Studies Of Lithium Chloride On Apoptosis And Its Mechanism Of Human Osteosarcoma U2-OS Cell

Background and purposesOsteosarcoma is a maligant tumor which originated from mesenchymal tissue. It frequently occurred in children and adolescents, the distal femur and the proximal tibia are the predilection sites. Furthermore, the tumor cells usually metastase to the lung brain and other tissues resulting in a bad recovery and high mortality rate. Before the70th, the main treatment method was amputation, but the five year suvival rate was less than20%. At present, through the new method, operation combined with adjuvant chemotherapy makes the five year suvival rate increase to about70%. But the commonly used chemotherapy drugs, such as cisplation(PDD) cyclophosphamide (MTX) have a great side effect in long term application. According to the latest study, the pathogenesis of osteosarcoma is closely related to the activation of oncogenes and the inactivation of tumor suppressor genes. Lithium is a kind of human body unessential trace elements, and it has a immune modulatory function. Studies shows that lithium has a broad spectrum antitumor effect either in vivo or in vitro, and it’s compounds form(lithium chloride) has been verified could inhibit leukemia K526HL-60cervical cancer and other tumor cells’proiferation and also could induce these cells to apoptosis in vitro cell experiments. Up to now, the study of lithium chloride on osteosarcoma U2-OS cells line has not been reported. This study is proposed to explore the mechanism of lithium chloride on U2-OS cells by observing it’s effects on cells’proliferation and apoptosis in vitro cell experiment. So as to explore a new approch for the pharmacologic treatment of osteosarcoma.Methods1. Cells culture:U2-OS cells were cultured at37℃in DMEM supplemented with10%hert-inactivated FBS in tissue culture flask under a humidified5%CO2atmosphere, applying to the experiment when they entered the logarithmic phase.2. MTT assay:the cultures were initiated in96-well paltes at a density of cells and treated for24h、48h、72h with various concentrations of LiCL respectively including five replicate for each concentrations. Cells were sequentially cultured for4h after lOul MTT were dropped into every well, furthermore, abandonning the culture media and adding200ul DMSO. Finally, determining the inhibition ratio of the cell proliferation by the microplate reader.3. Inverted fluorescent microscope observation of cell nucleus morphologic changes:different concentrations of LiCL were roled in the U2-OS cells to observe the growth of cell ragularly under inverted fluorescent microscope after24h.4. Flow cytometry analysis:the cultures were initiated in6-well paltes at a density of cells and treated for48h with various concentrations of LiCL, then gathering cells from control group, following by centrifugated with500-1000rpm/min washed once with PBS and harvested with trypsinization, finally stainning it. At4℃, data acqusition and analysis were carried out by a flow cytometry system.5. RT-PCR was used to detect the relative expression of the genes which were related to the apoptosis after the cells were roled with different concentrations of LiCL for48h.Results1. MTT shows that contrasting to the blank group, all medicine groups of LiCL could inhibit the growth of U2-OS cells, and the difference is of statistic significance (P<0.05). The inhibition ratio is increasing along with the dose and time revealling a dose-effect and time-effect relationship, the difference is of statistic significance (P <0.05). 2. Cell nucleus fluorescein stain shows that congtrasting to the blank group, the nucleus of U2-OS cells come to shrink and pyknosis by40mmol/L LiCL, and the level of nucleus stain is larger, the nucleus come to expansion and fragmentation by80mmol/L LiCL. This phenomenon is more evident in150mmol/L group, even appearring parts of apoptotic body.3. Flow cytomentry (FCM) shows that U2-OS cells were evidently induced into apoptosis and arrested in S checkpoint after being effected by dfferent concentions of LiCL.4. RT-PCR shows that the relative quntity of expression of Fas and Caspase-3genes obviously increases, and the difference is of statistic significance (P<0.05).Conclusions1. LiCL could noticeably inhibit the cells’proliferation and induce to apoptosis of human osteosarcoma U2-OS.2. The mechanism of LiCL inhibits U2-OS cells’preliferation may be closely related to the increase of expressiom of proapoptotic genes, Fas and Caspase-3.