Se-Methylselenocysteine Induces Undifferentiated Thyroid Cancer Cell Apoptosis And The Underlying Molecular Mechanisms

Objective: Thyroid cancer is one of the most common tumors in the endocrine system.The clinical mortality rate of undifferentiated thyroid cancer remains high due to its rapid growth,easy metastasis,high malignancy and poor prognosis.There is no effective clinical treatment for this disease now.Therefore,there is an urgent need to seek effective drugs or improve its efficacy for undifferentiated thyroid cancer.The object of this study is to evaluate the effect of Se-Methylselenocysteine(MSC)on the proliferation of Human thyroid cancer cells BHT101 as well as 8305 C cells and explored its underlying molecular mechanisms.This study constructs the experimental theoretical basis for inducing apoptosis of undifferentiated thyroid cancer cells preliminarily and provides new ideas for the treatment of undifferentiated thyroid cancer.Methods:1.The CKK-8 assay was used to detect the cell viability.Control groups and different concentrations of MSC groups were set up,the concentrations of MSC were25?M?50 ?M?100 ?M?150 ?M?200 ?M?400 ?M.The effects of different concentrations of MSC on proliferation of tumor cells were tested.The meaningful concentration groups were selected and followed up.2.Set up control groups and different concentrations of MSC groups 50 ?M?100?M?150 ?M,the flow cytometry was used to detect the effect of Se-Methylselenocysteine on cell cycle distribution,cell apoptosis and mitochondrial membrane potential.3.Set up control groups and different concentrations of MSC groups 50 ?M?100?M ? 150 ?M,The Hochest 33258 was used to detect the nuclear shrinkage,condensation and DNA fragmentation.4.Set up control groups and different concentrations of MSC groups 50 ?M?100 ?M? 150 ?M,The western blot was used to detect the apoptosis related protein and explore the underlying mechanism of Se-Methylselenocysteine on the BHT101 cells and 8305 C cells.5.The DCFH-DA assay was used to analyse the ROS in the BHT101 cells and8305 C cells.Results:1.CKK-8 assay showed that Se-Methylselenocysteine(MSC)inhibited the proliferation of anaplastic thyroid carcinoma cells in a dose depend manner.MSC inhibited the proliferation of cancer cell in the concentrations of 50 ?M,in the concentrations of 25?M,after 48 h and 72 h,it inhibited the proliferation of cells too.2.The flow cytometry showed that MSC induced periodic block of anaplastic thyroid carcinoma cells.The G1 phase was reduced at higher concentrations of 150 ?M from62.616 % to 34.085 % significantly,the S phase and G2 phase increased significantly from24.871 % and 12.513 % to 43.037 % and 22.868 % respectively.The presence of a distinct subdiploid peak(sub G1)was also observed from 20 % of 50 ?M to 60 % of 150 ?M,which was in a dose depend manner.3.The results of Hoechst 33258 staining showed that nuclear consolidation was induced by MSC,and the effect of MSC of 50,100 and 150 ?M was further confirmed by Anaxin-FITC PI.Anaxin V-FITC PI was further confirmed to increase the expression of BHT 101 apoptosis in the cells of 50,100 and 150 ?M by the expression of MSC after48 h.The apoptosis of BHT 101 cells increased from 15.3 % to 42.2 %,and similar phenomena was observed in the 8305 c cells,the apoptosis of 8305 c cells increased from10.8 % to 30.7 %.4.The expression of Cytc in cytoplasm and mitochondria was measured by Western blot,which showed that the expression of Cytc in mitochondria decreased after MSC,while the expression of Cytc in cytoplasm increased,suggesting that the apoptosis induced by MSC was related to mitochondria.It was also found that the expression of antiapoptosis protein Bcl-2 decreased after MSC,the expression of apoptosis protein Bax increased,and the expression of Active-Caspas3 and Cleved-PARP increased,showing a certain dose dependence.WB grey level statistics showed that the Bcl-2X decreased with increasingconcentrations of MSC,The results showed that the apoptosis induced by MSC was caspase dependent.5.PI3 K / Akt signal pathway test showed that MSC induced apoptosis by inhibiting PI3 K / Akt signal pathway.The phosphorylation of PI3K?Akt and m TOR were inhibited by MSC,and the archetypal expression was raised simultaneously,which in a dose depend manner.The ratio of p-PI3K/PI3K?p-Akt/Akt and p-m TOR/m TOR decreased along with the MSC increased.However,the expression level of the prototype and active form of GSK-3 ?(p-Tyr216)increased,the level of the inactivation form GSK-3 beta(P-Ser9)decreased,the expression level of Mcl-1 decreased,the expression of survivin decreased after MSC,the prototype of Bad increased and its phosphorylation decreased with the increase of MSC concentration.6.The DCHF-DA fluorescence probe detected that MSC could induce large amounts of ROS in tumor cells.The fluorescence of DCF after MSC treatment was detected by DHF-DA fluorescence probe.The higher the concentration of MSC,the higher the average fluorescence value,the higher the ROS was observed,which was in a dose depend manner.Conclusions: MSC inhibited the proliferation of human anaplastic thyroid carcinoma BH-101 cells and 8305 C cells significantly and caused cellular cycle block,MSC induced intracellular accumulation of ROS and then inhibited the activity of PI3K/Akt pathway,increasing mitochondrial membrane permeability,decreasing mitochondrial membrane potential and releasing apoptosis related factors,resulted in caspase-dependent apoptosis.MSC induced apoptosis of undifferentiated thyroid cancer cells BHT 101 and 8305 C,It was one of the promising drugs for undifferentiated thyroid cancer and deserved further study.This article also provided new ideas for the treatment of undifferentiated thyroid cancer.