| Background Hepatocellular carcinoma(HCC)is the sixth most common malignant tumor and the third leading cause of cancer-related death that seriously threatens human health worldwide.The majority of patients are diagnosed with HCC at an advanced stage,and the propo rtion of HCC cases that are actually suitable for surgical resection is less than 20%.Radiofrequency ablation(RFA),similar to surgical resection and liver transplantation,has been widely accepted as a curative treatment for small HCC lesions.Although surgical resection is the standard treatment for HCC,its application is limited because most patients present with severe liver dysfunction.Liver transplantation provides an alternative treatment for small HCC lesions.However,the shortage of donor liver tissues limits its application.Currently,RFA has been widely accepted as an alternative treatment for small HCC lesions in clinical practice,based on its advantages of minimal invasiveness,rapid recovery,lower cost,safety,reliable efficacy,repeatability,and shorter hospitalization time.However,one of the greatest problems with RFA is that insufficient RFA(IRFA)may lead to residual tumor progression and rapid local recurrence.Although IRFA has been reported to cause the malignant transformation of HCC via the epithelial-mesenchymal transition(EMT)or the hypoxia inducible factor-1α(HIF-1α)/VEGFA pathway,the specific molecular mechanisms remain poorly understood.Thus,an exploration of the mechanisms underlying the residual tumor progression and local recurrence of HCC after IRFA is very important.Based on accumulating evidence,autophagy plays a dual role in tumorigenesis and tumor development.On one hand,autophagy removes damaged or aged proteins and organelles in normal cells and inhibits tumorigenesis.On the other hand,autophagy enhances the tolerance of tumor to harsh environmental stress and promotes tumor cell survival.Sublethal heat stress has consistently been shown to induce autophagy in several human cancer cell lines,such as HeLa and A549 cells.Heat-induced autophagy was recently shown to improve survival and proteostasis in C.elegans.However,researchers have not yet determined whether sublethal heat stress induces autophagy in HCC cells,and the precise regulatory mechanisms underlying IRFA-induced autophagy remain largely unknown.After determining the LC3 expression patterns and the correlations between LC3levels and clinical prognosis,we discovered that autophagy was activated in HCC tissues after RFA and that the autophagy marker LC3 was an independent risk factor that predicted the recurrence of HCC after RFA.HCC cells were exposed to heat stress to simulate the microenvironment of the transition zone of RFA.Heat stress induced autophagy in HCC cells.After a series of functional assays,we clarified a novel mechanism by which heat stress induced autophagy to protect against heat-induced apoptosis in HCC via the ATP-AMPK-mTOR axis,suggesting that approaches targeting autophagy may be a promising strategy for improving the efficacy of RFA treatment for HCC.Methods(1)Tumor tissues and corresponding adjacent nontumor tissues were collected from130 patients with HCC undergoing surgical resection in the Department of Hepatobiliary Surgery,Southwest Hospital of Third Military Medical University(Army Medical University).Among these patients,100 only underwent surgical resection.The other 30cases were recurrent HCC after RFA treatment.At the same time,the normal liver tissues were collected from 5 liver transplant donors.Portions of the tissues were stored at-80℃ until the extraction of protein and mRNA.The remaining tissues were fixed with 4%paraformaldehyde for 48 h,and the tumor tissues were embedded and sliced at a thickness of 5μm for immunohistochemistry.Western blots were performed to detect the levels of the LC3 protein in HCC tissues and nontumor tissues,immunohistochemistry analyzed the location and intensity of LC3 expression in HCC tissues and nontumor tissues.(2)HCC cells were exposed to heat stress in a water bath set to different temperatures(37,43,45,or 47℃)for different durations(0,15,30,45,or 60 min)to simulate the microenvironment of the transition zone of RFA,and proteins were extracted at different time points(0,2,4,8,12,and 24 h)after heat stress.Western blot analyses were performed to detect the levels of the LC3-II and p62 proteins to identify the optical temperature,duration of heat treatment and detection time point after heat treatment.HCC cells were transfected with mRFP-GFP-LC3B adenovirus and subjected to the heat treatment.Then,cells were fixed with 4%paraformaldehyde for 15 min and analyzed using confocal microscopy(Zeiss,Germany).Levels of autophagy and autophagy flux were determined b y evaluating the subcellular localization and average number of GFP and mRFP puncta per cell.Transmission electron microscopy was used to identify and analyze the numbers and ultrastructures of autophagosomes and autolysosomes.(3)HCC cells were suspended and counted,and the cell density was adjusted to 1 x10~5 cells/ml.Next,100μl of cells were seeded into the wells of 96-well plates and cultured overnight to allow attachment.Then,cells were exposed to vehicle,CQ,heat treatment and a combination of CQ and heat treatment.After recovery at 37℃ for another 24 h,cell viability was evaluated using a Cell Counting Kit-8(CCK-8)(Dojindo,Japan).Cell proliferation was assessed using a Cell-Light?EdU Apollo?488 In Vitro Imaging Kit(RiboBio,Guangzhou,China).A FITC Annexin V/Dead Cell Apoptosis Kit(Invitrogen,USA)was used to detect the apoptosis of cells exposed to different treatments.Annexin V-positive cells represented apoptotic cells and were quantified using previously described methods.Western blots were performed to determine the levels of the LC3-II,p62,cleaved Caspase-3 and PARP proteins.Small interfering RNAs(siRNAs)targeting Beclin-1 or Atg5were purchased from Sangon Biotech Co.,Ltd(Shanghai,China).Then,these siRNAs targeting Beclin-1 or Atg5 were transfected into cells using Lipofectamine 3000(Invitrogen,USA).Western blot analyses were performed to detect the levels of the Beclin-1 and Atg5proteins and to evaluate the silencing efficiency of these siRNAs.Cells were exposed to siRNAs,heat treatment or a combination treatment.After recovery at 37℃ for another 24h,a FITC Annexin V/Dead Cell Apoptosis Kit(Invitrogen,USA)was used to detect the apoptosis of cells exposed to different treatments.Annexin V-positive cells represented apoptotic cells and were quantified as described previously.(4)HCC cells were suspended and counted,and the cell density was adjusted to 1 x 10~5cells/ml.Then,100μl cells were seeded into the wells of 96-well plates and cultured overnight to allow attachment.After 24 h,the cells were exposed to the heat treatment at47℃ for 30 min.After recovery for another 24 h,ATP levels were measured using the ATPlite Luminescence Assay Kit(6016943,PerkinElmer,USA)as described in a previous study.Relative ATP levels were normalized to the control.Western blot analyses were performed to detect the levels of the p-AMPK/AMPK,p-mTOR/mTOR,p-p70S6K/p70S6K and p-4E-BP1/4E-BP1 proteins.The effects of the AMPK activator AICAR and the AMPK inhibitor compound C on autophagy were analyzed in heat-treated cells to further confirm that AMPK was involved in autophagy induced by heat stress.Western blots analyses were performed to detect the levels of the p-AMPK/AMPK,p-mTOR/mTOR,LC3-II and p62proteins.(5)Five-week-old male BALB/c nude mice were obtained from Bioscience(Beijing,China)and maintained in the laboratory animal center of the Army Medical University(Chongqing,China).Animal studies were approved by the Institutional Animal Care and Use Committee of Southwest Hospital(Chongqing,China).SMMC7721 cells were pretreated at47℃ for 30 min or maintained at 37℃.After recovery for 24 h,cells(5 x 10~6 cells in 100μl of serum-free RPMI 1640 medium)were injected into the upper right flank of 24 nude mice.Then,these nude mice were randomly separated into the following four groups:vehicle,CQ,heat treatment,and a combination of CQ and heat treatment(n=6 mice per group).Starting on the second day,the mice in the vehicle and heat treatment groups received i.p.injections of 100μl of PBS thrice weekly,while the mice in CQ and combination treatment groups received i.p.injections of 60 mg/kg CQ in 100μl of PBS thrice weekly.Tumor sizes were measured every three days for 30 days,and the results were plotted.After 30 days,the tumors were harvested,photographed,weighed and then fixed with 4%paraformaldehyde for hematoxylin-eosin(H&E)and immunohistochemical(IHC)staining.Tumor sections were immunostained with a Histostain-Plus Kit(Zhongshan Biotechnologies,Beijing,China)according to the manufacturer’s instructions.IHC was performed to analyze the location and intensity of LC3,p62,Ki-67 and cleaved Caspase-3 staining,and the data were analyzed using Image-Pro Plus software(Media Cybernetics,MD,US).(6)Statistical analysis.All results were presented as the means±SEM of three independent experiments and were analyzed using SPSS 18.0 software(SPSS Inc.,IL,US).For quantitative data,significant differences between two groups were evaluated using Student’s t test,and significant differences among multiple groups were evaluated using one-way ANOVA.For categorical data,significant differences were evaluated using chi-square tests or Fisher’s exact tests.Differences with P values less than 0.05 were considered significant.Results(1)Levels of the autophagy marker LC3-II were increased in HCC cells compared to L02 cells.Increased levels of the LC3-II protein were detected in HCC tissues.Overexpression of LC3-II was detected in 77.3%(17/22)of HCC tissues compared to the corresponding adjacent nontumor tissues.The immunohistochemical analyses revealed increased LC3 expression in RFA groups compared with non-RFA groups,and LC3 was frequently located in the cytoplasm.The analysis of correlations between LC3 expression and clinical parameters indicated that increased LC3 expression was significantly correlated with recurrence after RFA(P<0.05),but not with other clinicopathological features,such as age,sex,HBV infection,Child-Pugh class,cirrhosis,serum AFP levels,tumor number,tumor size,vascular invasion,lymph node invasion and tumor differentiation.The Kaplan-Meier analysis revealed a higher local recurrence rate in RFA-treated patients with HCC presenting high LC3 expression than in patients with low expression of LC3(P<0.05).Based on these results,the LC3 expression level potentially represents a poten tial target for predicting recurrence and poor survival of RFA-treated patients with HCC.(2)SMMC7721 and Huh7 cells were exposed to sublethal heat stress at different temperatures for different durations,and then western blot analyses were performed to determine whether sublethal heat stress induced autophagy in HCC cells.The heat treatment increased LC3-II levels and decreased p62 levels in a temperature-dependent manner(P<0.001).Consistently,a heat treatment at 47℃ for various durations(0,15,30,45,or 60 min)also increased LC3-II levels and decreased p62 levels in a time-dependent manner(P<0.001).Furthermore,we utilized a tandem mRFP-GFP-LC3 adenovirus construct to confirm the induction of autophagy based on the formation of GFP-LC3 and mRFP-LC3 puncta,which indicate autophagosome formation.Heat stress induced the accumulation of GFP-LC3 and mRFP-LC3 puncta in the perinuclear region and cytoplasm in SMMC7721 and Huh7 cells in a temperature-dependent manner,suggesting that heat stress induces autophagy(P<0.001).Under an inverted optical microscope,heat-treated SMMC7721 and Huh7 cells contained numerous microscopic vacuoles compared to the control cells.Typical double-membraned vacuolar structures with the morphological features of autophagosome in heat-treated SMMC7721 and Huh7 cells were analyzed using a transmission electron microscope to confirm these findings.The number of autophagic vacuoles per cell was dramatically increased from less than 3 to 20 after the heat treatment(P<0.001).Thus,sublethal heat stress induces autophagy in HCC cells in a temperature-and time-dependent manner.This phenomenon was particularly obvious after a treatment at47℃ for 30 min.Therefore,a heat treatment at 47℃ for 30 min was selected for subsequent experiments.(3)We measured the level of SQSTM1/p62,a cargo protein recognized as a marker of autophagic flux,using western blotting to clarify whether the accumulation of autophagosomes induced by sublethal heat stress resulted from the induction of autophagosome formation or the inhibition of autophagosome degradation.When SMMC7721 or Huh7 cells were exposed to heat stress at 47℃ from 30 to 60 min,p62levels decreased,while LC3-II levels increased(P<0.001).When cells were exposed to heat stress in the presence or absence of the autophagy inhibitor CQ,LC3-II levels were further increased in the combination group compared with the group receiving the heat treatment alone,while p62 levels increased(P<0.001).We examined changes in the color of mRFP-GFP-LC3 puncta to confirm this finding.SMMC7721 and Huh7 cells were transfected with mRFP-GFP-LC3 adenovirus and then exposed to sublethal heat stress.If an autolysosome is formed upon the fusing of an autophagosome with a lysosome,GFP fluorescence,but not mRFP fluorescence,is attenuated under the acidic lysosomal conditions.When cells were exposed to heat stress and CQ,the number of GFP puncta was significantly increased,while the number of mRFP+/GFP-puncta decreased.This observation supports the hypothesis that autophagy induced by sublethal heat stress proceeds to the lysosomal degradation phase.Our findings indicate that the accumulation of LC3-II induced by sublethal heat stress results from the induction of autophagosome formation and not from the blockade of autophagosome degradation.(4)The autophagy inhibitor CQ was employed to disrupt lysosomal function and subsequently block autophagic degradation to determine the biological role of autophagy in heat-mediated apoptosis.Therefore,HCC cells were treated with PBS,CQ,heat stress or the combination of CQ and heat stress.CQ enhanced the heat-induced decrease in cellular proliferation,as indicated by decreases in cell viability and EdU incorporation(P<0.001).Consistent with this observation,heat-induced apoptosis was markedly increased in the presence of CQ,as indicated by an increased number of Annexin V-positive cells and the accumulation of cleaved Caspase-3 and cleaved PARP.Because the pharmacological autophagy inhibitor CQ might exert effects on lysosomes that are independent of autophagy,an alternative approach was applied to block the formation of autophagosomes via the knockdown of key autophagy genes,such as Beclin-1and Atg5,using siRNAs.First,we confirmed that the heat treatment increased Beclin-1,Atg5,Atg7 and LC3-II levels and decreased p62 levels.Furthermore,Beclin-1 and Atg5silencing significantly reduced Beclin-1 and Atg5 levels and markedly increased the heat-induced apoptosis of HCC cells(P<0.001).Collectively,heat-induced autophagy exerts a protective effect on heat-induced apoptosis in HCC cells.(5)Based on the results of the ATPlite luminescence assay,sublethal heat stress reduced the ATP levels in SMMC7721 and Huh7 cells(P<0.001).Western blot analyses revealed increased AMPK phosphorylation and decreased phosphorylation of the mTOR downstream targets p-p70S6K and p-4E-BP1 following exposure to sublethal heat stress in a time-dependent manner,suggesting that the ATP-AMPK-mTOR signaling pathway plays an important role in heat-induced autophagy.The effects of the AMPK activator AICAR and the AMPK inhibitor compound C on autophagy were analyzed in heat-treated cells to further confirm that AMPK was involved in autophagy induced by heat stress.Similar to heat stress,AICAR activated AMPK via phosphorylation at Thr172 and induced an increase in LC3-II levels and a decrease in p62 levels,whereas compound C blocked the effects of heat stress on AMPK activation and autophagy.Based on these results,the ATP-AMPK-mTOR signaling pathway is involved in heat-induced autophagy in HCC cells.(6)We established a xenograft tumor model using SMMC7721 cells to examine whether CQ potentiated the antitumor effect of heat treatment in vivo.SMMC7721 cells that had been pretreated at 47℃ for 30 min or maintained at 37℃ were subcutaneously injected into nude mice,which were treated with or without CQ,and tumor sizes were measured.By day 30,a significant difference was not observed between the vehicle and CQ groups(P>0.05).Tumor growth was moderately inhibited by the heat treatment but was more significantly inhibited by the combination treatment.An analysis of tumor weight at the end of the experiment indicated a statistically significant difference between the heat treatment and the combination treatment(P<0.001).IHC staining revealed that the heat treatment significantly increased the expression of LC3(a marker of autophagy)and decreased the expression of p62(a marker of autophagic flux)(P<0.001).Thus,the heat treatment induces autophagy and autophagic flux in vivo.In addition,compared with the heat treatment alone,the heat treatment plus CQ markedly increased cleaved Caspase-3levels but decreased Ki-67 expression(P<0.001).Taken together,targeting autophagy with CQ sensitizes cells to heat-induced apoptosis in vivo.Conclusions Taken together,the autophagy marker LC3 was overexpressed in HCC cell lines and HCC tissues,particularly in RFA-treated HCC tissues.Relatively high LC3 expression was associated with a high recurrence rate and poor prognosis,suggesting that LC3 potenti ally represents an effective biomarker for predicting the recurrence of HCC after RFA treatment.SMMC7721 and Huh7 cells were exposed to heat stress to simulate the microenvironment of the transition zone of RFA.Sublethal heat stress induced autophagy in HCC cells in a temperature-and time-dependent manner.Furthermore,the inhibition of autophagy by CQ or siRNAs targeting the autophagy-related genes Beclin-1 and Atg5 increased heat-induced apoptosis.The combination of CQ and the heat treatment significantly suppressed tumor growth both in vitro and in vivo.Mechanistically,we report for the first time that the ATP-AMPK-mTOR signaling pathway is involved in heat-induced autophagy.In conclusion,heat stress induces protective autophagy against heat-induced apoptosis in HCC via the ATP-AMPK-mTOR axis,suggesting that approaches targeting autophagy may be a promising strategy for improving the efficacy of RFA treatment for HCC. |
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