Effects Of Rapamycin And OP16on Esophageal Squamous Cell Proliferation And Akt/PTEN Expression

Purposes1. To observe the effect of rapamycin on cell proliferations and the expression ofp-Akt of esophageal squamous cell carcinoma (ESCC) cell lines.2. To provide a reasonable strategy for improving the sensitivity of ESCC cells torapamycin.3. To investigate the effects of new developed compound OP16on cell proliferationand Akt/p70S6K signaling pathway of ESCC.Methods1. Effects of rapamycin on cell proliferations and expression of Akt ofESCC cell lines.CCK-8assay was used to identify the effect of rapamycin on cell proliferation ofEC9706, ECa109, KYSE450, KYSE790and TE-1cells. Western blot assay was usedto detect the changes of p-Akt (Ser473) protein expression after ESCC cells weretreated with rapamycin or p70S6K siRNA.2. Effects of PTEN and rapamycin on the proliferation of EC9706celland Akt expressionThe protein expression of PTEN and p-PTEN (Ser380/Thr382/383) were detected byWestern blot. After EC9706cells were transfected with the recombinant vectorpcDNA3.1-PTEN, the effect of rapamycin and PTEN on cells proliferation andprotein expression of p-Akt (Ser473) were investigated by CCK-8and Western blot.The effect of wPTEN on tumor progression was detected in nude mice. 3. Effects of OP16on cell proliferation and protein expression of ESCCcell linesThe effects of OP16, a new compound developed by our laborotary, on cellproliferation and protein expression of p-Akt (Ser473), Akt1/2, p-p70S6K, p70S6K ofESCC cells were assessed by CCK-8assay and Western blot.Results1. Rapamycin inhibited the proliferations of EC9706, ECa109, KYSE450, KYSE790and TE-1cells. But proliferation inhibition rate was decreased when treated withhigher concentration of rapamycin in EC9706and TE-1cells. Protein p-Akt(Ser473) elevated in EC9706and TE-1cells when treated with rapamycin. RNAinterference-mediated knockdown of S6K1also resulted in an increase of Aktphosphorylation in vitro and in vivo.2. p-PTEN (Ser380/Thr382/383) was expressed in all5detected cell lines, but totalPTEN had no expression in them. EC9706cell proliferation was restrained andp-Akt was decreased when cells transfected with wPTEN. The similar resultscould be found in nude mice experiment.3. OP16inhibited the proliferation of ESCC cell in a concentration dependentmanner. OP16inhibited Akt phosphorylation expression and improved theexpression of p-p70S6K. OP16and rapamycin had synergy effects on theproliferation of ESCC cells and the protein expression in Akt pathway.Conclusions1. Rapamycin triggers a feedback mechanism resulting in the activation of Aktsignaling in EC9706and TE-1cells. PTEN can inhibit PI3K/Akt activation andimprove sensitivity of ESCC cells to rapamycin in vitro and in vivo.2. OP16and rapamycin downregulate key protein expression of p-Akt and p70S6Kand have a synergistic inhibition in ESCC cells.