Apoptosis Induced By Disodium Cantharidinate And Vitamin B6 Injection In Human Osteosarcoma MG-63

Objective:this study was designed to investigate human osteosarcoma MG-63 cells apoptosis induced by Disodium Cantharidinate and Vitamin B6 Injection and its molecular mechanisms.It may provid experimental foundation for osteosarcoma.Methods: osteosarcoma MG-63 cells were cultured in the environment of 37℃5% CO2 with modified Eagle medium (MEM)medium.The logarithmically growing MG-63 cells were used.1 The suppressive effects of Disodium Cantharidinate and Vitamin B6 Injection on the proliferaition of MG-63 cells were evaluated in vitro by MTT colorimetricassay. After cultured for several generations,the cells were digested and transferred into 96-well culture plates. after adherenced,add to Disodium Cantharidinate and Vitamin B6 Injection,culture 24,48,72 hours,add to MTT 10ul,incubation 4 hours,abandon to supernate fluid,add to DMSO, to swing for 5 min,determin OD492 with analysator,according to formula calculate inhibition ratio.2 Using light microscope observe the change of morphology: MG-63 cells were treated with different concentrations of Disodium Cantharidinate and Vitamin B6 Injection for 24,48,72 hours,the change of morphology was observed by inverted phase contrast microscope in different times and photographed.3 Flow cytometric analysis:MG-63 cells were treated with Disodium Cantharidinate and Vitamin B6 Injection for 24h.Experimental group and control group cells were harvested and washed twice with PBS.Cells were fixed with ice cold 70% ethanol at 4°C. Precipitates were digested with 0.5% pepsin after centrifugation.And then cells were resuspended in 0.5 mL propidium iodide(PI)/RNase A solution.Cells were incubated in the dark at room temperature for 15 min.The fluorescence emission of stained cells was measured with a flow cytometer.Data were analyzed with Multipcycle software.4 Morphology analysis by transmission electron microscopy: MG-63 cells treated with different concentrations of Disodium Cantharidinate and Vitamin B6 Injection for 24 h, and pre-fixed with 2.5% glutaraldehyde at 4°C.The cells were postfixed for 1 h with 1% osmium tetroxide,then dehydrated through a graded ethanol series,and embedded in Epon 812.The ultrastructure of cells was analyzed in ultrathin sections in a transmission electron microscope after the sections were stained with uranylacetate and lead citrate.5 Reverse transcription polymerase chain reaction (RT-PCR) were used to observed Survivin mRNA expression: The cells were collected after 24h of culture.Total RNA of MG-63 cells was extracted.Inverse transcription and amplification of Survivin mRNA were done using one-step method.The production was detected using electrophoresis method and then was analyzed using imaging system and analysis software.The relative amount of Survivin mRNA was accessed by the ratio of Survivin absorption factor andβ-actin absorption factor.Results: 1.MG-63 cells were treated with at various Disodium Cantharidinate and Vitamin B6 Injection concentra- tions for 24h,48h,and 72h respectively,the growth of cells was inhibited significantly in a time and dose-dependent fashion. The biggest inhibitory rate was (68.56±5.21)%.2 Inverted phase contrast microscope observed,comparing with control group cells,the growth of drug-treated group cells were inhibited,cell granulations were increased and thicken,some cells became round and Suspended in the medium,internal structure of cells were demolished, the cells of survival notably decreased.When the concentration reached to 5.0μg/ml,most of the cells lysised and could observe numerous cell debris.Electron microscope revealed the characteristic apoptosis aletrations,shrinking cellular morphology,integrity cell membr- ane,karyopycnosis,fragment of nucleus,chromatin condensation and margination,crescent nucleus,cytoplasmiec vacuoles.3 After cells were exposed to various Disodium Cantharidinate and Vitamin B6 Injection concentrations for 24h,The quantity of treated cells in G0/G1 phase increased but that in S,G2/M phase decreased. After cells were exposed to the concentration of 2.5μg/ml for 24h , MG-63 cells rates in G0/G1 phase were 75.46 ±1.72% whlie MG-63 cells rates in G0/G1 phase were 51.08±2.42% in control group. But that in S,G2/M phase decreased. The analysis of cellular DNA content by FCM showed that the apoptosis rates become higer with drug concentration increased.the apoptosis rates was 9.60±0.65%,which was significantly higher than control 1.09±0.18% (P<0.01).The analysis of cellular DNA content by FCM showed that there was a sub-G0/G1 peak in the graph of drug-treated groups(0.5μg/ml,1.0μg/ml,2.5μg/ml).That was a typical apoptotic peak,which was not shown in the graph of control groups. The result of RT-PCR showed that expression of Survivin mRNA was down-regulated.Conclusion:Disodium Cantharidinate and Vitamin B6 Injection can inhibit proliferation and induce apoptosis of MG-63 cells.Its possible molecular mechanisms might be related to modulation the expression of Survivin.The Precise mechanisms should be investigated further.