| Glioma is a malignant tumor derived from neuroepithelium. Statistical data discover that it account for approximately 40% of all human intracranial tumors. Its growth pattern is invasive. Total resection is difficult to be achieved. Mean survival span of patients with malignant glioma is just about 14 weeks. To date, combined therapy was always being adopted to treat glioma. Operation resection is a mainly method, chemotherapy and radiotherapy are always be used as auxiliary methods. But the effects of above methods are not satisfactory. Therefore, found a novel effective medicine to treat glioma become urgent and important. Arsenic Trioxide (As2O3) is the main component of traditional Chinese medicine- arsenicum sublimatum. In this experiment in vivo and in vitro we found that As2O3 can inhibit proliferation and induce apoptosis of SHG-44glioma cells and also explored primarily the mechanism of As2O3 on glioma cells. This experiment provided feasible animal experimental data for As2O being applied clinically on treating glioma in the future.The research work of this experiment listed as follow:1. Experimental study of inhibitory effect of As2O3 on human SHG-44 glioma cells in vitro.1.1 Inhibitory effect of As2O3 on the proliferation of cultured human SHG-44 glioma cellsMTT assay was performed to evaluate the proliferation inhibitory effect of As2O3 on SHG-44 glioma cells. Based on results of preliminary test, we calculated out by Excel technique the IC50 of As2O3 on SHG-44 glioma cells was 8.05μmol/L. Then we set different concentrations(0.5,1.0,2.0,4.0,8.0μmol/L)on cultured human SHG-44 glioma cells for 24h,48h,72h. Results showed that the Inhibit Rate (IR) of 8ummo/L group on 24h,48h,72h were 28.84±2.05%,50.77±2.81%,61.43±2.57% respectively which showed a time-dependent tendency. And the variability between each group were all significant (P<0.05).The Inhibit Rate (IR) of 0.5,1.0,2.0,4.0,8.0μmol/L groups on 48h were 13.02±1.31%,17.63±1.43%,30.91±2.93%,36.77±5.35%,50.77±2.81% respectively which showed a dosage-dependent tendency. And the variability between each group were all significant (P<0.05). About results showed that As2O3 could inhibit the proliferation of SHG-44 glioma cells and the inhibitory effect was in a dose-dependent and time dependent tendency.1.2 observation of inhibitory effect of As2O3 on SHG-44 glioma cells under inverted light microscopeCell morphology of SHG-44 glioma cells exposed to As2O3 were observed under inverted light microscope. SHG-44 glioma cells in control group showed adherent, cells grew densely and exhibited fusiform or polygon shape. SHG-44 cells slicked to the walls of culture plate firmly. Glioma cells treated with 1μmol/L As2O3 showed that the shapes of SHG-44 cells were irregular and cell volume began to reduce. The number of the treated cells was less than that of control, some round and contracted cells were found under light microscope. Glioma cells treated with 4,8 mol/L As2O3 showed that the number of SHG-44 glioma cells were significant lesser than that of control, and SHG-44 cells could not stick to the walls of culture plate firmly. Most cells died and broke down, and the left cells became round cells which were easily detached from the walls of culture plate and floated in the culture medium.1.3 and 1.4 SHG-44 glioma cell apoptosis observed by Giemsa and Hochest 33258 stainSHG-44 glioma cells capacity decreased significantly and the density become lower. Some cells shrinked to a round shape. Cells dispersed on culture plate, disperse or were observed with optical microscopy by Giemsa staining. In control group SHG-44 glioma cells grew densely and exhibited fusiform or polygon shape, cytoplasm showed pink color and cell nucleus took on blue. As2O3 treated group showed that cell volume began to reduce, cell number was less than that of control, some diffused distribution round cells were found, cytoplasm showed dense and dark stained, cell nucleus took on hyperchromatism of dark blue, nuclear fragmentation could be found and took on apoptosis morphology characters. 1.5 SHG-44 glioma cells apoptosis induced by As2O3 detected by flow cytometry.SHG-44 glioma cells were treated with different concentration(1,2,4,8μmol/L)As2O3 for 48h, and flow cytometry was used to assess the effect of As2O3 treatment on cell apoptosis. Result showed that apoptosis rate in 1,2μmol/L group had no significant variability compared with control group respectively(P=0.719 and 0.659). Apoptosis rate in 4,8μmol/L group had extremely significant variability when compared with control group respectively(P<0.01). SHG-44 glioma cells were treated with 8μmol/L As2O3 for 12h,24h,48h and the apoptosis rates of 36h,48h were significantly higher than those in control group (P<0.05).1.6 Effects of As2O3 on cell cycle of SHG-44 glioma cellsSHG-44 glioma cells were treated with different As2O3 concentration for 48h, and flow cytometry was used to assess the changes on cell cycle of As2O3 treatment. Results showed that SHG-44 glioma cells were treated with 4,8μmol/L As2O3 for 48h, the percentage of G0/G1 phase cell significantly decreased compared with control group(P<0.05). Cells into S phase of 1,2,4,8μmol/L groups increased significantly compared with control group(P<0.05), which indicated that As2O3 can block the cell in the S phase, and interfere the cell cycle progression.1.7 Effects of As2O3 on reactive oxygen species (ROS) level of SHG-44 glioma cellsThe ROS was measured by 2', 7'- Dichlorodihydrofluorescein Diacetate (DCFH-DA), assessed by flow cytometry on 48h. Results showed that the ROS level in SHG-44 glioma cells between 1μmol/L As2O3 group and control group was not significantly different (P=0.33). In 2,4,8μmol/L groups , the percentage of stained cells was significantly higher compared with control group(P<0.01). Comparison between groups showed that significant difference between each other.1.8 Effects of As2O3 on mitochondrial trans-membrance potential (MMP) of SHG-44 glioma cellsThe MMP of As2O3-treated SHG-44 glioma cell was labeled with Rhodamine123 (Rh-123), and assessed by flow cytometry. Results showed that MMP decreased in 2,4,8μmol/L As2O3 treated groups when compared with control group(P<0.05). The MMP decreased in SHG-44 glioma cells between 1μmol/L As2O3 and control group was not significantly different (P=0.161). As2O3 displayed a dose -dependent effect on MMP.1.9 Effects of As2O3 on Bcl-2 protein expression of SHG-44 glioma cellsThe expression of Bcl-2 protein was studied by western blot. Results showed that Bcl-2 protein expression decreased with As2O3 concentration increased, the effect was dose-dependent.Our in vitro study showed that As2O3 has cytotoxicity effect on cultured human SHG-44 glioma cells in vitro; can inhibit its proliferation; induce cell apoptosis; block the cell in the S phase and interfere the cell cycle progression; increase ROS level; destroying MMP; inhibiting the expression of Bcl-2. Overall, we consider that As2O3 as a new anti-glioma drug which have the potential to treat or even cure the malignant tumor of central nervous system, while its molecular mechanism of those effects needs to be further explored.2. Experimental study of inhibitory effect of As2O3 on human SHG-44 glioma cells in vivo2.1 Wistar rat bearing human SHG-44 glioma brain tumor model was established by stereotaxis technique and undertook MRI examination on 6th day. Results showed that 18 among 29 rats can be found abnormal signals on right brain parenchyma. The signals were equal or slightly lower than normal brain tissue. Achievement ratio of xenograft was 60%.2.2 Observation of general status and survival time of SHG-44 glioma model ratsOn the first day of implantation, there's one rat died. All the others were more or less showed signs of neurological distress, such as weakness, paralysis, epilepsy like tremor, hyperplasia, rotation along the long axis, et al. There 2 rats of control group died on the 2th,8th and 14th days post implantation and 3 died on 4th,18th days post implantation.2.3 The volume of tumor and inhibitory rateThe rats brain tissues were obtain after they were decapitated on the 21th day after implantation. Ectomized the surface brain tissue of right basal ganglia and get the tumors. The tumor volume of control group and As2O3 treated group were 87.13±38.98mm3, 10.86±7.92 mm3 respectively, the tumor volume of As2O3 treated group was significantly less than control group (p<0.01).2.4 Tumor pathology studyHE stain under microscope we found As2O3 treated group cell shirinkage and were stained with blue color while control group had no such changes.2.5 Tumor tissue ultramicroscopic observationUnder electron microscopy we saw As2O3 treated group cell show a early stage apoptosis changes such as chromatin marination,increased and aggregation of heterochromatin,formation of apoptotic body. Control group had no about changes.2.6 As2O3 concentration in brain tissueUsed the atomic absorption spectrophotometer to detected he concentration of As2O3 in brain tissues by cyanide atomic absorption protocol. The detection result of As2O3 concentration in the rats brain tissue was 1.53±0.62mg/Kg in As2O3 treated group and 0.040±0.014 mg/Kg in control group, the result indicated that As2O3 can pass through the blood brain barrier.Our in vivo study showed that As2O3 can inhibit human brain SHG-44 glioma cells proliferation, the inhibition effect was possibly correlative with the glioma cells apoptosis induced by As2O3,lower MMP,down regulate expression of Bcl-2 protein. While its molecular mechanism of those effect needs to be further explored.
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