| Objective:Glioma is the most common primary malignant tumor of the central nervous system,and glioblastoma(GBM)is the most common type.Its invasiveness and recurrence rate are significantly higher than those of other intracranial tumors.At present,the prognosis of GBM is poor,and the 5-year survival rate is about 5%.It is of great significance to find drugs and treatment strategies that can effectively inhibit GBM.Since ancient times,China has a tradition of using traditional Chinese medicine containing arsenic to treat a variety of diseases,and arsenic trioxide(ATO)is the main extracted component.How to enhance the anti-glioma effect of ATO,reduce adverse reactions and make it play an anti-tumor effect at a safe dose is worthy of further study.Low intensity ultrasound(LIUS)can cause reversible damage to the structure of cell membrane,improve the permeability of cell membrane,increase the concentration of chemotherapeutic drugs passing through the cell membrane,and instantly open the blood-brain and blood-tumor barrier to improve the permeability of vascular endothelial cells.The purpose of this study was to observe the effect of LIUS on enhancing the anti-GBM effect of ATO in vivo and in vitro,to verify its synergistic mode of inducing programmed cell death of glioma cells,and to explore the possible mechanism.Methods:In this study,GBM U87 MG,U251 cell lines and human astrocytes(NHA)were studied.In the in vitro part,ATO was first treated with different concentrations(2,4,6,8,10μM)and time(24,48,72 hours).LIUS acts at different intensities(50.4,83.4,142.0,290.0 m W/cm~2),1,3,and 5 minutes at different times,once every 24 hours,and acts for 72 hours.CCK8 was used to analyze the effects of ATO and LIUS alone on glioma proliferation and effects of NHA,screening of conditions for combined action.Then,CCK8,Ed U,flow cytometry and westernblot experiments were used to analyze the effects of combined application on glioma proliferation,apoptosis,epidermal growth factor receptor(EGFR),protein kinase B(Akt)and mammalian rapamycin target protein(mTOR).Epidermal growth factor(EGF),the phosphorylated substrate of EGFR,was added,and CCK8,Ed U,flow cytometry and westernblot experiments were used to verify whether the combined effect of ATO and LIUS enhanced the inhibition of EGFR phosphorylation,resulting in downstream Akt/mTOR changes,thereby affecting apoptosis and autophagy,and inhibiting the proliferation of gliomas.In vivo,the in orthotopic xenograft model of U87MG cell line in nude mice was established.14 days later,the nude mice were divided into two groups:intraperitoneal administration of ATO and extracranial irradiation of LIUS.After 28 days,the survival curve was drawn,and magnetic resonance imaging(MRI)and inductively coupled plasma mass spectrometry(ICP-MS)were used to analyze the difference of tumor size and arsenic content in brain tissue.The difference of protein expression in tissue was analyzed by immunohistochemical experiment.Results:1.The CCK8 experiment showed that when treated with ultrasound of142m W/cm~2 or above intensity alone(1,3,5 minutes),the glioma and NHA were inhibited to a certain extent in an intensity-and time-dependent manner.However,when the intensity was in 50.4m W/cm~2 and 83.4m W/cm~2 for 1,3,5 minutes,the inhibitory effect on the proliferation of glioma and NHA were not obvious.The inhibitory effect of ATO alone on glioma was concentration-and time-dependent.The glioma could be inhibited by 4μM for48 and 72 hours and 6,8,10μM for 24-72 hours,and the difference was statistically significant.However,at 2μM for 24-72 hours and 4μM for 24 hours,there was no significant inhibitory effect on U87MG and U251 cells.At the concentration of ATO in this study,there was no obvious inhibitory effect on NHA cells.Based on the experimental results of the single action of LIUS and ATO,on the premise of ensuring the safe concentration of ATO and the intensity of LIUS in human and normal brain tissue,we chose ATO(2μM)and LIUS(83.4m W/cm~2)which had no obvious inhibitory effect on glioma for 72 hours as the condition of combined application.2.Through the CCK8experiment,it was found that the combination of them had no obvious inhibition on NHA cells,but enhanced the anti-glioma effect of ATO,the relative increment rate(U87MG:60.3±6.2%,U251:73.1±1.7%).The Ed U test also showed that the ratio of Ed U positive staining cells in the combination group was lower than that in the other three groups,and the difference was statistically significant.There was no significant difference in the number of Ed U positive staining cells among control group,LIUS group and ATO group.LIUS can enhance the inhibition of glioma proliferation by ATO.3.The results of flow cytometry(Annexin V-FITC/PI)showed that there was no significant change in apoptosis induced by LIUS and ATO alone in U87MG,but the combination of LIUS and ATO could induce significant apoptosis in U87MG cell line(the apoptosis rate was 36.3±3.61%),and the difference was statistically significant.In U251,there was no significant difference in the number of apoptotic cells between the two groups.4.Western blot assay showed that the combination of LIUS and ATO could increase the expression of apoptosis marker protein Cleaved Caspase-3 in U87MG cell line,but there was no difference in U251cell line.Through the analysis of autophagy-related proteins LC3B and Beclin1,it was found that in U251,LIUS combined with ATO increased the expression of LC3B and Beclin1,while in U87MG,the expression of LC3B and Beclin1 decreased.At the same time,in U87MG and U251 cell lines,the combination of LIUS and ATO could increase Bax and decrease the protein expression of Bcl-2.On the other hand,the regulation of P53showed the opposite trend,and the difference of the above protein bands was statistically significant after gray quantization.5.Western blot assay showed that in U87MG and U251cell lines,the total protein expression of EGFR,Akt and mTOR did not change after the combination of LIUS and ATO,but the expression of phosphorylated EGFR,Akt and mTOR decreased,and the difference was statistically significant.ATO and LIUS alone had no significant effect on the expression of the above proteins.Therefore,the enhancement of the anti-glioma effect of ATO by LIUS may be related to the inhibition of EGFR phosphorylation which leads to the inhibition of downstream AKT/mTOR.6.Use the activation substrate EGF of EGFR and activate the downstream AKT/mTOR.CCK8and Ed U experiments showed that the inhibitory effect of LIUS and ATO on glioma was weakened by the introduction of EGF,and the activation of EGFR could reverse the inhibitory effect of LIUS and ATO on glioma.Flow cytometry also suggested that EGF could reduce the apoptosis of glioma cells induced by LIUS and ATO in U87MG.The results of western blot suggest that EGF can antagonize the inhibition of EGFR/AKT/mTOR pathway activation induced by LIUS and ATO.It can antagonize the expression of apoptosis-related proteins induced by LIUS and ATO.It also antagonized the expression of autophagy-related proteins in U251.7.The orthotopic xenograft model in vivo was further verified.Under the MRI,there was no significant difference in tumor size among control group,LIUS group and ATO group,but the tumor volume in ATO+LIUS combined group was significantly smaller than that in other groups.The measurement of arsenic in brain tissue by ICP-MS also showed that the content of arsenic in brain tissue of ATO+LIUS combined group(1466.3±102.4μg/kg)was significantly higher than that of ATO alone(649±71.6μg/kg).The survival rate of tumor-bearing mice was improved after combined application.The Immunohistochemistry showed that the staining intensity of Ki67,LC3B,p-EGFR,p-Akt and p-mTOR decreased in the combined group,but there was no difference among the other groups.The staining intensity of Cleaved Caspase-3 and P53 in the combined group was higher than that in the other groups.Conclusion:To sum up:1.After the action of LIUS combined with ATO,the drug effect was enhanced and the drug concentration in brain tissue was increased,which can make ATO play a certain anti-glioma effect under the safe dose of human body.2.The ATO+LIUS can inhibit proliferation by increasing apoptosis of U87MG and autophagy of U251.3.The mechanism is related to that ATO combined with LIUS reduces the phosphorylation activation of EGFR,thus inhibiting the downstream AKT/mTOR pathway. |
