in inhibiting the invasion and metastasis of gastric cancer through CFL1 mediated cytoskeleton remodeling.The purpose of this study is to establish the experimental basis for the development of anti-gastric cancer Chinese medicine targeting CFL1.Methods:MTT method was used to detect the activity of COE cells after 24 hours.Flow cytometry was used to detect the effect of COE on the proliferation and apoptosis of gastric cancer cells.Using JC-1 as a fluorescent probe,the mitochondrial membrane potential of gastric cancer cells after COE treatment was detected with the mitochondrial membrane potential detection kit.Apoptosis related proteins and proliferation related proteins were detected by western blot.Transmission electron microscopy was used to detect the effect of COE on the internal structure and microtubule structure of gastric cancer cells,including the changes of nuclear,mitochondrial and other subcellular structures.Western blot and PCR were used to detect the effect of COE on CFL1 protein and mRNA.At the same time,MG 132 proteasome inhibitor was used to observe the effect of COE on the degradation of CFL1 protein.The morphological changes of gastric cancer cells and the changes of microtubules were observed by laser confocal microscopy.Transwell test was used to detect the effect of COE on the invasion and metastasis of gastric cancer cells.Western blot and PCR were used to detect the expression of proteins and mRNA related to invasion and metastasis(MMP-2,MMP-9,TIMP-1 and TIMP-3)of gastric cancer cells by COE.Gelatinase assay was used to detect the effect of COE on the activities of MMP-2 and MMP-9.Western blot was used to detect the changes of invasion and metastasis related proteins(MMP-2 and MMP-9)and EMT related proteins(N-cadherin,vimentin and E-cadherin).Cytoskeleton staining and FAK immunostaining were used to detect the effect of COE on motility adhesion factors of gastric cancer cells.The subcutaneous tumor model of nude mice was used to observe the effect of COE on gastric cancer.TEM was used to observe the ultrastructural changes in the cytoskeleton of gastric cancer cells,such as the distribution and changes of microtubules and microfilaments.Results:MTT showed that the activity of gastric cancer cells treated with COE decreased significantly(P<0.05).The results of flow cytometry showed that the proliferation of gastric cancer cells decreased significantly after COE treatment(P<0.01).Annexin V-FITC staining showed that the apoptosis of gastric cancer cells increased after COE treatment(P<0.01)The mitochondrial membrane potential test showed that the J-aggregates decreased,and the monomer increased significantly after COE treatment.Western blot showed that the protein of PI3K,Akt,mTOR,P70S6K,Bcl-2 and Bcl XL decreased significantly(P<0.05),and the expression of Caspase-3 and Bax increased significantly(P<0.05).The results of transmission electron microscopy showed that after COE was applied to gastric cancer cells,the cell volume became smaller,the nucleus was smaller,and the chromosome was broken.Vacuoles appear in the cytoplasm.The mitochondrial structure of COE treated cells was destroyed and the cristae of mitochondria were not clear.After COE treatment of gastric cancer cells,the number of microfilaments around the cells decreased or disappeared,and the cytoskeleton partially dissolved or broken.Transwell experiment showed that the number of cells passing through the membrane of gastric cancer cells treated with COE was significantly reduced(P<0.05).After COE was applied to Lv-NC and Lv-siRNA-CFLl cells,the number of transmembrane cells in Lv-siRNA-CFL1 group were decreased significantly(P<0.001).Western blot results showed that the expression levels of TIMP-1 and TIMP-3 proteins were significantly increased in gastric cancer cells after COE treatment compared with the negative control group(P<0.05).The expression of MMP-2 and MMP-9 decreased significantly(P<0.05).The results of PCR showed that the mRNA of MMP-2 and MMP-9 increased slightly,but there was no significant difference(P>0.05),while the mRNA of TIMP-1 and TIMP-3 increased significantly(P<0.05).The results of gelatinase showed that COE could significantly inhibit the activities of MMP-2 and MMP-9 in gastric cancer cells.Western blot showed that the expression of CFL1,N-cadherin,vimentin,MMP-2 and MMP-9 protein decreased with the increase of COE concentration(P<0.05),while the expression of E-cadherin protein increased(P<0.05).The results of cytoskeleton staining and FAK immunostaining showed that the expression of FAK on the surface of gastric cancer cells induced by TGF-β was strong,and the expression of FAK on the surface of gastric cancer cells treated by COE was significantly reduced.Subcutaneous tumor transplantation in nude mice showed that the tumor volume of subcutaneous tumor transplantation in nude mice significantly decreased after COE gavage(P<0.05).The results of TEM showed that the cytoskeleton microfilaments in the negative control group were mainly distributed in the cytoplasm and cell membrane.The cytoskeleton microfilaments of TGF-β group were mainly distributed in the cell membrane.After COE treatment,the cytoskeleton microfilaments were mainly distributed in the cytoplasm,and a small number of microfilaments were distributed on the cell membrane.Conclusion:COE can obviously inhibit the proliferation of gastric cancer cells and promote the apoptosis of gastric cancer cells.The main mechanism may be the destruction of mitochondrial skeleton structure and the change of mitochondrial membrane potential to promote apoptosis.COE can significantly inhibit the expression of CFL1 in gastric cancer cells,which may be achieved by promoting the degradation of CFL1.COE can significantly inhibit the EMT and invasion and metastasis of gastric cancer cells,which may be achieved by inhibiting the cytoskeleton remodeling mediated by CFL1,so as to inhibit the invasion and metastasis of gastric cancer cells.