| ObjectiveTo explore the effects of baicalin on extranodal NK/T cell lymphoma(ENKTCL)cell growth and drug sensitivity,and to elucidate the molecular mechanism of baicalin regulating ENKTCL cell growth through FOXO3/CCL22 signaling pathway based on my previous research.Method1.Human ENKTCL cell lines SNK-6 and YTS were selected.After baicalin treatment,cell proliferation was detected by Ed U method,cell apoptosis was detected by FCM method,and the expressions of BCL2,Bax,FOXO3 and CCL22 were detected by Western blot.To investigate the effects of different concentrations of baicalin on the proliferation and apoptosis of the two cell lines.2.SNK-6 cells were transfected with FOXO3 si RNA interference plasmid or CCL22pc DNA overexpression plasmid to establish FOXO3 silencing and CCL22 overexpression models.The experiments were divided into control group,Baicalin group,FOXO3 si RNA interference plasmid transfection group,CCL22 pc DNA overexpression plasmid transfection group,and negative control group of interference and overexpression group respectively.Cell apoptosis was detected by FCM method,and BCL2,Bax,FOXO3 and CCL22 proteins were detected by Western blot to explore the molecular mechanism of baicalin on regulating the growth of ENKTCL cells.3.SNK-6 cells were inoculated subcutaneously into nude mice to establish tumor formation model.SNK-6 cells were treated with baicalin at a final concentration of20μmol/L,and 100μl cells containing 5×10~6cells were inoculated subcutaneously.The time of tumor occurrence in nude mice was recorded,and the tumor of each group was measured every 5 days to calculate the tumor volume.5 weeks after inoculation,the tumor was removed and weighed.The expression of CCL22 and FOXO3 protein in ENKTCL tissue of nude mices was measured by Western blot.The effect of baicalin on tumor growth in vivo was verified by animal experiments.4.SNK-6 and YTS cells were treated with baicalin combined with cisplatin.CCK-8method was used to determine the optimal action concentration and timing of cisplatin.Apoptosis was detected by FCM,and BCL2,Bax,cleaved caspase3,caspase3 protein expression was detected by Western blot.To investigate the effects of baicalin and cisplatin at different concentrations on the growth of SNK-6 and YTS cells,and to observe whether baicalin enhances the drug sensitivity of cisplatin through apoptosis mediated pathway.Result1.SNK-6 and YTS cells were treated with 5,10 and 20μmol/L baicalin there was a significant concentration-dependent increase in apoptosis rate(r=0.93 and 0.91)and decrease in survival rate(r=-0.98 and-0.99),respectively.After baicalin treatment,the BCL-2 protein expression in SNK-6 and YTS cells was decreased(P<0.001),while the Bax protein expression in SNK-6 cells was increased only when baicalin concentration was20μmol/L(P<0.001).Bax protein expression in YTS cells were all increased after baicalin treatment(P<0.001).The results showed that Baicalin inhibited proliferation and promoted apoptosis of ENKTCL cells by up-regulating Bax protein and down-regulating BCL-2protein.2.After baicalin treatment,the expression of FOXO3 protein in SNK-6 and YTS cells was significantly increased(P<0.01),while the expression of CCL22 protein was significantly decreased(P<0.001).After the FOXO3 si RNA interference plasmid was transfected into SNK-6 cells,the expression of FOXO3 protein in the transfection group was significantly decreased compared with the negative control group(P<0.01),and the expression level of CCL22 protein was significantly increased(P<0.01).After CCL22pc DNA overexpression plasmid was transfected into SNK-6 cells,the expression of CCL22 protein in the transfection group was significantly higher than that in the negative control group(P<0.05),while the expression of FOXO3 protein was not significantly changed.Both FOXO3 silencing and overexpression of CCL22 increased the protein expression of apoptosis suppressor BCL-2(P<0.001)and decreased the protein expression of apoptosis-promoting Bax(P<0.001).Cell apoptosis rate in FOXO3 si RNA interference plasmid transfection group and CCL22 pc DNA overexpression plasmid transfection group was significantly decreased compared with negative control group(P<0.001).It is suggested that baicalin is involved in the regulation of apoptosis of ENKTCL cells through FOXO3-mediated regulation of CCL22.3.The tumor formation model of nude mice showed that the tumor volume in the control group increased rapidly,reaching 372.5±106.1 mm~3at 35 days,and the tumor weight was 0.21±0.04 g.The average tumor volume in the experimental group was165.1±28.1mm3 at 35 days,and the tumor weight was 0.10±0.02 g,and the tumor volume was significantly reduced compared with the control group.The tumor weight was significantly lighter(P<0.05)than that of the control group.The expression of CCL22 and FOXO3 protein in ENKTCL tissue of nude mice were detected by Western blot.The results showed that the expression level of CCL22 protein in baicalin treatment group was significantly decreased compared with the control group(P<0.01),and the expression level of FOXO3 protein was significantly increased compared with the control group(P<0.05).These results indicated that baicalin inhibited tumor growth in nude mice by down-regulating CCL22 and up-regulating FOXO3.4.After the exploration of drug concentration in the early stage,the concentration of cisplatin was selected as 10μmol/L.When the concentration of baicalin and cisplatin were both 10μmol/L,a significant synergistic effect was observed.When baicalin concentration was 20μmol/L and cisplatin concentration was 10μmol/L,the results indicated that the combination of baicalin and cisplatin was mainly superposition with the increase of baicalin concentration.Compared with the monotherapy group,the proliferation rate of SNK-6 and YTS cells in the combination group was significantly decreased(P<0.001),and the apoptosis rate was significantly increased(P<0.001).For the apoptosis-related protein expression in SNK-6 and YTS cells,the anti-apoptosis-protein expression level of BCL-2in 10μmol/L baicalin+10μmol/L cisplatin group was decreased compared with that in10μmol/L cisplatin group(P<0.01,P<0.001).The expression level of the pro-apoptotic Bax protein was increased(P<0.05,P<0.01),cleaved caspase3 protein expression was significantly increased(P<0.001 for all),and compared with baicalin(10μmol/L)+10μmol/L cisplatin group(10μmol/L),cleaved caspase3 protein expression was increased(P<0.01,P<0.001).In SNK-6 cell lines,the expression level of Bax proapoptotic protein in 20μmol/L Baicalin+10μmol/L cisplatin group was higher than that in 10μmol/L cisplatin group(P<0.01).In YTS cell lines,only cleaved caspase3 protein expression was increased(P<0.001)in the 20μmol/L baicalin+10μmol/L cisplatin group compared with baicalin mono group at the same dose.Therefore,in the 10μmol/L baicalin+10μmol/L cisplatin group,compared with the 10μmol/L cisplatin group,the combination increased the expression of pro-apoptotic protein and down-regulated anti-apoptotic protein more significantly,suggesting that baicalin could enhance the anti-tumor effect of cisplatin by regulating apoptosis pathway.ConclusionBaicalin inhibits proliferation and promotes apoptosis of ENKTCL cells by up-regulating the expression of pro-apoptotic protein Bax and down-regulating the expression of apoptosis suppressor gene BCL-2,which is regulated by FOXO3/CCL22signaling pathway.At the same time,baicalin can enhance the drug sensitivity of cisplatin,providing a theoretical basis for a new clinical treatment strategy,especially for the adjuvant therapy of traditional Chinese medicine. |
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