| AimsTo investigate the sensitivity of xenograft nude mice of esophageal squamous cell carcinoma(ESCC)to Akt allosteric inhibitor MK2206 and molecular mechanism after down-regulating SGK3 expression.Methods1.Establishment of xenograft nude mice model of ESCC and pharmacodynamic evaluation of the anti-tumor effect of MK2206(1)ECa109-LV3-NC and ECa-LV3-SGK3 cell lines were cultured and subcutaneous inoculated to establish the xenograft nude mice model of ESCC.All nude mice were randomly divided into LV3-NC,LV3-SGK3,LV3-NC+MK2206 and LV3-SGK3+MK2206 group(n=5).LV3-NC and LV3-SGK3 group were treated with normal saline,LV3-NC+MK2206 and LV3-SGK3+MK2206 group were treated with MK2206.Drugs were intraperitoneal injected for 15 days at a concentration of 8mg/kg.(2)During the treatment,tumor length and short diameter were measured every day for calculating the tumor volume and plotting the tumor growth curve,the relative tumor volume rate and relative tumor growth rate were calculated at the end of treatment.(3)After the treatment,the nude mice were sacrificed by cervical dislocation method,and the organs and tumor tissues were collected for fixation and embedding.Then,H&E staining and TUNEL staining were performed on the tumor tissues to detect the apoptosis.2.Primary evaluation of drug toxicity at the current dosage.The potential toxicity of MK2206 to nude mice was preliminarily evaluated by body weight,mortality rate,blood routine coefficient,viscera coefficient,the contents of alanine transaminase,glutaminotransferase,creatinine and uric acid in serum of nude mice,as well as the H&E staining of heart,liver and kidney.3.Mechanism of MK2206 on esophageal squamous cell carcinoma based on SGK3 level in cells.The histones of ESCC were extracted by grinding with liquid nitrogen,and the expressions of proteins related to PI3K/Akt/mTOR signaling pathway and SGK3 protein in the xenografts of ESCC were detected by Western blot.Results1.According to the tumor growth curve,compared with the control group,the tumor growth became significantly slower in each treatment group,especially the LV3-SGK3+MK2206 group,in which tumor growth was the slowest and the volume of tumor was the smallest after treatment.By calculating the relative tumor volume,relative tumor growth rate and tumor inhibitory rate of each group,we also found that MK2206 alone had an inhibitory effect on the growth of ESCC,but had the strongest inhibitory effect on tumor with down-regulated expression of SGK3,indicating that the sensitivity of ESCC to MK2206 enhanced after SGK3 expression was down-regulated.2.H&E staining results showed that compared with LV3-NC group and LV3-SGK3 group,both groups treated with MK2206 emerged the characteristics of apoptosis,such as nuclear condensation and cytoplasmic vacuoles,especially in tumors with down-regulated SGK3 treated with MK2206.TUNEL staining results also confirmed that MK2206 had a stronger pro-apoptotic effect on tumors with down-regulated SGK3.3.During the treatment,there was no nude mice death in each group.After the treatment,there was no significant difference on body weight of nude mice,blood routine coefficient,viscera coefficient,aminotransferase,glutaminotransaminase,creatinine and uric acid between every group.Moreover,the pathological changes were not observed in the liver,heart and kidney according to the H&E staining results of them,suggesting that MK2206 had no significant toxicity to nude mice.4.MK2206 alone inhibited the expression of Akt,but enhanced the phosphorylation and expression of SGK3 and p70S6K,while after SGK3 expression was down-regulated,MK2206 inhibited posphorylated activation of Akt and SGK3 as well as p70S6K.These results suggested that the effects of MK2206 on ESCC cells were related to the compensatory activation of SGK3.ConclusionsDown-regulating SGK3 expression enhanced the sensitivity of the xenografts of ESCC to MK2206,the molecular mechanism was related to down-regulating SGK3 expression inhibiting the compensatory activation of MK2206 to SGK3. |
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