| Aims 1.To explore the changes and molecular mechanisms of cell sensitivity to PI3 K inhibitor LY294002 after down-regulating expression of Rictor in ESCC.2.To explore the relationship between the expression of SGK3 and the cell sensitivity to Akt inhibitor MK2206 and its mechanisms in ESCC.Methods The molecular mechanisms of down-regulating Rictor enhanced cell sensitivity to LY294002 in ESCC in vitro.1.The effects of LY294002 on the proliferation of ESCC cells were detected by CCK-8 assay,and the expression of related proteins in PI3K/Akt/m TOR pathway was detected by Western blot after cells were treated with LY294002.2.The results of our previous study showed that down-regulation of Rictor enhanced cell sensitivity to LY294002 in ESCC in vitro.In the study,the expression of related proteins in PI3K/Akt/m TOR pathway was detected by Western blot after cells with Rictor sh RNA were treated with LY294002.The anti-tumor effects and the molecular mechanisms of LY294002 on tumors from cells with Rictor sh RNA 1.The xenografts of nude mice were established by ECa109-Rictor-sh RNA and ECa109-control-sh RNA cell lines,and then the mice were treated with LY294002.The effects of LY294002 on tumor growth and apoptosis in vivo were detected by H&E staining and TUNEL assay.2.The total proteins of tumor tissues were extracted and the expression of related proteins in PI3K/Akt/m TOR pathway in vivo was detected by Western blot.The effects and molecular mechanisms of MK2206 on the proliferation of ESCC cells.1.CCK-8 assay was used to detect the effects of MK2206 on the proliferation of ESCC cells after ECa109 and EC9706 cells treated with MK2206 for 24 or 48 h.2.The total proteins of ECa109,EC9706,KYSE70,TE-1 and KYSE140 were extracted,and the expression level of SGK3 in the five ESCC cell lines was detected by Western blot.3.ECa109 and EC9706 cells were treated with 2 ?M MK2206 for 0 h,3 h,1 d,3 d and 5 d respectively,then the total proteins were extracted and the expression of Akt and SGK3 were detected by Western blot.Changes of cell sensitivity to MK2206 and molecular mechanisms after SGK3 was down-regulated in ESCC.1.ECa109 and EC9706 cells were transfected by three kinds of lentivirus carrying SGK3-sh RNA and the stably expressed ECa109-LV3-SGK3 and EC9706-LV3-SGK3 cells were screened by puromycin,and then the transfection efficiency was detected by Western blot.2.The ECa109 and EC9706 cells with the highest down-regulation efficiency of SGK3 were selected,respectively.The effects of MK2206 on proliferation,apoptosis,clone information and migration of cells with LV3-SGK3 were investigated by CCK-8 assay,flow cytometry,clone formation assay and wound healing assay.3.The ECa109 and EC9706 cells with LV3-SGK3 were treated with MK2206 for 0 h,3 h,1 d,3 d and 5 d,and the expression of Akt and SGK3 were detected by Western blot in ESCC.Results Down-regulation of Rictor enhanced cell sensitivity to LY294002 by off-setting the activation of Akt/PRAS40 pathway in ESCC in vitro.1.LY294002 inhibited proliferation of ECa109 and EC9706 cells.The IC50 of ECa109 and EC9706 cells were 45.62 ± 3.2 ?M and 48.03 ± 1.68 ?M at 24 h,19.17 ± 3.14 ?M and 16.47 ± 1.22 ?M at 48 h,respectively.2.LY294002 inhibited the PI3K/Akt/m TOR pathway.LY294002 decreased the expression of PI3Kp85?,p-Akt(Thr308)and p-p70S6K(Thr389)in a dose and time-dependent manner,but increased the expression of p-Akt(Ser473)and p-PRAS40(Thr246).3.Down-regulation of Rictor blocked the activation of Akt/PRAS40 pathway.The expression of Rictor in ECa109-Rictor-shRNA cells was significantly decreased.Down-regulation of Rictor enhanced the inhibitory effects of LY294002 on PI3K/Akt/m TOR pathway,and offset the activation of Akt/PRAS40 pathway in ESCC.Down-regulation of Rictor enhanced the inhibitory effects of LY294002 on the tumor growth of ESCC 1.Down-regulation of Rictor enhanced the growth-inhibiting and apoptosis-inducing effects of LY294002 on tumors.LY294002 inhibited the growth of tumor and down-regulation of Rictor enhanced the anti-tumor effects of LY294002;H&E staining and TUNEL assay showed that LY294002 promoted the apoptosis of ESCC cells,and down-regulation of Rictor enhanced the effects of LY294002 on the apoptosis of ESCC cells in vivo.2.The down-regulation of Rictor weakened the activation of LY294002 on Akt/PRAS40 pathway in vivo.Consistence with the in vitro study,the results of molecular mechanisms in vivo showed that down-regulation of Rictor could offset the activation of LY294002 on Akt/PRAS40 pathway in ESCC.The activity of Akt decreased and the expression of SGK3 increased after ESCC cells were treated with MK2206 for a long time.1.MK2206 could inhibit the proliferation of ESCC cells.The expression levels of SGK3 in the five ESCC cell lines were different;MK2206 could inhibit proliferation of ECa109 and EC9706 cells,the IC50 of ECa109 and EC9706 were(25.37±1.40)and(25.58±1.41)?M at 24 h,were(11.25±1.05)and(14.15±1.15)?M at 48 h,respectively.2.The activity of Akt was inhibited but the expression of SGK3 was promoted after cells treated with MK2206 for a long time.Expressions of p-Akt(Ser473),p-Akt(Thr308)and p-PRAS40(Thr246)were decreased,and the expressions of p-p70S6K(Thr389),SGK3 and p-SGK3(Thr320)were up-regulated after cells the long-term treatment with MK2206.Down-regulation of SGK3 enhanced the sensitivity to MK2206 of ESCC cells 1.Down-regulation of SGK3 enhanced the cell sensitivity to MK2206 in ESCC.The interfer efficiency in ECa109-LV3-SGK3-home-1077 and EC9706-LV3-SGK3-home-1071 cells was the highest,respectively.After SGK3 was down-regulated in ECa109 and EC9706 cells,MK2206 inhibited cells in G1 and G2 phase better,and the inhibitory effects of MK2206 on the cell proliferation,clone formation and cell migration were also enhanced compared with control cells.2.When cells were treated with MK2206 for a long time,the expression levels of p-Akt(Ser473),p-Akt(Thr308),p-p70S6K(Thr389),SGK3 and p-SGK3(Thr320)were inhibited in ECa109-LV3-SGK3 and EC9706-LV3-SGK3 cells.Conclusios 1.The down-regulation of Rictor enhanced the cell sensitivity to LY294002 by offset the activation of Akt/PRAS40 pathway induced by LY294002 in ESCC.2.Down-regulation of SGK3 increased the cell sensitivity to MK2206 by inhibiting the compensatory activation of MK2206 to SGK3 in ESCC. |
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