| As one of the most common seen primary tumor of the central nervous system,gliomas accounts for 40%-60%of the adults intracranial tumors with a five-year survival rate less than 38%,while in the subset of glioblastoma (GBM),the rate is less than 5%.Surgical removal is regarded as the first line treatment with radiotherapy,chemotherapy and genotherapy as alternative treatment.Chemotherapy was promoted in the last 30 years and has demonstrated its powerful usefulness in management of many kinds of cancers. Up to now,many promising new drugs are under research.Unfortunately,in the field of glioma,these drugs have failed to show brilliant antitumor effects. Therefore,scients focus on developing new drugs to benefit patients with glioma.Arsenic's reputation as a therapeutic agent was enhanced within the past 30 years.In 1970's,arsenic trioxide(As2O3)was used by Chinese clinicians in the treatment of myelocytic leukemia and got a high remission rate,which showed the world its power in managing malignant diseases.In September,2000,the Food and Drug Administration of U.S.approved arsenic trioxide for the treatment of relapsed or refractory acute promyelocytic leukemia(APL).Similar to As2O3,realgar belongs to arsenical compounds with a lower toxicity(realgar LD50＞10g/kg vs.As2O3 LD50＜50mg/kg)which could be used in oral administration.Realgar plays an important role in the treatment of hematologic cancers but few studies were reported on realgar in other solid cancers.Although the precise mechanism of arsenic trioxide action is unknown,a variety of studies in vitro suggest that promotion of apoptosis and induction of differentiation are the key mechanisms which may contribute to its anticancer effects in vivo.Moreover,changes in cell oxidation,DNA damage and inhibition of angiogenesis also take effect on killing cancer cells. In order to interpret the possibility of application of realgar in the treatment of glioma,we investigated the rols realgar play on glioma cells in vitro and in vivo and the possible mechanism.Objectives:Rat glioma cell line C6 was used to observe the effects of realgar in vitro and a C6 nude mice subcutaneous animal model was built to explored the effects of realgar on glioma and its possible mechanisms in vivo.Methods:1.The growth characteristics of rat C6 glioma cell①Cell CutureRat C6 glioma cells were cultured in high glucose DMEM medium supplemented with 10%fetal bovine serum,100U/L penicillin,100U/L streptomycin in a incubator with a condition of 37℃,5%CO2 and saturated humidity.②Observation of Morphology and Growth CharacteristicsPhase-contrast microscope was used to observe the morphology and growth of C6 glioma cell.And the adherence rates after passage in every point of time were counted to indicate the growth characteristics.Cells in log growth phase were collected by digestion and then planted in 3 pieces of 6 hole-plate in the density of 1×105/ml.After 2h,4h,6h,8h, 10h and 12h of plantation,the cells were digested and counted(three times to get the mean)to get the adherence rates.③Growth CurveThe growth rate of cells was determined by MTT in 96-well plate and a growth curve was drawn with the time as abscissa and absorbance as ordinate. The cells in the 24-well plates were digested and counted and a growth curve was drawn with the time as abscissa and the logarithm of cell number as ordinate.The cell-doubling time was calculated.2.Effects of realgar in vitro①Preparation of realgar solutionThe realgar solution was made according to references of Bai etc. Atomic absorption spectrometry was used to detect the concentration of arsenic which was used as the drug's concentration.②Determinaition of the working concentration of realgarC6 cells were in log growth phase were collected and cultured with different concentrations of realgar,and the growth inhibition rates of at 24h and 48h were performed by MTT.The realgar's concentrations varied from 5mg/L to 100mg/L(5mg/L,10mg/L,25mg/L,50mg/L,100mg/L).PBS was used as negative control and tissue culture medium as blank control.After treated by realgar for 24h and 48h,ELISA reader was used to get the absorbance value at A570nm wavelength,and cell survival rate rate were calculated as well as and growth inhibition.The IC50(inhibitory concentration 50)of the realgar solution was determined via constructing a dose-response curve.According to the results of IC50,25mg/L was assigned as the working concentration of realgar in the following study.③Ultrastructure of C6 cells treated with realgarCells in log growth phase were collected and treated with realgar solution for 24 and 48 hours.Then these cells were fixed in glutaraldehyde and slices for transmission electron microscope were made.Electron microscope was used to observe the ultrastructure changes of C6 cells.④Detection of early-stage apoptosisCells in log growth phase were collected and cultured for 24h after transfer before interefered by realgar.By Annexin V-FITC-PI staining method was used to label C6 cells treated with realgar for 24h and 48h.Early-stage apoptosis in cells were detected by flow cytometry(FCM). ⑤Detection of advance-stage apoptosisSlides of C6 cells treated with realgar for 24h and 48h were prepared. TUNEL method was used to detect the advanced-stage apoptosis in cells Positive cells were counted under microscope to get the apoptosis index.⑥Detection of Bax mRNA,Bcl-2 mRNA and c-myc mRNAThe expressions of apoptosis-related gene such as Bax mRNA and Bcl-2 mRNA and oncogene as c-myc mRNA of C6 cells treated with realgar for 24h and 48h were detected by RT-PCR.⑦Detection of Bax protein,Bcl-2 protein and c-myc proteinProtein levels of Bax,Bcl-2 and c-myc of C6 cells treated with realgar for 24hrs and 48hrs were detected by Western Blotting.⑧Detection of cell proliferative activityThe PCNA(proliferating cell nuclear antigen)expression of C6 cells treated with realgar for 24h and 48h were detected by immunohistochemical method to get the cell proliferative activity.3.Realgar in vivo:①Preparation of animal model and observation of tumorsA 0.5 ml single cell suspension with 105 cells was subcutaneous transplanted in the buttock of Balb/c nude mice.The sizes of tumors were detected by ruler everyday.The mice were randomly divided into three groups which were realgar treated groups,negative control with PBS and blank control without any treatment.They were taken into the expriments when the tumor reached approximately 5mm in diameter.②When C6 mice models were successfully achieved.Growth curve were drawn.Animals were killed in the 11th day after treatment and tumors were seperated for further study.③Verification of the glioma cell line by microscope and GFAP testingStructures of tumor tissues were observed after stained by HE. Immunohistochemical method of GFAP was performed for observation of glial cells,whose character is the expression of GFAP. ④Ultrastructural changes of tumors after treatmentA transmission electron microscope were used to observe ultrastructural changes of the tumors.⑤Detection of apoptosisTUNEL method was applied to the paraffin sections from the three groups. Apoptosis index counts were performed under microscope.⑥Detection of levels of protein and mRNA of Bax,Bcl-2 and c-mycWestern Blotting and RT-PCR standard procedures were performed for the detection of proteins and mRNAs of Bax,Bcl-2 and c-myc seperately.4.Statistical analysisSPSS for Windows 11.5 were used for all the statistical analysis.All of the measurement data was expressed in the format of "(?)±s".T test,variance analysis and nonparametric test were used according to distinct kinds of results(sample size,homogeneity of variance,and test of normality).The difference between the groups treated withχ2 test..A P level less than 0.05 was considered as statistiacally significant.Results:1.The growth characteristics of rat C6 glioma cell:①In pre-experiment,the growth curve we obtained confirmed the stability of the C6 cells that we have obtained.Under phase-contrast microscope,it indicated that adherence cells were shuttle or triangle shape in uniform size;they arranged densely with strong light refraction and smooth membrane without tuber;nucleus is circular and small nucleolus with more karyokinesis.Adherence rate in 2h,4h,6h,8h,10h and 12h was 22%,32%,75%,80%,75%and 82%,respectively.Cells were qualified for further experiments.②Growth curve by MTT:the cell were planted in 96 hole-plate with the density of 4×103/hole and 24 hole-plate with 2×104/hole.From 24th hour after planted,cells entered in log growth phase lasting 3 days which was qualified for further experiments. 2.Realgar in vitro:①By atomic absorption spectrometry,the concentration of realgar solution was 1034mg/L.②Statistical difference was observed between blank control and each group treated with different concentration of realgar for 24h and 48h(P＜0.05). No statistical difference was observed between 5mg/L and 10mg/L;no statistical difference was observed in 10mg/L,25mg/L and 50mg/L(P＞0.05). Differences between 100mg/L and any one of other groups was statistically significant(P＜0.05),which showed good cell inhibition.The IC50of realgar solution is calculated via the mathematic model which is 28mg/L and 26mg/L for the 24h and 48h respectively.③Ultrastructure of C6 cells treated with 25 mg/L realgar for 24h changed and apoptotic bodies were observed under transmission electron microscope,.④After Annexin V-FITC-PI staining,C6 cells treated with 25mg/L realgar for 24h was detected by FCM for early-stage apoptosis.Apoptosis rate for 24h and 48h is respectively 10.13±3.14%and 41.04±17.44%.⑤Results of TUNEL showed that positive rate of cells treated with realgar for 24h and 48h was respectively 8.7±1.83%and 12.2±5.75%.The difference between these two groups was statistically significant(P＜0.05).⑥RT-PCR showed that compared with controls,the mRNA expression of c-myc and Bcl-2 of cells treated with realgar for 24 hours were significantly inhibited.After 48 hours,the mRNA expression of c-myc and Bcl-2 were further inhibited.The expression of apoptosis Bax mRNA was enhanced with incubating time.⑦The protein levels of c-myc and Bcl-2 of cells treated with realgar for 24 hours were decreased and the protein levels of Bax increased. Compared to cells treated for 24 hours,the protein levels of c-myc and Bcl-2 of cells treated with realgar for 48 hours were decreased and the protein levels of Bax increased. ⑧Immunohistochemical staining showed no difference between PCNA of cells treated with realgar and controls.3.Realgar in vitro:①Tumors in Balb/c nude mice treated with realgar grew at a significantly lower speed compared with blank controls and negative controls (P=0.007).Further analysis showed that the difference between blank control and realgar was significant(P=0.005),as well as the difference between PBS negative control and realgar(P=0.006).Moreover,the difference goes on with study's preceeding.②Identification of pathology of tumors:Tissues of tumors expressed GPFA,which indicated that subcutaneously transplanted tumors were gliomas. Cells with GPFA positive in the group of realgar were significantly more than in blank control(P＜0.05).③Under microscope,compared with blank control and PBS negative control,cells of tumor treated with realgar were disperse with irregular arrange with obvious necroses.Under the electron microscope,,there were more cells with apoptosis with pyknosis of nucleuses and high dense masses scattered in nucleuses in cells of tumor of the treated group.Meanwhile,there were abundant organelles and the chromatins distributed symmetrical in cells of tumor of blank control.No apoptosis bodies were found in blank control.④TUNEL results indicated that positive rate of cells treated with realgar,the blank control group and the negative control group was 5.17±0.42,1.62±0.21 and 1.76±0.33 respectively.The diffences between realgar treated group and any one of the two control groups were statistically significant(P＜0.05).⑤Protein and mRNA expression of Bax,Bcl-2 and c-mycWestern Blotting and RT-PCR showed that compared with blank control and negative control,the realgar treated group expressed lower mRNA and protein levels of Bcl-2 and c-myc,but higher mRNA and protein levels of Bax. All of the differences were statistically significant(P＜0.05). Conclusion:①Realgar could inhibit glioma C6 cells both in vitro and in vivo, which has no relationship with cell proliferative activity.Apoptosis inducing was the main mechanism for realgar to inhibit cells.②Upregulation of the expression of apoptosis associated gene Bax and downregulation of anti-apoptosis gene Bcl-2 and oncogene c-myc played important roles in apoptosis induced by realgar.
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