| Background Arsenic Trioxide(ATO, As2O3) is the earliest Chinese medicine to treat tumor in human being,and it has a variety of pharmacological effects as reported. The antitumor effect of As2O3becomes a focus in recent years, however, the research on the effect on the carcinomas of central nervous system is very less, so this experiment is designed to evaluate the antitumor effect of As2O3on rat glioma cell line C6,and the anti-glioma effect of As2O3in combination with cisplatin(CDDP) is performed.This experiment is explained by two parts in this paper:The first part is to explore the inhibitory effect of As2O3in combination with CDDP on rat glioma cell C6in vitro. The second part is to discuss the inhibitory effect of Aas2O3in combination with CDDP on rat glioma cell line C6in vivo.The first part The Inhibitory Effects of Arsenic Trioxide in Combination with Cisplatin on rat Glioma Cell C6in vitroObjective This part is to investigate the As2O3in combination with CDDP effect of inhibitory proliferation, cycle arrested and induced apoptosis on rat glioma cell C6in vitro.Methods The proliferation inhibition efficiency of As2O3,CDDP and As2O3in combination with CDDP on rat glioma cell C6in vitro was assessed in different concentrations by MTT assay. Specimens were divided as control group, As2O3group, CDDP group and As2O3in combination with CDDP group according to the results of MTT.The cells growth status of C6were observed under an inverted microscope, and cell cycle distribution and apoptosis ratios of C6cells were detected by flow cytometry.The relative protein of C6cells were detected by Western blot.Results The results detected by MTT assay were as follows:The inhibitory proliferation ratios of C6cells after24h were2.28±0.16%,15.32±0.53%and34.42±0.48% respectively in different concentrations of(3.125,6.25,12.5μmol/L) As2O3.The inhibitory proliferation ratios of C6cells after24h were8.63±0.22%,21.32±0.51%and31.59±0.79%respectively in different concentrations of (2.5,5,10μmol/L)CDDP. The inhibitory proliferation ratios of C6cells after24h were20.28±0.51%,55.43±0.73%and76.33±0.38%respectively in different concentrations of (3.125+2.5,6.25+5,12.5+10μmol/L) AS2O3in combination with CDDP, compared with the ratios of control group, and the difference was significant statistically (P<0.05).The C6cells status were observed under the inverted microscope:C6cells were dead partially in CDDP group and AS2O3in combination with CDDP group, especially AS2O3in combination with CDDP group was outstanding.Compared with the control group and AS2O3group, C6cells grew more slowly and were less in CDDP group and AS2O3in combination with CDDP group.The difference of C6cells status was not obvious between the control group and AS2O3group.The cell cycle distribution detected by flow cytometry showed:The C6cells were treated by control,As2O3,CDDP and AS2O3in combination with CDDP after24hours, and the proportions of cells in G2phase were3.01±0.32%,9.93±0.41%,40.18±0.73%and60.18±0.56%respectively, and AS2O3group.CDDP group and AS2O3in combination with CDDP group compared with the control group, the difference of proportions was significant statistically (P<0.05).The cell apoptosis ratios detected by flow cytometry showed:The C6cells were treated by control,As2O3,CDDP and AS2O3in combination with CDDP after24hours, and the cell apoptosis ratios were2.39±0.03%,4.61±0.02%,14.59±0.12%and36.14±0.39%respectively,and CDDP group and AS2O3in combination with CDDP group compared with the control group, the difference of proportions was significant statistically (P<0.05).The difference of apoptosis ratios was not obvious between AS2O3group and control group statistically (P>0.05).The relative protein detected by Western blot showed:The C6cells were treated by control,As2O3,CDDP and AS2O3in combination with CDDP after24hours, and the expression level of Caspase-3protein were10.15±3.17%,15.38±2.56%,30.69±2.42%and41.56±1.43%respectively. The difference of expression level of Caspase-3protein was not obvious between AS2O3group and control group statistically (P>0.05), and CDDP group and AS2O3in combination with CDDP group compared with the control group, the difference of expression level of Caspase-3protein was significant statistically (P<0.05).The C6cells were treated by control,As2O3,CDDP and AS2O3in combination with CDDP after24hours, and the expression level of PTEN protein were10.19±3.16%,13.38±2.56%,25.69±2.41%and35.56±1.44%respectively. The difference of expression level of PTEN protein was not obvious between AS2O3group and control group statistically (P>0.05), and CDDP group and AS2O3in combination with CDDP group compared with the control group, the difference of expression level of PTEN protein was significant statistically (P<0.05).The C6cells were treated by control,As2O3,CDDP and AS2O3in combination with CDDP after24hours, and the expression level of PI3K protein were55.19±3.27%,43.38±2.58%,15.69±2.41%and10.56±1.44%respectively.The difference of expression level of PI3K protein was not obvious between AS2O3group and control group statistically (P>0.05),and CDDP group and AS2O3in combination with CDDP group compared with the control group, the difference of expression level of PI3K protein was significant statistically (P<0.05).The C6cells were treated by control,As2O3,CDDP and AS2O3in combination with CDDP after24hours, and the expression level of Akt protein were65.19±3.27%,53.38±2.58%,25.69±2.41%and15.56±1.44%respectively.The difference of expression level of Akt protein was not obvious between AS2O3group and control group statistically (P>0.05), and CDDP group and AS2O3in combination with CDDP group compared with the control group, the difference of expression level of Akt protein was significant statistically (P<0.05).Conclusion AS2O3combined with CDDP is able to inhibit the proliferation, of glioma cell line C6,and the proliferation inhibitory effect is taken action may by arresting the G2 phase,up-regulating Caspase-3and PTEN expression level and down-regulating PI3K and Akt expression level and inducing C6cells apoptosis.The second part The Inhibitory Effects of Arsenic Trioxide in Combination with Cisplatin on rat Glioma Cell C6in vivoObjective This part is to investigate the AS2O3in combination with CDDP effect of inhibitory proliferation and induced apoptosis on rat glioma cell C6in vivo.Methods The proliferation and apoptosis effects of control, As2O3,CDDP and AS2O3in combination with CDDP on rat glioma cell C6in vivo were assessed by HE assay.The expression level of Caspase-3and IκB-α of control, As2O3,CDDP and AS2O3in combination with CDDP on rat glioma cell C6in vivo were measured by immunohistochemical methods,and the rats’ life span was observed.Results The results detected by HE assay were as follows:The proliferation inhibitory effect of glioma cell C6in vivo were treated by control,As203,CDDP and AS2O3in combination with CDDP after7days.The result of the control group was as follow:Lots of glioma cell C6were gathering, and cytoblasts were big and hyperchromatic,and cytoplasmic was less and red by dyed,and glioma cells C6death by apoptosis and necrosis were not obvious. Some nuclears of glioma cell C6were enrichment or cracking,and cytoplasm was increasing and red by dyed,and somas were obvious taper in AS2O3group,CDDP group and AS2O3in combination with CDDP group.Particularly,there were los of tumor necrosis tissue in AS2O3combined with CDDP group.The results detected by immunohistochemical methods were as follows:The glioma cells C6were treated by control,As2O3,CDDP and AS2O3in combination with CDDP in vivo after7days, and the expression level of Caspase-3were0.52±0.08%,1.15±0.09%,2.54±0.14%and5.61±0.22%respectively.The expression level of Caspase-3was difference between AS2O3group and cortrol group statistically (P<0.05), and CDDP group and AS2O3in combination with CDDP group compared with the control group, the difference of expression level of Caspase-3was significant statistically (P<0.001). The glioma cells C6were treated by control,As203,CDDP and AS2O3in combination with CDDP in vivo after7days, and the expression level of IκB-α were3.47±0.19%,2.29±0.12%,1.04±0.11%and0.48±0.12%respectively.The difference of expression level of IκB-α was not obvious between AS2O3group and control group statistically (P>0.05), and CDDP group and AS2O3in combination with CDDP group compared with the control group, the difference of expression level of IκB-α was significant statistically (P<0.05).In orthotopic glioma model, in combination with CDDP, AS2O3can significantly prolong survival in tumor-bearing rats (compared with the control group, P<0.05).Conclusion AS2O3in combination with CDDP is able to inhibit the proliferation of glioma cell line C6,and the proliferation inhibitory effect is taken action may by up-regulating Caspase-3and down-regulating IκB-α expression level and inducing C6cells apoptosis,and prolong survival in tumor-bearing rats. |
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