| ObjectiveGefitinib,a wide used,oral,targeted inhibitor of the EGFR kinase,is approved for the treatment of non-small cell lung cancer patients as first-line therapy.However,serious liver dysfunction causes some patients to discontinue the medication,even die.The molecular mechanisms responsible for these effects remain largely unknown.Previously,we showed that gefitinib caused hepatotoxicity via inducing hepatocyte apoptosis,and autophagy may be involved in this process.Therefore,we aim to study the role of autophagy in gefitinib-induced hepatotoxicity,and further explore the relationship between autophagy and apoptosis.Finally,we hope to find a new strategy to intervene gefitinib-induced hepatotoxicity,expand the breadth and depth of its clinical applications.MethodsPart 1.The Effect of autophagy in gefitinib-induced hepatotoxicity(1)Establishment of ICR mouse model for gefitinib-induced hepatotoxicity.The liver toxicity of gefitinib was assessed by liver organ index,serum hepatic enzyme activity of alanine aminotransferase(ALT),aspartate transaminase(AST)and lactic dehydrogenase(LDH),hematoxylin and eosin(HE)staining of liver sections;(2)Immunohistochemistry staining and TUNEL staining was used to confirm the occurrence of hepatocyte apoptosis in liver sections;(3)SRB assay was used to detect proliferation inhibition of gefitinib in normal human liver cell line HL7702(L02);(4)Apoptosis was evaluated by western blot,DAPI staining and PI/Annexin-V staining for flow cytometry;(5)Autophagy was detected by immune transmission electron microscopy(TEM),western blot,imununohistochemistry staining and acridine orange(AO)staining in vivo and in vitro;(6)Pan-caspase inhibitor Z-VAD-FMK was used to inhibit gefitinib-induced apoptosis,the autophagy was detected by western blot,PI staining for flow cytometry,and the proliferation of L02 was detected by SRB assay;(7)Atg5 was silenced by small RNA interference technique,and gefitinib-induced apoptosis was investigated by western blot,PI staining for flow cytometry,and the proliferation of L02 was detected by SRB assay;(8)Transgenic mouse model with hepatocyte-specific deletion of Atg7 was established through cre-loxp system.Liver organ index,serum hepatic enzyme activity,HE staining and immunohistochemistry staining were applied to confirm the relationship between autophagy and gefitinib-induced hepatotoxicity.Part 2.The mechanism of gefitinib-induced autophagy in mediating apoptosis(1)TMT quantitative proteomic profiling identifies was applied to find out the target proteins of gefitinib-induced autophagy;(2)The lysosomal inhibitor chloroquine(CQ)was used to verify the possible substrate proteins in autophagic degradation induced by gefitinib;(3)In case of the off-target effect,small RNA interference techniques were used,it was confirmed that gefitinib induced autophagic degradation of the substrate protein COX6A1 by western blot;(4)Counted the number of mitochondria in hepatocytes according to the results of electron microscopy,the changes of mitochondrial were observed by the mitochondrial protein Hsp60 Tomm20 by western blot.(5)The expression of COX6A1 protein in cytoplasm and mitochondria was detected by western blot.(6)Western blot was used to detect the COX6A1 protein level in L02 cells with different concentration and time of gefitinib;(7)Western blot was used to detect the COX6A1 protein level in the liver tissue of ICR mice treated with gefitinib;(8)Hepatocytes was overexpressed of COX6A1 by transfected COX6A1 plasmid,and gefitinib-induced apoptosis was investigated by western blot,PI staining for flow cytometry;(9)The mRNA level of COX6A1 was detected using real-time PCR(RT-PCR);(10)Hepatocytes were exposed to gefitinib in the presence or absence of cycloheximide(CHX),and the synthetic protein level of COX6A1 was measured by western blot analysis;(11)Hepatocytes were exposed to gefitinib in the presence or absence of MG 132 and western blot was used to analyze the role of ubiquitin proteasome pathway;(12)Hepatocytes were exposed to gefitinib in the presence or absence of 3-MA and detected the level of COX6A1 by western blot;(13)Atg5 or Atg7 knockout MEF cells or hepatocytes were used to detected the level of COX6A1 by western blot.Part 3.The mechanism study of gefitinib-induced autophagy and its intervention strategies(1)SRB assay was used to detect proliferation inhibition of gefitinib,afatinib and erlotinib in L02 cells,and western blot was used to detect the COX6A1 protein level after treatment with gefitinib,afatinib and erlotinib;(2)Epidermal growth factor receptor(EGFR)was silenced by small RNA interference technique,western blot was used to detect the COX6A1 protein level and the role of EGFR in gefitinib-induced autophagy and apoptosis;(3)The protein level of polo-like kinase 1(PLK1)after gefitinib treatment was detected by western blot;(4)The mRNA level of PLK1 was detected by using RT-PCR;(5)Immunohistochemistry staining was used to detect the colocalization of liver injury and PLK1 expression;(6)The happening course between liver injury and PLK1 expression was determined by serum hepatic enzyme activity and western blot in ICR mouse model;(7)Silencing or overexpression of PLK1 protein by siRNA or plasmid in hepatocytes,and the relationship between PLK1 and autophagy was investigated;(8)Western blot was used to detect the role of PLK1 inhibitor BI-2536 in gefitinib-induced autophagy;(9)PI staining for flow cytometry was used to detect the role of PLK1 inhibitor BI-2536 in gefitinib-induced apoptosis;(10)The protective effect of PLK1 inhibitor BI-2536 in gefitinib-induced hepatotoxicity was studied in ICR mice model by liver organ index,serum hepatic enzyme activity,western blot,HE and immunohistochemistry staining;(11)SRB assay was used to detect antitumor effect of gefitinib combined with PLK1 inhibitor BI-2536 in PC9、H1299 and H460.ResultsPart 1.The effect of autophagy in gefitinib-induced hepatotoxicity(1)Gefitinib induced hepatocyte apoptosis in vitro and in vivoWe treated ICR mice with gefitinib for 4 weeks.Consistented with clinical reports,gefitinib caused liver dysfunction in vivo.Liver organ index was increased by 1.3 times than control group.The serum hepatic enzyme activity of ALT,AST and LDH of gefitinib treated group were increased 2-10 fold than control group.Eosinophilic change was detected in liver sections treated with gefitinib by HE staining.The expression of cleaved-PARP(c-PARP)protein was increased in injure liver sections detected by immunohistochemistry.The results of TUNEL staining also showed that the green fluorescence intensity was significantly increased in liver sections of gefitinib-treated mice compared with control group.These results suggested that gefitinib-induced apoptosis may be the cause of liver injury.SRB staining confirmed that gefitinib significantly inhibited the proliferation of L02 cells,and the IC50 was 19.9 μM.The results of PI/Annexin-V staining for flow cytometry showed that hepatocyte apoptosis ratios were increased in the concentration-and time-dependent manner,while the necrosis ratio did not change.DAPI staining exhibited nuclear chromatin condensation,fragment and apoptotic bodies after gefitinib treatment.Western blot results showed a significant increase in apoptosis-related protein PARP cleavage.The above results confirmed that gefitinib induced hepatocyte apoptosis in vivo and vitro,and causing liver toxicity.(2)Gefitinib induced hepatocyte autophagy in vitro and in vivo simultaneouslyWe employed TEM to detect the presence of autophagic vacuoles.Gefitinib increased endogenous conversion of LC3-I to LC3-II levels in liver tissues.The expression of LC3 protein was increased in injure liver tissue sections detected by immunohistochemistry.Autophagic vacuoles was also detected by TEM in L02 cells treated with gefitinib,.Treatment with gefitinib induced the accumulation of AO in the cytoplasmic vacuoles in a concentration-and time-dependent manner.Hepatocytes were treated with gefitinib(0,5,10,20 μM)24 hours or gefitinib(20 μM)for 0,6,12,24 hours,and the percentage of AO positive cells were 14.01 ± 3.22%,19.78 ± 1.51%,21.34 ± 5.97%,37.46 ± 4.07%and 14.88 ± 0.63%,16.76 ± 3.80%,24.40 ± 1.95%,40.01 ± 7.67%respectively.The conversion of the LC3(LC3-Ⅰ to LC3-Ⅱ)was also observed after gefitinib treatment in L02 in a concentration-and time-dependent manner.All results were confirmed that gefitinib induced hepatocyte autophagy in vivo and in vitro while inducing apoptosis.(3)Gefitinib-mediated autophagy leaded to hepatocyte apoptosis,which in turn result in liver toxicityTo further comfirmed the role of autophagy and apoptosis in gefitinib-induced hepatotoxicity,the pan-caspase inhibitor Z-VAD-FMK was used.PI staining for flow cytometry shown that the apoptotic rate was 52.95 ± 20.70%in gefitinib group and 20.59± 18.50%in combination group.But SRB assay and western blot results shown that inhibition of hepatocyte apoptosis did not affect the occurrence of autophagy and the proliferation of L02.However,with gefitinib treatment,c-PARP protein was decreased in Atg5 knockdown group detected by western blot.The survival rate was reduced in The Atg5 knockdown group from 55.12 ± 4.58%to 86.41 ± 0.81%compared to native control group detected by SRB assay.The apoptosis rate was reduced in The Atg5 knockdown group from 57.22 ± 11.25%to 24.72 ± 4.17%compared to native control group detected by PI staining for flow cytometry.Hepatocyte-specific deletion of Atg7 could block autophagy and reverse gefitinib-induced hepatotoxicity by liver organ index,serum hepatic enzyme activity,HE and immunohistochemistry staining.These data indicated that gefitinib-induced apoptosis was stimulated by autophagy.Part 2.The mechanism of gefitinib-induced autophagy in mediating apoptosis(1)TMT quantitative proteomic profiling identifiesWestern blot results showed that chloroquine(CQ)inhibited the conversion of LC3-Ⅰ to LC3-Ⅱ,indicating that the autophagylase and its inclusion protein degradation was inhibited.Then the sample of control,gefitinib and gefitinib + CQ groups were analyzed by a large-scale proteomic analysis.In total,6,585 proteins were identified,among which 6,448 proteins were quantified.The fold-change cutoff was set when proteins with quantitative ratios above 1.3 or below 1/1.3 are deemed significant.B2M,COX6A1 and TUBB8 were the top candidates from the TMT analysis.Furthermore,inhibition of autophagy by silencing Atg5,only COX6A1 protein level could be reversed.According to the results of electron microscopy,the number of mitochondria had no significant change between the groups of control and gefitinib.Western blot showed that gefitinib did not affect the mitochondrial protein Hsp60 and Tomm20 protein levels.The level of COX6A1 protein in mitochondria was significantly decreased after gefitinib treatment by western blot.Western blot showed that the COX6A1 protein level downregulated as concentration and time dependent manner treated with gefitinib.The level of COX6A1 protein in the liver of ICR mice was also decreased dected by western blot.The all results indicated that COX6A1 might be the substrate protein in gefitinib-mediated autophytic degradation.(2)COX6A1 was involved in regulation of gefitinib-mediated apoptosisCOX6A1 overexpression significantly reduced c-PARP protein level and hepatocyte apoptosis ratio induced by gefitinib detected by western blot and PI staining for flow cytometry.These results shown that COX6A1 maight be a key reason for gefitinib-induced apoptosis.(3)Gefitinib promoted the autophagic degradation of COX6A1We are encouraged to further address whether autophagy mediates the degradation of COX6A1.RT-PCR was perfomed to confirm that gefitinib had little influence on the mRNA of COX6A1.Hepatocytes were exposed to CHX and/or gefitinib at different time points and estimated the expression levels of COX6A1,results shown that gefitinib also had little influence on the protein synthesis level.The degradation of COX6A1 was not occurred through ubiquitin-proteosome pathway by MG132(a specific proteasome inhibitor)treatment.Selective PI3K inhibitor 3-MA was used to inhibit autophagy,and western blot shown the down-regulation of COX6A1 induced by gefitinib could be reversed.Moreover,western blot shown that knock-out of Atg5 or Atg7 in MEF cells or hepatocytes could block the down-regulation of COX6A1 induced by gefitinib.Part 3.The mechanism of gefitinib-induced autophagy and its intervention strategies(1)EGFR was not involved in gefitinib-activated hepatocyte autophagy To investigate whether gefitinib-mediated COX6A1 autophagic degradation is a target effect,the EGFR inhibitors erlotinib and afatinib were used.The concentration of erlotinib and afatinib in hepatocytes was determined by SRB staining,and western blot showed that COX6A1 protein was downregulated only in hepatocytes treatment with gefitinib,whereas the COX6A1 protein level was unchanged after treatment with erlotinib and afatinib.Western blot showed that silence EGFR did not downregulate COX6A1 protein level compared with gefitinib group.And silenced EGFR did not induce significant autophagy compared with gefitinib group,and also did not affect the autophagy induced by gefitinib.And silenced EGFR also did not cause significant apoptosis.(2)PLK1 was involved in gefitinib-induced hepatocyte autophagyWestern blot showed that PLK1 protein level increased in concentration-and time-dependent manner.The expression of PLK1 protein was increased in injure liver tissue sections detected by immunohistochemistry.The data of serum and liver tissues of ICR mice at different time suggested that gefitinib-induced liver dysfunction after gefitinib treated for 3 weeks,while the PLK1 protein was increased after gefitinib treated for 2 weeks.Western blot showed that silencing PLK1 significantly inhibited gefitinib-induced autophagy and degradation of COX6A1.But overexpression of PLK1 protein in hepatocytes could enhance gefitinib-induced autophagy and degradation of COX6A1.These results showed that PLK1 was involved in gefitinib-induced hepatocyte autophagy,which in turn caused gefitinib liver toxicity.(3)PLK1 inhibitor BI-2536 could protected liver toxicity of gefitinibPrevious results suggested that inhibition of PLK1 could block autophagy and thereby reduce gefitinib-induced liver toxicity.We used PLK1 inhibitor BI-2536 in combination with gefitinib,western blot showed that BI-2536 could inhibit the conversion of LC3-Ⅰ to LC3-Ⅱ and degradation of COX6A1.BI-2536 could inhibit gefitinib-induced apoptosis of hepatocytes by PI staining for flow cytometry.In ICR mouse model,BI-2536 blocked autophagy,apoptosis and reversed gefitinib-induced hepatotoxicity detected by liver organ index,serum hepatic enzyme activity,HE and immunohistochemistry staining.To further validate the clinical feasibility of PLK1 inhibitor as the gefitinib liver toxicity intervention strategy,three tumor cells PC9,H1299 and H460 were analyzed,SRB assay showed that gefitinib combined with BI-2536 had little affect on the anti-tumor efficacy of gefitinib.These results indicated that BI-2536 had potential clinical application for mitigating the liver injury induced by gefitinib without influencing its anticancer activityConclusionGefitinib-induced autophagy was the key event in its apoptosis depedent hepatotoxicy.Autophagy promoted the degradation of mitochondrial-associated protein COX6A1,which in turn caused hepatocyte apoptosis.PLK1 was involved in the activation of gefitinib-induced autophagy,and PLK1 inhibitor BI-2536 could inhibit autophagy to improve the liver function of gefitinib without influencing its anticancer activity.This study illustrated the mechanism of gefitinib induced liver toxicity,which provides an effective and feasible theoretical basis for managing the clinic applications of gefitinib and protecting its hepatotoxicity. |
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