Selenocystine Inhibits The Growth Of Human Glioma Cells Through Induction Of Mitochondria-mediated Apoptosis

Objective：To examine the effects of selenocystine (SeC) on the proliferation of human glioma cellsand to elucidate the apoptotic signaling pathways explore the possible mechanism.Methods:（1） MTT assay was employed to determine the growth inhibition of SeC on humanglioma cells, including U87, U251and SWO-38cells.（2） Flow cytometry was used to analyze the effect of SeC on the cell cycledistribution.TUNEL-DAPI co-staining assay was used to confirm the inducing ofapoptosis.（3） The change in mitochondrial membrane potential in cells exposed to SeC wasexamined by flow cytometry using a mitochondria-selective JC-1dye. Cellularimmunofluorescence detection of cleaved caspase-3，cleaved caspase-9. The proteinlevels of caspase and Bcl-2family members in SWO-38cells treated withselenocystine at various concentrations (5,10,20μΜ) were examined by Westernbloting.Results:1. The effects of Selenocystine on the proliferation of human glioma cell linesThe results of MTT assay show that compared with the control group,selenocystine significantly inhibited the proliferation of U87and SWO-38humanglioma cell, even at the concentration of10μΜ. However, SeC showed lowereffection on U251cells. The inhibition rates rose with the dosage of Selenocystineincreasing, which indicated Selenocystine inhibited the proliferation of humanglioma cell U87, U251and SWO-38cells in a dose-dependent manner. Theinhibition effect in SWO-38cells was more significant than U87and U251cells. 2. The effects of Selenocystine on the cell cycle and apoptosis of SWO-38cellsThe alternation of the cell cycle was measured by flow cytometry in SWO-38cells treated with Selenocystine at different concentrations (5,10and20μΜ) for24h. The data showed that the percentage of the cells at S phase cells increased andG2/M phase decreased, in response to SeC treament. Selenocystine blocked the celltransition from S phase to G2/M phase in dose-independent manner and inducedS-phase arrest. The result of TUNEL assay showed that SWO-38cells exhibitedtypical morphological change of apoptotic cells under fluorescent microsocope,after treated with selenocystine at various concentrations (5,10,20μΜ) for48h.Moreover, we showed that selenocystine induced a dose-dependent increase in thesub-G1cell population, which reached7.27%and15.44%after treatment with5μM and20μM SeC for24h, respectively. Selenocytine induced SWO-38gliomacells apoptosis.3. The effect of Selenocystine on apoptosis in SWO-38cells and its possiblemechanismsSeC causes the depletion of mitochondrial membrane potential by regulatingthe expression of Bcl-2family proteins. Using JC-1as a marker of△ψ m, flowcytometric studies revealed that SeC induced a dose-and time-dependent loss of△ψm. Western bloting analysis revealed that SeC treatment suppressed theexpression of pro-survival Bcl-2family proteins, such as Bcl-2、Bcl-xL and Mcl-1,and moderately increased the expression levels of pro-apoptosis Bcl-2familyproteins, such as Bax、Bim、 Bak.Conclusion:1. Selenocystine inhibited the growth of human glioma cell U87, U251and SWO-38in a dose-and time-dependent manner.2. Selenocystine induced apoptosis in SWO-38human glioma cells via themitochondrial apoptotic pathway. The anti-tumor mechanisms are involved in pro-survival Bcl-2family inhibition and pro-apoptosis Bcl-2family induction,disrupting mitionchondrial membrane potential and release of cytochrome c, whicheventually leads to the activation of caspases-9and caspases-3.