Research On The Anti-metastasis Effects Of Celastrus Orbiculatus Extracts In Suppressing Cancer Stem-like Cell By Inhibiting DJ-1 In Human Esophageal Squamous Carcinoma

IntroductionEsophageal squamous cell carcinoma is one of the major diseases that seriously endanger the health of our people.In 2015,the number of new cases and deaths of esophageal cancer accounted for more than 10%of the total population.Cancer stem cells(CSCs)refers to a small amount cells in tumor possess the stem cell properties such as infinite proliferative potential and self-renew,and resulted in metastasis,recurrence,and resistance to chemotherapy and radiation therapy.Celastrus orbiculatus(Celastraceae),a folk medicine in China,has been used to treat many diseases,such as arthritis and other inflammatory diseases.In recent years,many studies have confirmed Celastrus orbiculatus ethyl acetate extract(COE)has showed significant antitumor activities.In the previous study,the COE was found to have effects of inhibiting the cell proliferation and inducing apoptosis and cell cycle arrest of ESCC cells.However,it is not clear whether the COE can inhibit the invasion and metastasis of esophageal squamous cell carcinoma,and there are few studies on whether the COE can inhibit tumor stem cells.In this study,we investigated the effects of COE on cell migration and invasion abilities,and established a model of CSCs using the SFM culture.Then the proteomics technique was used to explore the target proteins of COE on CSCs in ESCC.DJ-1 was one of the protein targets.Molecular cloning technology was used to construct the high expression and low expression cell model of the target protein.The significance and possible mechanism of DJ-1 in ESCC were further analyzed with clinical ESCC samples and in vivo and in vitro experiments.We investigated the effects of COE on ESCC stem like cell in vitro and tumor metastasis abilities in vivo and explored the underlying molecular mechanism.Therefore,the present findings suggest that COE might have potential therapeutic effects against ESCC.The findings will be described in four parts as follows.PART I:Celastrus orbiculatus extracts inhibit the invasion,migration of ESCC and proliferation of CSCs via DJ-1 in human esophageal squamous cell carcinoma.Objective:To observe the effects of COE on invasion,migration abilities and EMT process of human esophageal squamous cell carcinoma cells.To test the effects of COE on mRNA level of stem cell transcription factors.To find the potential targets of the COE on tumor stem cell model,and explore the mechanism of COE on inhibiting human esophageal squamous cell carcinoma metastasis.Methods:Human esophageal squamous cancer cell lines ECA-109 and TE-8 were cultured in vitro.Different concentrations(10,20,40,80,160,320 μg/mL)of the COE were added to detect the effect of proliferation on cells for 24 h,48h,72h by MTT method.20,40,80 μg/mL of COE were used in subsequent experiment.The effect of the COE on cell morphology was observed by an ordinary microscope.The transwell assay was performed to examine the abilities of cell invasion and migration with the COE treatments.The expressions of EMT-related proteins(E-cadherin,N-cadherin,Vimentin)in cells were detected by Western blot.RT-qPCR assay was used to detect the effect of COE on the mRNA expression levels of stem cell transcription factor SOX2,OCT4 and Nanog in cells.Serum free medium(SFM)suspension culture was applied to enrich the cancer stem like cell in human esophageal squamous carcinoma cells ECA-109,and expressions of CD90,CD133,stem cell factor SOX2,Nanog and OCT4 and tumorigenic ability of cells on nude mice were examined to identify the cancer stem cell properties.MTT assay wsa used to detect the effect of COE on the proliferation of stem cells.More importantly,proteomic technology was applied to search for the protein targets of COE on cancer stem-like cells in ESCC.Results:Different concentrations of the COE showed different degrees of inhibition of cell growth on ECA-109 and TE-8 cells in a concentration and time-dependent manner(P<0.05).The 24h IC50 of COE on ECA-109 and TE-8 cells were139.17±13.95 μg/mL and 153.74±10.72 μg/mL respectively.20,40,80 μg/mL of the COE and positive control drug 5-Fu 32 μg/mL were used in ECA-109 and TE-8 cells for 24 h in subsequent experiments.With the increase of concentration of COE,numbers of cells were decreased and cells morphology changed to apoptosis.Transwell assay results confirmed that COE could inhibit the migration and invasion of ECA-109 and TE-8 cells in a concentration-dependent manner(P<0.05).In addition,E-cadherin expression gradually increased and the expressions of N-cadherin and Vimentin gradually decreased with the increase of COE concentration(P<0.05),indicating that COE could inhibit the process of EMT.RT-qPCR results suggested that the COE could reduce mRNA levels of stem cell transcription factors SOX2, Nanog and OCT4.Human ESCC stem-like cells(ECA-109-S)were successfully enriched as the cells could form spheres in SFM culture.The expressions of CD90 and CD 133 on the surface of ECA-109-S cells at day 14 were higher than that in serum culture.RT-qPCR confirmed that the expression of stem cell transcription factor SOX2,Nanog and OCT4 in spheres cells were higher than that in serum culture(P<0.05).In vivo experiment confirmed that the lower orders of magnitude(2×104,2×103,2×102)of ECA-109-S cells also formed subcutaneous transplantation tumor in the back of nude mice,and tumors volume in 2×104 ECA-109-S group were significantly greater than the 2×106 wild ECA-109 group.The above results suggested that the tumor stem cell model was successfully established.The MTT assay results showed that COE could inhibit the proliferation(P<0.05).6 protein points were found in two-dimensional electrophoresis.Five proteins were successfully identified by mass spectrometry analysis.The five proteins were alpha-tubulin,Stomatin(EPB72)-like 2,Prohibitin1,Parkinson’s protein 7(PARK7,also named DJ-1),heat shock protein 27(HSP27)respectively.Conclusion:COE could inhibit proliferation,invasion,metastasis,EMT and stem cell transcription factors of ECA-109 and TE-8 cells.COE could inhibit the ECA-109-S proliferation and sphere formation.The potential protein targets of COE on ECA-109-S may be alpha-tubulin,Stomatin(EPB72)-like 2,DJ-1,prohibitinl and HSP27.PART Ⅱ:DJ-1 promotes cell proliferation and metastasis in esophageal squamous cell carcinoma through Wnt/β-catenin signaling pathwayObjectives:This study was designed to explore the role and mechanisms of DJ-1 in tumour cell proliferation and metastasis in ESCC and its underlying mechanisms.Materials and Methods:The expression levels of DJ-1,vimentin and E-cadherin in 84 human ESCC tissue specimens were measured via immunohistochemistry.An epithelial-mesenchymal transition(EMT)model was established by the addition of TGF-β1.DJ-1 overexpression and knockdown were implemented with a lentiviral vector,and a series of assays was performed in cell and animal models.XAV939 was used to block the Wnt/β-catenin signalling pathway.A 5-ethynyl-2’-deoxyuridine(EdU)staining assay and colony formation assay were performed to determine the cell proliferation.A transwell assay was used to assess the migration and invasion abilities of the cells.Protein expression in the cells were measured by Western blot.Furthermore,a nude mouse intraperitoneal xenograft model was used to observe the metastasis of tumour cells in vivo,and the expression of various proteins in tumour tissues was assessed by immunohistochemistry.Results:In 84 cases of ESCC specimens,the expressions of DJ-1 were closely associated with lymph node metastasis and distant metastasis,and expressions of Vimentin and E-cadherin were closely associated cell differentiation,tumor infiltration depth,lymph node metastasis and distant metastasis(P<0.05).The expression level of DJ-1 was positively correlated with Vimentin expression,(r=0.739,P<0.001),and negatively correlated with E-cadherin(r=-0.770,P<0.001).DJ-1 expression were increased obviously in EMT model induced by TGF-β1 compared with normal ECA-109 cells(P<0.05).ECA-109-LV-DJ-1 cells had higher proliferation,colony formation,migration,and invasion abilities than the control group ECA-109-LV-con,and the expression of E-cadherin was decreased while expression of N-cadherin and Vimentin increased in ECA-109-LV-DJ-1 cells(P<0.05).The expression level of LRP6,p-LRP6 and β-catenin in the Wnt/β-catenin signaling pathway was increased,and the expression level of Axin1 was decreased(P<0.05).However,after silencing the expression of DJ-1,cell proliferation,colony formation,migration,invasion abilities were inhibited(P<0.05).E-cadherin expression level increased,and the expression of N-cadherin and Vimentin decreased compared to the control group(P<0.05).The expression level of LRP6,p-LRP6 and β-catenin was decreased,while the expression level of Axinl was increased(P<0.05).Inhibitor of the Wnt/β-catenin pathway XAV939 could take the edge off cell proliferation,colony formation,migration,invasion abilities significantly compared to ECA-109-LV-DJ-1 cells(P<0.05).XAV939 decreased the expressions of N-cadherin,Vimentin,LRP6,p-LRP6 and β-catenin,and increased the E-cadherin and Axinl expression(P<0.05).In vivo experiment results showed ECA-109-LV-DJ-1 cells had a stronger metastasis ability while the ability weakened in ECA-109-LV-siRNA-DJ-1 cells(P<0.05).In addition,the expression of DJ-1,Vimentin and β-catenin increased,and E-cadherin decreased in metastatic tumor tissues formed by ECA-109-LV-DJ-1 cells(P<0.05).However,in LV-siRNA-DJ-1 group,DJ-1,Vimentin and p-catenin decreased,and E-cadherin increased(P<0.05).Conclusions:DJ-1 promoted proliferation,invasion,metastasis and the EMT in ESCC via activation of Wnt/β-catenin signal pathway.Our results suggested DJ-1 could represent a promising therapeutic target for the prevention and treatment of ESCC metastasis.PART III:DJ-1 is essential for maintaining cancer stem cell-like phenotype through mitochondrial function in esophageal squamous carcinomaObjective:To detect the role of DJ-1 in maintaining cancer stem cell-like phenotype in human esophageal squamous cell carcinoma cells,and search for its potential mechanism.Methods:The expression levels of DJ-1,SOX2 and Nanog in 84 cases of ESCC tissues were detected by immunohistochemistry.Their correlation with clinicopathological features and correlations of DJ-1 with SOX2 and Nanog were analyzed respectively.The cancer stem cell model was constructed by SFM culture and lentiviral vector and siRNA were used to construct DJ-1 high expression and low expression cell model.Sphere formation assay was used to detect abilities of cell sphere formation.RT-qPCR analysis was used to test the mRNA levels of SOX2,Nanog and OCT4.Mito-Tracker Red staining was applied to test mitochondria mass.Fluorescence observation and flow analysis were used for the determination of mitochondrial membrane potential.Transmission electron microscopy was applied to count the number of mitochondria in cells and observe morphology and structure of mitochondria.ATP detection kit was used to detect total ATP in the cell.Total content of ROS in cells were marked with DCFH-DA and analyzed by flow cytometry analysis.Protein levels in cells were detected by western blot.The model of subcutaneous transplantation of nude mice was established with ECA-109-LV-DJ-1,ECA-109-LV-siRNA-DJ-1 and the corresponding negative control cells.Tumor volume growth curves were drawn,and tumors weight were recorded.Immunohistochemical method was used to detect the expressions of DJ-1,Nanog and PGC-1α in subcutaneous transplantation tumor.Results:In 84 cases of human ESCC specimens,the expression of SOX2 closely associated with tumor infiltration depth,lymph node metastasis and distant metastasis,and Nanog expression and tumor cell differentiation,tumor infiltration depth,lymph node metastasis and distant metastasis(P<0.05).DJ-1 was positively correlated with the Nanog(r=0.685,P<0.001)and SOX2(r=0.620,P<0.001).The cells overexpressing DJ-1 has stronger stem cells properties,such as sphere formation ability,increased expression of cell surface marker CD90 and CD 133,stem cell transcription factor Nanog,OCT4 and SOX2,enhanced tumorigenic ability in vivo(P<0.05).However,above abilities and expressions were weakened or decreased after cutting down the expression of DJ-1 in cells(P<0.05).Cells in SFM culture had a higher mitochondria mass,higher mRNA expression of PGC-1α,more ATP production and higher mitochondrial membrane potential(P<0.05).And there was no obvious change in the total content of ROS in the sphered cells compared to wild ECA-109 cells.The mitochondria mass,levels of PGC-1α mRNA and ATP production and counts of mitochondria increased in ECA-109-LV-DJ-1 cells compared to ECA-109-LV-CON cells(.P<0.05).On the contrary,in ECA-109-LV-siRNA-DJ-1 cells,these mitochondrial related indicators decreased(P<0.05)significantly except the mitochondria number,but the mitochondria appeared obvious damage,which was characterized by the swelling of mitochondria,disordered arrangement of ridge.In ECA-109-siRNA-DJ-1 cells,the ROS level increased significantly(P<0.05).Conclusion:DJ-1 maintains cancer stem cell-like phenotype through mitochondrial function in esophageal squamous carcinoma.PART IV:Celastrus orbiculatus extracts inhibits cancer stem-like cell invasion and metastasis via DJ-1 mediated mitochondriopoiesis in esophageal squamous carcinoma.Objective:To observe the effect of the COE on the invasion metastasis of cancer stem-like cell in ESCC and explore its possible mechanism.Methods:The cells were divided into negative control group,ECA-109-S group and ECA-109-S plus 20,40,80 μg/mL COE and 5-Fu groups respectively.Transwell assay was conducted to detect cells invasion and migration abilities of each group.Western blot was used to detect the influence of the COE on the expression of EMT related proteins(E-cadherin,N-cadherin,Vimentin)in cells.Transmission electron microscopy was used to observe the mitochondrial structure and morphology and count the number of mitochondria in cells.Mito-track staining was applied to the analysis mitochondria mass.Flow analysis was used for the determination of mitochondrial membrane potential.Western blot was used to detect DJ-1 and mitochondrial related protein in each group cells.2×104 ECA-109-S cells were injected into the abdominal cavity of the nude mouse(n=25)and 2×106 wild ECA-109 cells were also injected as a cell control(n=5)to establish the abdominal metastatic tumor model.The 15th day after the injection,the nude mouse with ECA-109-S cells were randomly divided into control group(containing 1%DMSO saline solution),low dose of COE(10 mg/kg),medium dose of COE(20 mg/kg),high dose of COE group(40 mg/kg),Gimeracil and Oteracil Potassium Capsules(GOPC)group(10 mg/kg).Each group contained 5 nude mice.The nude mice were fed with the corresponding dose drugs once a day.The general conditions of the nude mice were observed daily during the experiment.At the end of the treatment,all nude mice were sacrificed,and the peritoneal metastasis nodules were observed and numbers of tumor were counted.Immunohistochemical method was applied to detect the expressions of DJ-1,E-cadherin,Nanog and PGC-1α in the tumor tissues.Results:Migration and invasion ability of ECA-109-S cells were obviously stronger than the ECA-109 cells,and COE could suppress ECA-109-S migration and invasion ability,in a concentration dependent manner(P<0.05).The expression of E-cadherin was decreased in ECA-109-S compared to ECA-109 while decreased with COE treatments,and N-cadherin and Vimentin expressions were opposite(P<0.05).Transmission electron microscopy results showed that 80μg/mL COE could reduce the number of mitochondria in ECA-109-S cells(P<0.05)and change the structure of the mitochondria.Mitochondria were swelling and further experiments showed that their intima were disappeared and the ridges were in disorder.Mito-Tracker Red staining showed that 80 μg/mL COE significantly reduced mitochondrial mass(P<0.05).Different concentrations of COE(20,40,80 μg/mL)could decrease the mitochondrial membrane potential of ECA-109-S,and reduce DJ-1 and mitochondria related proteins(PGC-1α,HSP60,VDAC)expressions(P<0.05).In vivo experiment results showed the activity and growth status of nude mice in each dose group were significantly better than cell control and negative control group.The incidence of tumor in the omentum,mesentery,abdominal wall,diaphragm and liver in drugs treated groups were significantly lower than that in the negative control group(P<0.05),while the incidence of tumor in the cell control group was higher than the negative control group(P<0.05).Immunohistochemical results showed that the COE could obviously increase the expression of E-cadherin and decrease expressions of DJ-1,Nanog and PGC-1α protein in transplanted tumor tissue.Conclusion:COE could inhibit the invasion and metastasis of cancer stem-like cell in ESCC,and its mechanism may be related to the regulation of DJ-1 mediated cancer stem cells mitochondriopoiesis by COE.