| Objective:Malignant gliomas are the most common primary tumors of the central nervous system,characterized by rapid progression,susceptibility to recurrence,and short survival.Currently,clinical surgical resection,and radiotherapy,and chemotherapy are important means for the treatment of brain gliomas.Regarding chemotherapy,the application of chemotherapeutic drugs often leads to cytotoxicity and rapid drug resistance in patients,affecting their survival and quality of life.Low-frequency ultrasound can lead to noninvasive,transient,reversible,and local opening of the blood-brain barrier and thus enhance drug penetration and absorption,providing a window for glioma treatment.Previous studies have shown that the TGF-β1/Smad signaling pathway is closely related to the biological behavior of glioma cells.However,the underlying mechanism remains unclear.In this study,through in vivo and in vitro validations,we explored the associations of TGF-β1/Smad signaling with proliferation,migration,and invasion of glioma cells.Further,we explored how low-frequency ultrasound in combination with microbubbles can enhance the inhibitory effect of curcumin,an inhibitor of the TGF-β1/Smad signaling pathway,on glioma cells.Methods:1.1 Each type of the U251,U87,and C6 cells was divided into five groups:blank control,curcumin alone;ultrasound+curcumin;ultrasound+microbubble agent;and ultrasound+microbubble agent+curcumin(combined treatment).Different treatments were applied to each group.For the blank control group,no curcumin was added and no ultrasound irradiation was applied;For the curcumin alone group,only curcumin was added;For the ultrasound+curcumin group,curcumin was added followed by ultrasound irradiation for 30 s;For the ultrasound+microbubble agent group,microbubble agents was added followed by ultrasound irradiation for 30 s;For the combined treatment group,microbubble agents and curcumin were added followed by ultrasound irradiation for 30 s;For all the groups applicable,the final concentrations of curcumin and microbubble agents were 15μmol/L and 1-5×10~6/ml,respectively;For all the groups that were treated with ultrasound irradiation,the ultrasound intensity used was142.0 m W/cm~2.After the treatment,the morphological change of cells in each group was observed under the microscope;The apoptosis rate of cells in each group was assessed by flow cytometry with PI staining;The proliferation rate of cells in each group was measured using the CCK8 assay;The migration and invasion abilities of cells in each group were measured using the scratch assay and Transwell invasion assay,respectively;The expression levels of VEGF,NCAM and integrinsαvβ3 in cells in each group were examined using both the q RT-PCR and Western blot.1.2.U251,U87,C6 three cell lines were sub-grouped respectively.Blank control:no treatment,rh TGF-βGroup:rh TGF-βadministered alone(10 ng/ml),and the combination group:treated with the addition of microbubbles(microbubble measurements:1-5×10~6/ml)and curcumin(15μMol/L)was followed by ultrasound irradiation for 30 s,combined treatment with+rh TGF-βGroup:given rh TGF-βAfter factoring in microbubble agents and curcumin were added followed by ultrasound irradiation for another 30 s,the inhibitor ITD-1 group:Smad2/Smad3 phosphorylation inhibitors(5μmol/m L).The levels of p-Smad2/Smad3,VEGF,NCAM and integrinsαvβ3 in cells of each group were detected using q RT-PCR and Western blot.2.The C6 cells were cultured and expanded to a sufficient number.Then the cells were resuspended to a density of 5×10~7cells/ml in serum-free DMEM.Twenty-five healthy Wistar rats weighing 180±13 g were anesthetized with 10%chloral hydrate(0.3 ml/100g,i.p.).A cavity was created by craniotomy in each rat and C6 glioma cell suspension was injected according to the guidance of a stereotaxic instrument.The rats were divided into five groups(5 in the control group,4 in the other groups,and 4 were failed for tumor cell engraftation).Seven days after surgery,the rats in the ultrasound+curcumin group,ultrasound+microbubble agent groupand combined treatment group and received low-frequency ultrasound treatment for 30 s,ultrasound+curcumin group,combined treatment group received curcumin via tail vein injection,in the ultrasound+microbubble agent group and combined treatment group received microbubble via tail vein injection.The no treatment group(control group)added nothing and were not applied to ultrasound irradiation.Tumor size and growth at the inoculation site were evaluated by MRI on the 1st-,7th-and 14th-day after treatment.After the rats were sacrificed,the tumor tissues and peritumoral tissues were collected.The distribution,arrangement,and necrosis of tumor cells in each group were evaluated by hematoxylin and eosin(H&E)staining.Expression levels of VEGF,NCAM and integrinsαvβ3 in tumor cells of each group were detected by immunohistochemistry(IHC)staining.Results:1.1.Changes in cell morphology:Normal glioma cells of each cell type in the control group showed spindle or polygonal morphology with clear cell borders and reticular connection with surrounding cells.Compared to the control group,the proliferation rates of glioma cells in other groups were inhibited to different degrees;the cells had smaller sizes,with variable morphology,decreased synapses,enlarged intercellular spaces,and fragmented nuclei.The combined treatment group had the most significant changes in these features.Changes in cell proliferation rate:Compared with the control group,the proliferation of glioma cells in the other groups was inhibited(P<0.05).However,in each treatment group,the cell proliferation inhibition rate in the combined treatment group was significantly higher than that in the other groups(P<0.05,P<0.0001,respectively).Changes in apoptosis rate:Compared with the control group,the apoptosis rates of cells were significantly higher in other groups(P<0.05,P<0.0001)except for the ultrasound+microbubble agent group.Moreover,the apoptosis rate of cells in the combined treatment group was significantly higher than that in the other groups(P<0.05,P<0.0001).Migration and invasion abilities:The migration and invasion abilities of cells in each group were different.More specifically,the migration and invasion abilities of cells were significantly reduced in all treatment groups compared with the control group(P<0.05,P<0.0001).The migration and invasion ability was significantly reduced in the combined treatment group compared with other treatment groups(P<0.05,P<0.0001).The expression levels of VEGF,NCAM and integrinsαvβ3 were significantly decreased in cells of all treatment groups compared to the control group(P<0.05,P<0.0001).Moreover,compared with the other treatment groups,the expression levels of these genes in the combined treatment group were significantly higher(P<0.05,P<0.0001).1.2.Expression levels of P-Smad2/Smad3,VEGF,NCAM and integrinsαvβ3 after secondary grouping of the three cell lines:The expression levels of P-Smad2/Smad3,VEGF,NCAM and integrinsαvβ3 were significantly higher in the rh TGF-βgroup(P<0.0001)and decreased in the combination treatment and inhibitor ITD-1 groups(P<0.05,P<0.0001),compared with the control group.While control and combination treatment+rh TGF-β,combination treatment group,and inhibitor ITD-1 group no significant difference was observed in expression(P>0.05).2.Observation of tumor volume:Twenty-five rats were injected with glioma cells,of which 21 formed transplanted tumors with a tumorigenesis rate of 84%.In following 14days after treatment,the tumors grew slower in the other groups compared with the control group(P<0.05,P<0.0001).Among all the treatment groups,the tumors grew slowest in the combined treatment group(P<0.05).Observation by HE staining:Compared with the control group,the distribution of tumor cells decreased in the treatment group.Specifically,the arrangement of tumor cells in the treatment groups was loose,and large sheets of tumor cells showed features including necrosis,nuclei fragmentation and lysis.Among all the treatment groups,the combined treatment group showed the most obvious alterations.Immunohistochemistry:The expression levels of VEGF,NCAM-L1 and integrinsαvβ3 in tumor cells were significantly decreased in the treatment groups compared with the control group(P<0.05,P<0.0001).The expression levels of VEGF,NCAM-L1 and integrinsαvβ3 in tumor cells were higher in the curcumin group and curcumin+ultrasound group than the combined treatment group(P<0.05,P<0.0001).Conclusions:In summary,1)Levels of VEGF,NCAM and integrinsαvβ3 are related to tumor cell proliferation,migration,and invasion,and utrasound irradiation combined with microbubble agents can enhance the inhibitory effect of curcumin on their expressions.This combination provides a new avenue for the effective treatment of glioma cells.2.P-Smad2/Smad3 is a key factor in the TGF-βsignaling pathway.Its downstream signaling molecules,including VEGF,NCAM-L1 and integrinαvβ3,were significantly higher when p-Smad2/Smad3 was overexpressed.Combined treatment of xxx and xxx could reverse this overexpression.When p-Smad2/Smad3 was inhibited,the expression levels of these downstream factors were comparable with that of the combined treatment group.Therefore,p-Smad2/Smad3 may be the mediator by which ultrasound combined with microbubble agents use to enhance the inhibitory effect of curcumin on glioma. |
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