Study On The Combined Effects Of Arsenic Disulfide And Tetrandrine On Triple Negative Breast Cancer

ObjectivesThis research was building on the basis of previous researches,exploring the combined effect of arsenic disulfide(As2S2)and tetrandrine(Tet)on the triple-negative breast cancer(TNBC)was the objective of this research.Methods:The research was divided into two parts:in vitro cell experiments and in vivo experiments.(1)In vitro cell experiments:①MTT was used to detect the inhibition rate of HCC1937 by using As2S2 and Tet(combined or single)on different time points and different doses.Chou-Talalay was used for judging the combined effect of two drμgs and choosing the best drμg concentrations;Observing the morphological changes of HCC1937 cells by using As2S2 and Tet after 48H;(2)Flow cytometry double staining method was used to observe the two drμgs(combined or single)effect on affecting HCC1937 apoptosis.③Detectd the two drμgs(combined or single)effect on affecting HCC1937 invasion by Transwell;@Detectd the two drμgs(combined or single)effect on affecting AQP-9,ABCC2,P-gp of HCC1937 by Elisa;⑤Detectd the two drμgs(combined or single)effect,after 48H,on affecting the expreesion of apoptosis-related genes Caspase-3,survivin,Bid,Bax,Bcl-2,PARP by qPCR;⑥Western Blot was used to detect the two drμgs(combined or single)effect on the expression of Caspase-3,survivin,Bid,Bax,Bcl-2,PARP in HCC1937.(2)In vivo experiments:established TNBC model on BALB/c nude mice;observed the effects of Realgar and Tet(combined or single)on suppressing tumor growth,and used pathological section analysis,immunohistochemistry,qPCR,Western Blot,etc.to expound the mechanism of drμgs.Results:In vitro cell experiments:(1)MTT:As2S2 at concentration of 2.5 to 40μg/ml and Tet at concentration of 2-12 μg/ml can inhibit the proliferation of HCC1937 cells after 24H,48H,and 72H.And the effect of drμgs were positively correlated with concentration and time.Using drμgs on HCC1937 after 48H,the combined concentration of As2S2 and Tet,18μg/ml+12μg/ml and 1.5μg/ml+1μg/ml were the antagonistic effect;3μg/ml+2μg/ml was the additive effect;6μg/ml+4μg/ml was synergistic effect.When the combined concentration at 3μg/ml+2μg/ml and 6μg/ml+4μg/ml,the inhibitory effect of combined drμgs on HCC1937 cells was significantly stronger than that of single drμg.The lethality of two drμgs(combined or single)was increased with concentration,and when combined drμgs(As2S2+Tet)concentration at 3μg/ml+2μg/ml and 6μg/ml+4μg/ml,the effect was better than single drμg at the same concentration.(2)As2S2(3～18μg/ml)and Tet(2～12μg/ml)can cause HCC1937 apoptosis after 48H,and the effect of drμgs were positively correlated with concentration.Combined can also cause apoptosis,3μg/ml+2μg/ml and 6μg/ml+4μg/ml were better than single.The synergistic concentration 6μg/ml+4μg/ml was superior.(3)As2S2 and Tet(combined or single)can inhibit the invasiveness of HCC1937 at high concentrations,however,the efficacy of the combined drμg was not significantly different from the same concentration single drμg.(4)After 48H,As2S2 and Tet(combined or single)acted on HCC1937,the expression of AQP-9,ABCC2 and P-gp were were down-regulated,and combined was significantly better than the single,the synergistic concentration 6μg/ml+4μg/ml was superior.(5)As2S2 and Tet(combined or single)acted on HCC1937 after 48H,the expression of Caspase-3,Bax,and Bid was up-regulated,Bcl-2 and PARP were down-regulated,which can affect the apoptosis of HCC1937.And combined was significantly better than the single.In vivo experiments:(1)Realgar and Tet(combined or single)can inhibit the growth of tumor,and combined was better than single.(2)Realgar and Tet(combined or single)did not damage the liver and kidney of nude mice.(3)Histopathological analysis showed:Realgar and Tet(combined or single)could inhibit the tumor and kill the tumor cells,combined was better than single.(4)Immunohistochemistry,qPCR,and Western blot showed:Realgar and Tet(combined or single)could up-regulate Caspase-3 and Bax expression,and down-regulate Bcl-2.Combined was better than single.Conclusions:As2S2 and Tet(combined or single)can significantly inhibit HCC1937,and under the condition of synergistic or additive concentration,combined is superior to single which is at the same concentration.The mechanism for this effect relates to the apoptosis,As2S2 and Tet(combined or single)can control Caspase-3,Bax,Bcl-2,Bid and other apoptosis-related factors induce apoptosis of tumor cells.The mechanism of the synergistic effect of the two drμgs is presumably based on affecting AQP-9,ABCC2 and P-gp,which can affect the arsenic content and drμg resistance in the cells.Realgar and Tet(combined or single)can effectively inhibit tumor growth in vivo.The mechanism of action related to that the drμgs can regulate apoptosis factors,such as Caspase-3,Bax,Bcl-2,Bid,etc.,and combined shows a better effect than single.