Relationship Between SGK3 Expression Level And AZD5363 Sensitivity Of Esophageal Squamous Cell Carcinoma Cells

AimsLong-term application of Akt inhibitor AZD5363 caused compensatory activation of SGK3 and reduced the sensitivity of esophageal squamous cell carcinoma cells to AZD5363,investigate the relationship between SGK3 expression level and sensitivity of ESCC cells to AZD5363.MethodsEffect of AZD5363 on ESCC cells and molecular mechanism of drug resistance induced by long-term applicationThe effects of Akt inhibitor AZD5363 on proliferation and apoptosis of ESCC cells were detected by CCK-8 assay and Annexin V-FITC/PI staining assay.The effect of long-term application of AZD5363 on proliferation of ESCC cells was used to detect by CCK-8assay,the expression of Akt and SGK3 was detected by Western blot after ESCC cells were treated with AZD5363.Molecular mechanism of AZD5363 affecting SGK3 activity1.Proteins that may activate SGK3 were analyzed using the protein interaction database REACTOME.The expression of the analyzed protein was detected by Western blot after ECSS cells were treated with AZD5363.2.Ca²⁺concentration was detected after ECa109 cells were treated with AZD5363 for 1 d.3.The binding of Akt with Ca²⁺regulatory protein IP3R1 in ECSS cells was detected by co-immunoprecipitation（Co-IP）assay,the expression of IP3R1 protein was detected by Western blot after ESCC cells treated with AZD5363.Relationship between SGK3 expression level and sensitivity of ESCC to AZD5363 and molecular mechanism1.EC9706 and ECa109 cells were infected with three SGK3-sh RNA lentivirus expression vectors（sh1,sh2,sh3）and negative control vector（NC）,respectively.The cell lines with stable expression were screened by purimycin,and the interfere efficiency of SGK3 was detected by Western blot.2.The effects of AZD5363 on proliferation,apoptosis,clone information ability and migration of ESCC cells with down-regulated SGK3 were investigated by CCK-8 assay,clone formation assay,wound healing assay,cell cycle assay and apoptosis assay.3.ESCC cells with down-regulated SGK3 were treated with AZD5363,and the expression of related proteins in Akt/p70S6K pathway and SGK3 were detected by Western blot.ResultsAZD5363 inhibited ESCC by inhibiting the activity of Akt,but obtained resistance by activated SGK3 after a long time1.AZD5363 could inhibit the proliferation of EC9706 and ECa109 cells.The values of IC₅₀ to ECa109 for 24 h and 48 h were（33.36±1.52）and（17.67±1.24）μM,to EC9706 were（33.84±1.53）and（20.86±1.32）μM,to Het-1A were>300μM and（66.66±1.82） μM,respectively,indicated that AZD5363 had good selectivity to cancer cells.2.After ESCC cells were treated with 10,20 and 40μM AZD5363 for 48 h,the apoptotic percentage of cells were（12.1±1.84）%,（20.75±0.78）%and（33.15±5.44）%for ECa109,and（10.95±0.21）%,（20.1±2.40）%and（29.3±1.98）%for EC9706,respectively,suggesting that AZD5363 could promote the apoptosis of ESCC cells in a concentration-dependent manner.3.After EC9706 and ECa109 cells were treated with 20 or 30μM of AZD5363 for 1-7 d,the cell proliferation results showed the sensitivity of cells to AZD5363 significantly decreased after 5 d at 30μM and even obtained resistance on the 7th day,indicating that ESCC cells were resistant to AZD5363 after a long-term application.4.With the treatment time of AZD5363 prolonging,the activity of Akt protein in ESCC cells decreased in a time-dependent manner,but the expression levels of SGK3 and p-SGK3 increased gradually,suggesting that long-term application of AZD5363 might activate SGK3.The molecular mechanism of AZD5363 activating SGK31.Bioinformatics analysis results confirmed that PI（3）P produced by h Vps34 could bind to the PX domain of SGK3.Also,AZD5363 could promote the expression of h Vps34 protein in ESCC cells.2.Ca²⁺concentration increased after ECa109 cells treated with AZD5363 for 1 d.The results of co-immunoprecipitation（Co-IP）showed that there was an interaction between Akt and IP3R1,and the expression of IP3R1 was significantly increased by AZD5363.These results suggested that the activation of SGK3 by AZD5363 might be related to the IP3R1/Ca²⁺/h Vps34 pathway.Down-regulation of SGK3 enhanced the sensitivity of ESCC cells to AZD53631.Western blot results confirmed that sh3 in ECa109 and EC9706 cells had the best interference efficiency,so ECa109-sh3 and EC9706-sh3 and their NC cells were used for subsequent experiments.2.Compared with NC cells,AZD5363 had more obvious inhibitory effects on the proliferation,clone formation,migration and inducing effects on apoptosis of ECa109-ah3 and EC9706-sh3 cells,as well as more cells were retarded in G0/G1 or G2/M Phase,suggesting that down-regulation of SGK3 could improve the sensitivity of ESCC cells to AZD5363.Molecular mechanism that down-regulation of SGK3 expression enhancing the sensitivity of ESCC cells to AZD5363Compared with NC cells,AZD5363 did not promote the expression of SGK3 and p70S6K in ECa109-sh3 and EC9706-sh3 cells,suggesting that down-regulation of SGK3eliminated the activation of SGK3 and p70S6K by AZD5363.ConclusionsAZD5363 activated SGK3 through the IP3R1/Ca²⁺/h Vps34 pathway to induce the resistance of ESCC cells to it.Down-regulation of SGK3 can eliminate the activation of SGK3 and p70S6K by AZD5363 and thus enhance the sensitivity of ESCC cells to AZD5363.