Effect Of Autophagy On The Anti-esophageal Squamous Cell Carcinoma Activity Of Sulforaphane And Its Molecular Mechanism

AimsTo investigate the effects of inhibiting autophagy on the anti-tumor efficiency of SFN to ESCC and molecular mechanisms.Methods1.Effects of SFN on the proliferation,clone formation and apoptosis of ESCC cells and molecular mechanisms(1)The effects of SFN on proliferation,colon formation and apoptosis of ESCC cells were detected by CCK-8 assay,colony formation assay and flow cytometry,respectively.(2)Effects of SFN on expression of anti-apoptotic protein Bcl-2,pro-apoptotic protein BAX and Cleaved-caspase 9 in ESCC cells were detected by Western blot.2.Effects of SFN on autophagy of ESCC cells and molecular mechanisms(1)The effects of SFN alone or combined with CQ on the expression of LC3-? and p62 in ESCC cells were detected by Western blot and immunofluorescence,then mRFP-GFP-LC3 adenovirus infection was used to detect the autophagy flux of ESCC cells were treated with SFN.(2)Expression of autophagy-related proteins Atg5 and Atg7 was detected after ESCC cells were treated with SFN by Western blot.Proteins of nucleus and cytoplasm were extracted,and effects of SFN on expression of Nrf2 in cytoplasm and nucleus were detected by Western blot.The effects of ML385 alone or combined with SFN on the autophagy proteins LC3 and p62 were detected by Western blot.(3)CCK-8 assay and colony formation assay were used to detect the effects of ML385 on proliferation and clone formation of ESCC cells.And the anti-proliferative of SFN alone or combined with CQ and serum-free starvation,respectively,were detected by CCK-8 assay.3.Effects of inhibiting autophagy on anti-tumor efficiency of SFN on ESCC and molecular mechanisms(1)The effects of autophagy inhibitor CQ and SFN alone or in combination in ESCC cells on cell proliferation and clone formation were detected by CCK-8 assay and colony formation assay.And the anti-proliferative effects of down-regulating Beclin-1 upon SFN treatment in ESCC cells were detected by CCK-8 assay and colony formation assay.(2)The effects of SFN alone or combined with CQ on the expression of Bcl-2,BAX and Cleaved-caspase 9 were detected by Western blot,and the effects of SFN alone or combined with CQ on the expression and distribution of Nrf2 protein were detected by Western blot and immunofluorescence.4.Effects of SFN combined with CQ on growth of ESCC xenografts and molecular mechanisms(1)The ESCC xenograft mice models were established to detect the effect of SFN alone and combined with CQ on tumor growth,and the tumor suppression rate and relative tumor growth rate were calculated.(2)The effect of SFN alone or combined with CQ on cell apoptotic in ESCC xenograft was detected by H&E staining and TUNEL assay.The expression of LC3,p62,Nrf2,HO-1,Bcl-2,BAX and Cleaved-caspase 9 in tumor tissues from nude mice was detected by Western blot.(3)The drug safety was evaluated by body weight changes,relative organ weights,haematological parameters and H&E staining of liver and kidney tissues.Results1.SFN inhibited ESCC activities by activating caspase pathway(1)SFN inhibited the proliferation and clone formation as well as induced apoptosis of ESCC cells.(2)SFN increased the expression of Cleaved-caspase 9 in ESCC cells,but not Bcl-2 and BAX.2.SFN induces autophagy in ESCC cells by activating Nrf2 signaling(1)SFN increased expression and accumulation of LC3-? and reduced p62 protein level in ESCC cells.Moreover,degradation of p62 in ESCC cells exposed to SFN was blocked while LC3-? accumulation and LC3-positive puncta increased in the presence of lysosomal inhibitors CQ,indicating that SFN might induce cell autophagy.In addition,mRFP-GFP-LC3 adenovirus experiment confirmed that SFN significantly increased the number of autophagosomes and autolysosomes in ESCC cells.The above results indicated that SFN could induce cell autophagy by promoting autophagy flux in ESCC cells.(2)The expression of Atg5 and Atg7 has no changes after ESCC cells were treated with SFN,indicating that SFN had no relationship with the initial stage of autophagy.SFN alone increased expression of Nrf2 in nucleus,and thus activated Nrf2,SFN combined with Nrf2 inhibitor ML385 decreased LC3-? and increased p62 level,indicating that SFN could induce autophagy by activating Nrf2.(3)ML385 could inhibit the proliferation and clone formation of ESCC cells,which indicates that the activation of Nrf2 by SFN is an unfavorable factor for its antitumor activity,and CQ enhanced while serum-free starvation impaired the proliferation-inhibiting effects of SFN.These data suggested that activation of Nrf2 by SFN may cause cytoprotective autophagy.3.Autophagy inhibition improves antitumor responses of ESCC cells to SFN(1)CQ enhanced the proliferation-inhibiting and apoptosis-inducing effects of SFN on ESCC cells,indicating that CQ could promote sensitivity of ESCC cells to SFN.In addition,down-regulation of Beclin-1 also promoted the proliferation-inhibiting effects of SFN on ESCC cells.The above results indicated that inhibition of autophagy could enhance the antitumor efficiency of SFN to ESCC cells.(2)SFN alone increased the expression of Cleaved-caspase 9 in ESCC cells,but not Bcl-2 and BAX,while SFN combined with CQ could synergistically reduce the expression of Bcl-2 and increase Cleaved-caspase 9 level in ESCC cells.SFN promoted the expression of Nrf2 in cell nucleus,while CQ markedly inhibited the activation of SFN to Nrf2 when cells were treated with CQ and SFN combination,suggesting CQ could neutralize the activation of Nrf2 by SFN and enhance the activation of caspase pathway by SFN.4.CQ enhances the inhibitory effect of SFN on the growth of ESCC xenografts(1)SFN significantly inhibited tumor growth and induced cell apoptosis,but when combined with CQ,it has a stronger inhibitory effect on tumor growth and more obvious apoptosis inducing effects on cells.(2)The combination of SFN and CQ inhibited the expression of Bcl-2 protein and further promoted the expression of Cleaved-caspase 9.In addition,SFN combined with CQ inhibited the expression of Nrf2 and HO-1.(3)There were no significant changes in body weight,relative organ weights,haematological parameters of nude mice in each group,indicating that CQ and SFN had no obvious toxicity at the therapeutic dosage.ConclusionsAutophagy inhibitor CQ could enhance the sensitivity of ESCC cells to SFN in vitro and in vivo by enhancing the activating effects of SFN on caspase pathway and neutralizing the activation of SFN to Nrf2 in ESCC.