| Human glioma is the most common primary tumor of the nervous system,which exhibits the most invasive and malignant properity.The average survival time is only 12-15 months.Surgery,radiotherapy and chemotherapy have been widely used in clinical practice to prolong the life of glioma patients.However,glioma remains a fatal disease with poor prognosis.Due to the resistance and serious side effects to conventional chemotherapy,single chemotherapy is no longer suitable for the only therapy of human glioma.Because of the high invasive and the unclear surrounding tissue boundary,it is difficult to cut the whole glioma tissue by surgery,and these residual tumor tissue is the main factor of late tumor recurrence.Due to the growth position and the current radiotherapy method,traditional radiotherapy can not accurately locate and may damage the surrounding normal tissue.Therefore,new treatment strategies are urgently needed nowdays.That is,it can not only accurately locate the glioma,but also reduce the serious side effects.Nowdays,combined chemo-radiotherapy as a novel therapy has been widely developed and used in clinic for treating kinds of human cancers.However,the mechanism remains elusive.Radiotherapy is an effective and cost-effective strategy because of its accurate positioning and less side effects,which was widely used in cancer treatment.In particular,it plays a key role in local disease control.As one of the preferred permanent implanted radioactive sources,radioactive 125I particle has an average energy of 27.4-31.4 keV with an effective radius of 1.7 cm.Radioactive 125I particle implantation has been widely used for cancer treatment due to its high precision and low incidence of complications.Radioactive 125I has achieved great success in the therapy of huaman cancers.However,the anti-radiation and side effects of radioactive 125I particles are still the main problems leading to the loss of therapeutic effect.Studies have shown that combined chemo-radiotherapy can provide better treatment outcomes for better disease control and significantly improve the quality of life of cancer patients.Therefore,design and development of new combined chemo-radiotherapy strategies to overcome drug resistance and side effects has become the focus of clinical oncology.However,the underlying mechanisms remain elusive.Salinomycin(SAL)as a polyether antibiotic was mianly isolated from the Streptomyces albus species of bacterium.SAL has always been used as an anticoccidial agent in the poultry industries.Gupta et al for the first time in 2009 conducted a high-throughput screening of 16,000 compounds against human breast cancer stem cells(CSCs),and the results indicated that SAL showed novel anti-cancer potential against human cancers.SAL exhibits broad-spectrum anticancer properties,including antiproliferation,anti-angiogenesis,autophagy and apoptosis,which was mediated by the induction of reactive oxygen species(ROS)overexpression,DNA damage and mitochondrial dysfunction and regulation of MAPKs,AKT,Wnt/p-catenin,Notch,FOX03a and STAT3/Skp2 signaling pathways.Additionally,SAL has been identified as potent chemosensitizer which can effectively enhance the anticancer activities of several chemotherapeutic drugs,such as doxorubicin,etoposide,gemcitabine and antimitotic drugs,like docetaxel,paclitaxel,colchicine and vinblastine.Several studies also demonstrated that the combined treatment of radiation with SAL is a promising strategy to enhance the anticancer efficacy of radiotherapy and overcome the radioresistance of human nasopharyngeal and breast carcinoma.However,the anticancer potential and mechanism of combined treatment of 125I seeds and SAL have not been explored.Herein,in the present study,a chemoradiation model of 125I seeds and SAL in vitro and in vivo was designed,and the enhanced anticancer efficiency and mechanism were also evaluated in human glioma.Purpose1.To explore the anticancer effect and molecular mechanism of combined treament of 125I and SAL against human glioma cells in vitro.2.To explore the anticancer effect and molecular mechanism of combined treament of 125I and SAL against human glioma cells in vivo.MethodsU87 and U251 cells were cultured in vitro.Cells were randomly divided into control group,SAL-treated group,125I-treated group and 125I+SAL-treated group.Cell morphology was detected by phase contrast microscopy.Cell viability under different treatment conditions were detected by trypan blue staining.Cell apoptosis and cell cycle distribution were detected by flow cytometiy after PI staining.Intracellular ROS and superoxide were detected by DCFH-DA and Mito-SOX,respectively.Annexin v-PI kit and TUNEL-DAPI kit were used to detect early and late apoptosis,respectively.Western blotting method was used to detect the changes of relevant protein factors.Effects of radioactive 125I particles or/and SAL on glioma cells in vivo were detected by xenotransplantation in nude mice combined with immunohistochemistry.Results1.Detection of cell morphology:compared to the control cells and cells treated by single agent,the combined treatment cells showed significant loss in cell number,cell shrinkage,loss the contact of cell-to-cell,increase of apoptotic body.The combined treatment of 125I and SAL significantly inhibited the cells viability.2.Cell activity detection:Compared with the normal control group and the separate treatment group,the cell activity of the 125I combined with SAL group was significantly reduced,with statistically significant differences(P<0.05).The results showed that 125I combined with SAL inhibited the growth of glioma cells in vitro.3.Detection of cell apoptosis:compared to the control cells and cells treated by single agent,flow cytometry showed that combined treatment of 125I and SAL induced enhnced apoptosis.Annexin V-PI and TUNEL-DAPI showed that combined treatment of 125I and SAL triggered early apoptosis and late apoptosis,respectively.Western blotting indicated that combined treatment of 125I and SAL activated caspase-3.These results all suggested that combined treatment of 125I and SAL can more efectively induce apoptosis in human glioma cells in vitro.4.Detection of ROS and superoxide anion:compared to the control cells and cells treated by single agent,DCFH-DA and Mito-SOX results revealed that combined treatment of 125I and SAL caused more accumulation of ROS and superoxide anion.Western blotting demonstrated that combined treatment of 125I and SAL triggered significantly DNA damage,as convinced by the phosphorylation of Ser15-p53 and Ser139-histone.Addition of glutathione(GSH)effectively attenuated combined treatment-induced apoptosis,DNA damage and cell killing.These results indicated that combined treatment of 125I and SAL could induce ROS-mediated DNA damage.5.Detection of MAPKs and PI3k/AKT:compared to the control cells and cells treated by single agent,combined treatment of 125I and SAL significantly activated MAPKs,as convinced by the activation of p-p38,p-ERK and p-JNK.But inactivated PI3k/AKT,as convinced by the inactivation of p-AKT.Addition of AKT inhibitor(LY294002)significantly enhanced combined treatment-induced cell killing.Addition of ERK inhibitor(U0126)significantly inhibited combined treatment-induced cell killing.These results found that combined treatment of 125I and SAL could inhibit glioma cells growth in vitro through regulating MAPKs and PI3k/AKT.6.Detection of anticancer effect in vivo:compared to the control group and groups treated by single agent,combined treatment of 121I and SAL significantly inhibited the tumor weight and tumor volume.Mechanism investigation revealed that combined treatment of 125I and SAL inhibited cell proliferation and angiogenesis,induced apoptosis,triggered DNA damage and inhibited AKT expression,which were consist with that of cell level.These results found that combined treatment of 125I and SAL could inhibit glioma growth in vivo.Conclusion1.Combined treatment of 125I and SAL induced enhanced cell growth inhibition in glioma cells through induction of cell apoptosis.2.Combined treatment of 125I and SAL triggered enhanced DNA damage by ROS generation,and dysfuantioned the expression of MAPKs and PI3K/AKT.3.Combined treatment of 125I and SAL more effectively inhibited tumor grwoth in vivo,which was consist with that of cell level. |
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