Pharmacodynamic And Mechanical Study Of OP16,an Analogue Of Oridonin, Combined With Rapamycin In Esophageal Squamous Cell Carcinoma

Aims1.The primary aim was to explore the in vitro effects and molecular mechanisms of OP16 combined with rapamycin on proliferation,apoptosis and autophagy of ESCC cells.2.The next aims were to detect the in vivo effects and molecular mechanisms of OP16 combined with rapamycin on tumor growth and apoptosis of ESCC xenografts,and to evaluate preliminarily the potential toxicity induced by OP16 and rapamycin.Methods1.The anti-proliferative effects of OP16 combined with rapamycin in ESCC cells were detected by colony formation assay and CCK-8 assay,and the expressions of relevant proteins in m TORC2/Akt(Ser473)and PI3K/Akt(Thr308)pathway were detected by Western blot.2.The pro-apoptotic effects of OP16 alone or combined with rapamycin in ESCC cells were detected by Annexin V-FITC/PI staining,and the expressions of relevant proteins in mitochondrial apoptotic pathway were detected by Western blot.3.The effects of OP16 on the expression and cellular distribution of LC3 and SQSTM1/p62 in ESCC cells were explored by Western blot and immunofluorescence.Subsequently,the effects of OP16 on the autophagic flux in ESCC cells were detected by Western blot,m RFP-GFP-LC3 adenovirus assay and transmission electron microscope.Then the relationship between the autophagy and apoptosis induced by OP16 was explored by PI staining and CCK-8 assay.Finally,the molecular mechanisms underlying the autophagy induced by OP16 were detected by Western blot.4.The ESCC xenograft mice models were established to detect the growth-inhibitory effects of OP16 combined with rapamycin in ESCC xenografts.And the in vivo pro-apoptotic effects of OP16 combined with rapamycin in ESCC cells were detected by H&E staining and TUNEL assay in ESCC xenografts.The expressions of relevant proteins in m TORC2/Akt(Ser473),PI3K/Akt(Thr308)and mitochondrial apoptotic pathway were detected by Western blot.And the potential toxicity was evaluated preliminarily by the mortality,body weight changes,haematological parameters,relative organ weights and H&E staining of liver and kidney tissues in nude mice.Results1.OP16 combined with rapamycin could synergistically inhibit colony formation of ESCC cells.OP16 combined with RAD001 could synergistically suppress proliferation of ESCC cells.OP16 alone could inhibit the expressions of p-Akt,PI3 K p85? and PI3 K p110?,and promote the expressions of p-Rictor in ESCC cells.OP16 combined with rapamycin could diminish rapamycin-induced activation of p-Akt,PI3 K p85? and PI3 K p110?,and reverse rapamycin-induced inhibition of p-Rictor in ESCC cells.2.OP16 alone could trigger apoptosis of ESCC cells.OP16 combined with rapamycin could synergistically trigger apoptosis of ESCC cells.OP16 alone could inhibit the expression of Bcl-2,and promote the expressions of Bax,cleaved caspase-9 and cleaved caspase-3 in ESCC cells.OP16 combined with rapamycin could synergistically inhibit the expression of Bcl-2,and synergistically promote the expressions of Bax,cleaved caspase-9 and cleaved caspase-3 in ESCC cells.3.OP16 could promote the expression and cellular accumulation of LC3,and inhibit the expression of SQSTM1/p62 in ESCC cells.OP16 could activate autophagic flux by promoting autophagosome formation in ESCC cells.Inhibition of apoptosis by Z-VAD-FMK could not decrease the OP16-induced death of ESCC cells.Activation of autophagy by starvation treatment could significantly increase the OP16-induced death of ESCC cells.Inhibition of autophagy by chloroquine or Beclin-1 knockdown could significantly decrease the OP16-induced death of ESCC cells.OP16 had on obvious effects on the expression of Atg5,Atg7 and Atg12.Activation of Akt phosphorylation by SC79 could significantly decrease the OP16-induced LC3-II expression in ESCC cells.4.OP16 combined with rapamycin could synergistically inhibit the tumor growth,and synergistically trigger in vivo cell apoptosis in ESCC xenografts.The effects of OP16 combined with rapamycin on the expressions of relevant proteins in ESCC xenografts were consistent with the results in vitro.OP16 combined with rapamycin had on obvious effects on body weight changes,haematological parameters and relative organ weights,and had no obvious hepatorenal toxicity in xenograft mice.Conclusions1.OP16 combined with rapamycin in vitro could synergistically regulate proliferation,apoptosis and autophagy of ESCC cells,which may through the molecular mechanisms that OP16 and rapamycin could regulate the m TORC2/Akt(Ser473),PI3K/Akt(Thr308)and mitochondrial apoptotic pathway.2.OP16 combined with rapamycin in vivo could synergistically regulate tumor growth and cell apoptosis of ESCC xenografts,and the molecular mechanisms were consistent with the results in vitro.No obvious toxicity or adverse effect was observed in nude mice during the combined treatment in therapeutic doses.