| BackgroundMalignant gliomas(WHO grade III and IV) are the most common primary brain tumors constituting approximately 60–70 % of all brain tumors.The current standard therapy for newly diagnosed malignant gliomas is maximal surgery(if feasible)followed by radiotherapy and chemotherapy. Temozolomide(TMZ) is one of the effective chemotherapeutic agents for the treatment of malignant gliomas. TMZ achieves cytotoxicity mostly by methylating the O6 position of guanine. Moreover,it is generally incurable and responds poorly to chemotherapy. Besides,the side effects associated with chemoradiotherapy has aroused attention, including myelodysplastic syndrome, leukemia and severe bone marrow suppression.Granulocyte–Macrophage Colony-Stimulating Factor(GM-CSF) is a cytokine whose best-known functions are to stimulate survival,proliferation and differentiation of hematopoietic myeloid cells.The availability of GM-CSF has enabled several therapeutic applications in pathological conditions where the number and/or functional capacity of leukocytes is sub-optimal including in patients with malignant tumors,in order to reduce the duration and severity of leukopenia in cancer therapy. However, research of application of GM-CSF and TMZ on high grade glioma experimental has not yetbeen reported.ObjectiveThis experiment aims to studying TMZ destruction on high-grade glioma cells after intervention of GM-CSF and trying to explore its mechanism.Materials and methodsData collection of high grade glioma patients. 41 patients with glioma who received surgical treatment were collected in neurosurgery ward of henan province people’s hospital from September 2013 to August 2015.Glioma tumor samples were performed pathologic examination, MGMT protein expression detected by immunohistochemical staining, and MGMT promoter methylation status detected by methylation-specifific PCR with the patients giving informed consent. Briefly, tumor samples were dissociated into single cell suspension using mechanical methods. All cells were plated and maintained at 37°C in a humidified incubator with 5% CO2.Cells were passaged when the alignment reached to 90%, and were used to experimenting after the fourth generation of passage.Selection of high grade glioma cells and GM- CSFR detection. Six cases of high grade glioma cells were selected according to the pathology, MGMT expression,MGMT promoter methylation status and cell culture conditions. Cells were passaged when the alignment reached to 90%, and GM-CSFR were detected by immunofluorescence after the third generation of passage.Time-cell survival curve. TMZ group, GM-CSF group, TMZ +GM-CSF group and Blank group(MTT, dimethyl sulfoxide,medium) were set.The final concentration of GM-CSF and TMZ was respectively 50 ng/ml and 100μmol/L. We assayed cell viability after cultivation of 24 h, 48 h, 72 h, 96 h.Cell viability assays by MTT. Control group, TMZ group, GM-CSF group,TMZ +GM-CSF group and Blank group(MTT, dimethyl sulfoxide,medium) were set. The cells firstly incubated with GM-CSF( 50 ng/ml) for 12 h before TMZ(100μmol/L) was added. Cell viability were assayed after cultivation of 72 h..Cell cycle assays. Only control group and GM-CSF group were set. The cells were allowed to stabilize for 6 h, and then incubated with 50ng/ml concentrations ofVI GM-CSF for 48 h. The analyses were performed by flow cytometry.Cell apoptosis rate assays. Control group, TMZ group, GM-CSF group and TMZ +GM-CSF group were set.Glioma cells were incubated with 50ng/ml concentrations of GM-CSF for 12 h, and then 100μmol/L TMZ was added. Cells were collected and then deal with Apoptosis Detection Kit after 72 h. The analyses were performed by flow cytometry.ResultsThere were two cases of anaplastic astrocytoma and four cases of glioblastoma multiforme according to the results of pathology. Based on the results of immunohistochemical and evaluation standard, positive expression MGMT protein in1 case, strong positive expression in 1 case, weakly positive expression in 1 case and negative expression of MGMT protein in other 3 cases. MGMT promoter methylation and unmethylation of cells have 3 cases respectively. Immunofluorescence show that GM-CSFR expressed in the high grade glioma cells.Time-survival curves of glioma cells. The inhibition rate of both TMZ group and GM-CSF+TMZ group on tumor cells reached to the maximum after 72 h and the survival rate decreased to the minimum.Cell viability assays MTT assay suggested that compared with the control group, GM-CSF group h ad increased cell viability in varying degrees,and the viability of TMZ group and GM-CSF+TMZ group both lower significantly.In three cases of cells(MGMT gene methylation), the cell viability of the GM-CSF+TMZ group [(67.67±1.16),(68.13±1.06),(68.42±1.73)]were significantly lower than that of the corresponding TMZ group [(90.00±1.73),(82.33±1.53),(82.67±2.11)](P=0.000,P=0.000,P=0.001). However, there was no significant difference between the two groups in another three cases(MGMT gene unmethylated)(P>0.05).Cell cycle In all 6 cases of primary glioma cells, GM-CSF treated group showed signific ant reduction in the fraction of cells in G1 phase(35.32±1.69,26.47±4.74,57.33±2.79, 33.78±3.42, 34.10±2.90, 19.41±1.61)with concomitant increase in S phase(55.87±4.07,46.91±4.99,35.28±3.34,35.73±6.05,56.79±2.76,50.78±1.51)(P<0.05)Cell apoptosis rate Compared with control group, cell apoptosis rate of GM-CSF group were reduced in all 6 cases.For the MGMT methylation cells, the apoptosis rate in GM-CSF+TMZ group( 19.91±1.93, 25.33±2.57, 32.15±2.85)was higher than that in the corresponding TMZ group(10.79±0.60,15.15±1.74,22.27±1.89)( P=0.000, P=0.000,P=0.007),which coincided with MTT assay results.However,apoptosis rate had no obvious change between TMZ group and GM-CSF+TMZ group in other 3 cases of MGMT methylation cells(P > 0.05).Conclusions:1.The inhibition rate of TMZ on high-grade glioma cells increased gradually and the survival rate decreased over time after the intervention of GM-CSF.2.GM-CSF Enhances destruction of TMZ on High-grade Glioma Cells with MGMT gene promoter methylation,while the effect is not ideal on glioma cells with MGMT unmethylation.3.GM-CSF could induce high-grade glioma cells entering the cell cycle ra pidly, which could enhance the lethal effect of TMZ on glioma cells with MG MT gene promoter methylation... |
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