Research On The Efficacy And Mechanism Of Celastrus Orbiculatus Targeting Tumor-associated Macrophages In Glioblastoma

IntroductionGlioblastoma multiforme(GBM)is one of the most aggressive and malignant brain tumors in humans.Gliomas account for 25%of the primary adult central nervous system tumors.Substantial intermingling of tumor cells with normal brain tissue creates a significant challenge in treating GBM.Surgery cannot completely remove the large number of tumor cells produced by GBM.Also,genetic heterogeneity leads to aberrant activation of multiple signaling pathways in the tumor microenvironment.These reasons lead to very poor prognosis and survival rates.Most therapeutic approaches targeting glioma cells have failed.An alternative strategy is to target the tumor microenvironment(TME).The development of a drug targeting TME is greatly significant in prolonging the survival of patients with glioblastoma and improving the prognoses.Celastrus orbiculatus Thunb.a traditional Chinese medicinal herb of the Genus Celastraceae Celastrus,is a deciduous wood plant and widely distributed in its native environment.The stalk,roots,leaves and fruits have been used as folk medicines against cancer diseases.A variety of ingredients have been separated from it's stalk,root,leaves and fruits by modern technology,such as terpene,triterpene,flavonoids,organic acids,steroids,tannin,etc.The effects of the Celastrus orbiculatus ethylacetate extract(COE),including anti-tumor,anti-inflammatory,antiviral,antibacterial and antioxidant,have been widely confirmed.However,there is currently no report on COE to treat glioma.In this study,we investigated the effects of COE on inhibiting the migration and invasion of human glioblastoma cells and the underlying molecular mechanism.We also examined the effect of COE on inhibiting proliferation,inducing apoptosis of glioblastoma in vitro,and its possible molecular mechanism.The role of CSF-1/IL-34/CSF-1R axis in the tumor microenvironment was researched in a U251 cell co-culture with M2-like macrophage cell model.The molecular mechanism of indirect inhibition of glioblastoma cell proliferation and induced apoptosis through the tumor-associated macrophages(TAMs)was expounded.The results indicate that COE affects both the glioblastoma cells and the TAMs in the tumor microenvironment.This will provide new therapeutic strategies and a theoretical basis for developing new multi-target glioma drugs to target the tumor microenvironment.This study will be described in three parts,as follows.Part I:Celastrus orbiculatus extract inhibits the migration and invasion of human glioblastoma cell lines U87 and U251 in vitroObjective:Gliomas are highly aggressive tumors of the nervous system,and current treatments fail to improve patient survival.To identify substances that can be used as treatments for gliomas,we examined the effect of Celastrus orbiculatus extract(COE)on the invasion and migration of human glioblastoma U87 and U251 cells in vitro.Methods:The effects of COE on cell viability and adhesion were tested using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and cell adhesion assay,respectively.The effects of COE on cell migration and invasion were assessed by a wound-healing assay and transwell migration and invasion assays.The effects of COE on the expression of epithelial-mesenchymal transition(EMT)-related proteins and matrix metalloproteinases(MMPs)were evaluated using western blot and gelatin zymography,respectively.Finally,the effect of COE on actin assembly was observed using phalloidin-tetramethylrhodamine isothiocyanate labeling and confocal laser scanning microscopy.Results:Compared to that of the control group,the proliferation of U87 and U251 cells in COE-treated group were significant inhibited by COE in a dose and time dependent.We found that COE inhibited the adhesion,migration,and invasion of U87 and U251 cells in a dose-dependent manner.COE reduced N-cadherin and vimentin expression,increased E-cadherin expression,and reduced MMP-2 and MMP-9 expression in U87 and U251 cells.Furthermore,COE inhibited actin assembly in U87 and U251 cells.Conclusions:COE attenuates EMT,MMP expression,and actin assembly in human glioblastoma cells,thereby inhibiting their adhesion,migration,and invasion in vitro.These findings suggest that COE could be used as a treatment for glioblastoma.Part ?:Effect of Celastrus orbiculatus Extract on Cell Proliferation and Apoptosis of the human Glioblastoma U251 cell in vitroObjective:We aimed to observe the effect on different concentrations of Celastrus orbiculates extract(COE)on the proliferation and apoptosis on U251 cells,(a glioblastoma cancer cell line),as well as explore the possible molecular mechanism.Methods:U251 cells in logarithmic growth phase were divided into control group and COE-treated group.MTT assay and BrdU incorporation experiment were used to observe the inhibitory effect on cell viability and cell proliferation,respectively.Annexin V/PI double staining was used to detect the effect of COE on U251 cell apoptosis.Ultrastructural changes of U251 cells after COE treatment were observed under transmission electron microscope,and the expression of apoptosis marker proteins Bcl-2/Bax and Caspase-3 were detected by western blot.Results:Compared to that of the control group,the proliferation of U251 cells in COE-treated group were significant inhibited by COE(P<0.05).COE induced the early apoptosis and ultrastructural changes of U251 cells.Apoptotic bodies were also observed under transmission electron microscope.The expression of B-cell lymphoma-2/Bcl-2 Associated X Protein(Bcl-2/Bax)and Caspase-3 were changes depending on the concentration of COE.Conclusion:COE can inhibit the proliferation of U251 cells by down-regulating the expression of Bcl-2/Bax and increasing the expression of Caspase-3 protein activity,thereby inducing cell apoptosis.This suggests that COE can be used as a drug candidate for glioma treatment.Part III:Effect of Celastrus orbiculatus extract on Proliferation and Apoptosis of Glioblastoma by CSF-1/IL-34/CSF-1R axis which express on tumor-associated macrophages.Objective:To investigate the expression and clinical significance of CSF-1/IL-34/CSF-1R axis in human glioblastoma tissue,we observed the effects of Celastrus orbiculatus extract(COE)on the proliferation and apoptosis of co-culture U251 cells in vivo through the targeting of the CSF-1/IL-34/CSF-1R axis by the tumor-association macrophages(TAMs).We also studied the effect of COE in inhibiting the pathological process through the tumor microenvironment(TME)with the xenotransplantation nudo mouse model.Methods:Immunohistochemistry and real-time fluorescence PCR were used to examine the CSF-1,IL-34 and CSF-1R mRNA levels in 45 cases of glioblastoma specimens and 8 cases of control groups.THP-1 cells were treated with 320 nM PMA for 24h to differentiate into macrophages,and then treated with IL-4 to induce the cells to M2-like macrophages.We can observe the morphological changes of cells and detect the cell surface marker changes by flow cytometry.Distribution of expression of CSF-1,IL-34,CSF-1R in co-culture system was determined by Immunofluorescence staining.The mRNA expression of CSF-1,IL-34,CSF-1R in M2-like macrophages in co-culture systems,treated with different concentrations(20,40,80,160?g/mL)of COE,were analyzed using real-time PCR.The IL-34 knock out THP-1 cell line was constructed by CRISPR/CAS9 technology.Differentiation of the IL-34KO THP-1 cells into IL-34KO M2-likemacrophages was induced by PMA and IL-34,and verified by fluorescence microscopy.Proliferation and apoptosis of different group U251 cells was detected by flow cytometry(Annexin V,FITC-PI double staining and BrdU incorporation)after treatment with 80 ?g/mL COE or Temozolomide(TMZ)?M.To evaluate the tumor growth and inhibitory effects of COE,cultured and mixed U251 cells and wild M2 or IL-34 KO M2 cells(2:1)were inoculated into the flank of BALB/c nude mice.After the nude mice model was established,the mice were randomly divided into the control group and COE treatment group.Then,the mice were intragastrically given COE for 21 days.The tumor dimensions were measured every three days and tumors were excised and weighed after 21 days of treatment.Result:Immunohistochemistry and RT-PCR results showed that the expression of CSF-1R,IL-34 and CSF-1 in tumor tissue was much higher than that in the control group.CSF-1/IL-34/CSF-1R widely express in tumor.Macrophage recruitment occurs from surrounding tissue to the tumor.The PMA-treated THP-1 macrophages(CD68 positive)exhibited significant expression of M2 macrophage surface marker CD 163.Co-culture M2 macrophage with U251 cell,the CSF-1,IL-34,CSF-1 R were only detected on M2-like macrophages by immunofluorescence staining.Treatment of the co-culture system with different concentration of COE,indicate that CSF-1/IL-34/CSF-1R axis is activated when COE affects the U251 cell.The mRNA level of IL-34 expression synchronously with COE,while differently with CSF-1 and CSF-1R.These observations suggest that IL-34 is a CSF-1 R independent ligand in TAMs.The co-cultured U251 cell proliferation ratio is 31%,which is higher than the Non-co-cultured U251 cell proliferation ratio of 21%.M2 macrophages showed a protective effect on tumor cells.There is no statistical significance between U251 cell proliferation ratio of co-culture U251+IL-34KOM2+COE group(19.4%)and co-culture U251+IL-34KOM2+TMZ(17.2%).It is suggested that COE does not affect the proliferation of glioblastoma by the CSF-1/IL-34/CSF-1R pathway,especially by the IL-34 ligand.At the same time,the apoptotic ratio of the co-culture U251+IL34 KO M2+COE group is 12.41%,while the apoptotic ratio of the co-culture U251+M2+COE group is 15.9%,and the apoptotic ratio of the U251+IL-34 KO M2 co culture +TMZ group is 8.26%.The apoptotic ratios are all significantly different.This shows that COE can interfere with the apoptosis of GBM by affecting TAMs,and the target is IL-34.The results of the experiment in vivo also showed that COE could direct and indirect inhibit the growth of glioblastoma.Conclusion:This investigation provided evidence that COE inhibits the apoptosis of U251 cells through the Tumor associated macrophages in Tumor microenvironment,targeting IL-34.