| Hepatocellular carcinoma(HCC)is a primary liver cancer that develops from hepatocytes and is responsible for approximately 90% of cases.According to the Global Cancer Incidence,Mortality and Prevalence(GLOBOCAN)report for 2020,HCC is the sixth most common cancer globally and the third leading cause of cancer-related deaths.The poor prognosis of HCC is due to its asymptomatic nature during the early stages,leading to late diagnosis,and its resistance to traditional chemotherapy and radiotherapy.Early diagnosis of HCC,when the tumor is less than 5 cm in size,provides treatment options such as liver transplantation and surgical resection.Unfortunately,most patients are diagnosed at advanced stages as the disease is typically asymptomatic in its early stages.Advanced hepatocellular carcinoma(HCC)can be managed using various treatment methods such as radiofrequency ablation(RFA),transarterial chemoembolization(TACE),tyrosine kinase inhibitors(TKIs)and immunotherapy.However,despite the availability of these treatment options,they are not able to significantly prolong the lifespan of patients due to the emergence of therapy resistance and disease recurrence.Hepatocellular carcinoma(HCC)is one of the most common malignancies with a poor prognosis.Radiotherapy(RT)is widely applied in HCC patients,but its efficacy is limited due to inherent or acquired radioresistance.Recently,evidence has showed that the cancer metabolic switch from aerobic respiration to glycolysis(predominance in the Warburg effect)promotes radioresistance in several cancer types.However,how the cancer metabolic switch regulates radiation reactivity in liver cancer remains unclear.Aberrant metabolism is a major hallmark of cancer.In order to gain a survival advantage,cancer cells flexibly develop abnormal metabolic networks through different pathways.A core component is the Warburg effect(aerobic glycolysis as the predomiXnance).Although glycolysis is less efficient at producing ATP than aerobic respiration,in-termediate glucose metabolism between aerobic respiration and aerobic glycolysis can shunt to anabolic pathways such as lipid synthesis(adipogenesis),amino acid produc-tion,and nucleotide synthesis,thereby promoting tumor cells’ sustainable proliferation and progression and avoiding the occurrence of apoptosis.Moreover,high-level aerobic glycolytic metabolism also helps cells to avoid massive reactive oxygen species(ROS)accumulation without compromising the cancer-cells energy demand and thus keeping them away from overloaded oxidative stress,which is another main cause of apoptosis.Therefore,glycolysis is often used as the metabolic basis in tumors.Alt-hough several studies have reported radioresistance with high rates of glycolysis,the exact metabolic mechanism of ionizing radiation(IR)responsiveness remains unclear.IR triggers various forms of cell death,including apoptosis.After IR treatment second messengers and the damaged DNA could mediate apoptosis.However,how cancer metabolites are involved in IR-induced apoptosis remains elusive.The family of cathepsins is involved in overall protein turnover and specific cellular processes,such as apoptosis,antigen presentation,and prohormone processing.Despite being implicated in cancer development and the processing of neurotransmitters,the role of CTSH,a ubiquitously expressed lysosomal cysteine protease,in cancer apoptotic process is rarely reported.To solve the elusive questions above,here,we outline a comprehensive CTSHinvolved metabolic mechanism to regulate apoptosis and radioresistance,suggesting novel therapeutic strategies in radiation sensitization via disrupting cancer metabolism.Objective:The aim of this study is to observe the impact of CTSH on cell metabolism and radioresistance in hepatocellular carcinoma(HCC)cells and tumor models.Highthroughput analysis methods such as single-cell sequencing,transcriptome sequencing,and protein mass spectrometry,followed by bioinformatics analysis were used to investigate the signaling pathways and corresponding targets that are regulated by CTSH in tumor metabolism and cell death.Further experiments and validation are conducted using techniques such as immunofluorescence co-localization,flow cytometry,and Western blot to explore the regulatory mechanisms of CTSH on liver cancer metabolism and subsequent cell death,as well as its impact on radiation sensitivity.In addition,we aim to verify the clinical relevance of the experimental results through bioinformatics analysis.Methods:(1)HPA and TCGA databases,as well as the TNMplot platform were used to detect the expression of CTSH in liver cancer tissues and other tissues,and its correlation with liver cancer prognosis,as well as its impact on prognosis.(2)The human liver cancer cell lines MHCC97 H,Hep G2,and Huh7 were used as research subjects.Human embryonic kidney cells 293 T are used as engineering cells.(3)Genetic engineering methods were used to construct CTSH knockdown vectors and establish corresponding stable knockdown cell models.(4)An X-ray linear accelerator(model: X-RAD 320 ix,Precision X)was used to perform ionizing radiation on cells and experimental animals.Irradiation conditions: voltage: 320 kV,current: 12.5 m A,target-skin distance: 50 cm,dose rate: 3.0 Gy/min.(5)Flow cytometry after Trypan blue staining and Annexin V staining were used to detect cell death;colony formation assay is used to detect cell radiation sensitivity;DIOC6 staining followed by flow cytometry is used to detect cell mitochondrial membrane potential.(6)Immunofluorescence co-localization experiments were performed to detect the subcellular localization of each research protein and its expression activity.(7)Western blotting was used to detect changes in protein expression levels.(8)Single-cell sequencing was used to detect changes in tumor cell transcriptome levels after irradiation in rats.(9)PCR array was used to detect changes in gene expression levels before and after irradiation of selected genes,and transcriptome sequencing is used to detect changes in cell m RNA levels after CTSH knockdown.(10)Mass spectrometry(MS)was used to detect changes in individual protein levels after CTSH knockdown.(11)GO and KEGG enrichment analyses were used to determine the correlation between the changing genes and biological processes or pathways under each treatment condition,while GSEA enrichment analysis was used to determine the pathways or biological processes that undergo significant changes under each treatment condition.Results:1.Radiation-inhibited tumor growth and induced apoptosis in vivoFirst,the IR treatment was applied to rats bearing an orthotopic tumor in the liver.Compared with the control group,tumor growth was significantly inhibited in IR treatment groups,and this effect was more obvious when the dose was added to 9 Gy×3fraction(Figure1 a-b;Supplement Figure 1 A).Then,to further detect the mechanism of tumor inhibition induced by IR,transcriptome sequencing was conducted.The most significantly increased genes were collected(Attachment File 2).Biological process(BP)enrichment analysis showed that the up-regulated genes after IR were highly related with cell death,especially apoptosis(Figure1 c-d).In addition,with KEGG enrichment analysis,apoptosis and the TNF pathway seemed to be closely related to these genes(Figure1 e).Consistently,immunohistochemical(IHC)staining showed positive results of Caspase3 and apoptosis-inducing factor(AIF)after IR,which confirmed the occurrence of apoptosis(Figure1 f).2.CTSH participated in radioresistance regulation of HCC cellsDue to the non-homology between human and rat models [29],to further confirm the change of genes in HCC cells,a PCR array was performed on MHCC97 H and Hep G2 cell lines(Figure 2 a-c).As transcription always happens earlier than translation,we examined at 1 and 6 hours after IR,and expression profile were established(Figure2 b-c).The genes expressed consistently with in-vitro sequencing result(Attachment file 1 and 2)were found(marked with * in Figure 2 b-c).Meanwhile,trypan blue staining flow cytometry verified cell death after IR.Inhibitors of apoptosis,ferroptosis,necrosis,and autophagy pretreatment were applied to HCC cells,and IR-induced cell death was found reversed by the pretreatment of ZVAD(commonly used as an apoptosis inhibitor).With the pretreatment of ZVAD,the value of the MHCC97 H cell-death signal peak was significantly reduced after IR(Figure 2 d right),while in other groups the rescue effect was not evident(Supplemental Figure 2 A).Previous results had already informed us of the involved genes in vivo(Fig 1 c-e).After filtering out genes expressed inconsistently with the in vivo results or those already well reported [22,30-32],we mainly focused on CTSH and FAS(Supplemental Figure 2 B).Then,due to the low abundance of FAS and the inconsistency of CTSD expression(Figure 2 e;Supplemental Figure 2 C),we abandoned FAS and selected CTSH for further study.To further understand the influence of CTSH,a CTSH knockdown model was established in Hep G2 cells(Figure 2 f),and the radio-sensitivity was examined by colony formation assay.The results shown that knockdown(KD)of CTSH significantly increased the radio-sensitivity of Hep G2 cells(Figure 2 g-h).Consistently,MHCC97 H cells showed a significantly higher radio-sensitivity compared with Hep G2(Fig2 i-j),with a lower CTSH expression after IR stimulation(Figure 2 e).Taking these results together,we realized that CTSH participated in the maintenance of radioresistance in HCC cells.3.Restrained glycolysis and promoted aerobic respiration inhibited radioresistance of Hep G2 cellsTo further understand how CTSH performs in regulating HCC metabolism and radiation resistance,proteome mass spectrometry(MS)was performed.As the MS results showed,360 proteins were up-regulated and 430 were down-regulated after CTSH knockdown(Figure 3 a-b).Further enrichment analysis showed the up-regulated proteins mainly localized in the mitochondria(Figure 3 c).In addition,these upregulated proteins were related to mitochondrial function and aerobic respiration,while the down-regulated ones were associated with biosynthesis and metabolic processes(Figure 3 d-e).As mitochondria are known to play an important role in metabolism and apoptosis [33] and that the cancer metabolic switch is important for cancer cell fate [34],we wondered whether CTSH was affecting cell fate by regulating the metabolic switch.Remarkably,more detailed GSEA results confirmed our hypothesis—after CTSH knockdown the glycolytic metabolism was inhibited while the aerobic respiration was promoted(Figure 3 f-g).Important molecules for glycolysis such as HK2,PFKL,PKM,and LDHA were all down-regulated.Furthermore,key factors of the tricarboxylic acid cycle(TCA),such as CS,OGDH,and IDH,and oxidative phosphorylation(OXPHOS)promoting factors,such as AIFM1 and CYC1,were up-regulated(Figure 3 h-j).At the same time,mitochondrion pyruvate carrier MPC1 increased,indicating an increase in mitochondria pyruvate intake.Thus,enhanced TCA and OXPHOS formed a strong upregulation of the aerobic respiration cascade(Figure 3 h-j).Considering the reported association of glycolysis with radioresistance,the above results indicate that CTSH knockdown inhibited the radioresistance of Hep G2 cells by perturbing glycolysis and XIV reversing the cancer metabolism to aerobic respiration(Figure 3 k).4.Knockdown of CTSH and enhanced aerobic respiration promoted radiation-induced apoptosis via IAP inhibition and AIF signalAs the involvement of CTSH in apoptosis was found by our bioinformatic analysis(Fig 1 D-E),to directly confirm this,an annexin V-PI staining flow cytometry was performed.The results showed that apoptosis was significantly promoted by CTSH knockdown in both IR and NC conditions(Figure 4 a-b).Referring to our previous results(Fig 2 d)and the up-regulation of AIFM1(an apoptosis-inducing factor)(Fig 3j),to figure out which signaling CTSH knockdown occurred during the proapoptotic process,we analyzed the MS results by GSEA.The results suggested a promotion of apoptosis;among these,many apoptotic-related genes were affected after CTSH knockdown(Figure 4 c-d).Thus,to figure out the target of CTSH apoptotic regulation through mitochondrial signaling,an intersection of apoptosis and mitochondrial-related genes was taken;a total of 14 genes,including 9 up-regulated and 5 down-regulated,were listed(Figure 4 e-f).Among them,HTRA2 and DIABLO were up-regulated(Figure 4 g-h),and they were both reported to promote the apoptotic process as inhibitors of apoptosis(IAPs)[35-37].An immunofluorescence co-localization assay further verified our findings;compared to the blank-load transfection group(Sh Vec)and the control group(NC),HTRA2 and DIABLO increased in the CTSH knockdown group and inhibited the activity of the IAPs(XIAP and Survivin)in irradiated CTSH knockdown cells,and the inhibition of the IAPs occurred within both the cell nucleus and the cytoplasm(Figure 4 i-k;Supplement Fig3).Accumulating data support that tumor suppression may be achieved by inhibiting glycolysis and promoting OXPHOS(part of the aerobic respiratory chain)[35].As a killer protein,besides promoting OXPHOS,the up-regulated AIFM1(Figure 4 g)is also known to induce DNA fragmentation during the apoptotic process [38-41],suggesting a proapoptotic effect based on the up-regulated aerobic respiration cascade.Furthermore,the increased(activation of)caspase family(Caspase9 and Caspase3 cleavage)expression finally confirmed these connections(Figure 4 l).From these results,we realized that the proapoptotic effect of CTSH knockdown is carried out through promoted IAP inhibition and enhanced aerobic respiration,and this process is executed by HTRA2,DIABLO,and AIF signaling(Figure 4 m).5.CTSH knockdown changed mitochondrial membrane permeability and stability in proapoptotic signalingApoptosis includes intrinsic(mitochondrial)and extrinsic(death receptor)mechanisms.As previous results had suggested the involvement of mitochondrial genes(Figure 3 d-e;Figure 4 c-d),we decided to focus on mitochondrial dysfunction and apoptosis for further study.Remarkably,the GSEA results showed an increased cascade of the transmembrane transport of mitochondria and cytochrome c release(Figure 5 a).Among those,VDAC forms a channel through the mitochondrial outer membrane and allows the diffusion of hydrophilic molecules.It opens at low or zero membrane potential and closes at potentials above 30-40 m V [42].Fam162 A,FIS1,and OPA1 were reported to be involved in proapoptotic factor release(such as cytochrome c),caspase activation(such as CASP9),and mitochondrial permeability transition induction [43-45].Interestingly,after CTSH knockdown,all these genes were upregulated(Figure 5 b).Besides glycolysis,HK2 also plays a role in maintaining the integrity of the outer mitochondrial membrane and preventing the release of apoptogenic molecules from the intermembrane space and subsequent apoptosis [33].In cancer cells,it binds to and inhibits VDAC to suppress mitochondrial function while stimulating glycolysis [46],and it has also been observed to be down-regulated(Figure3 j).All these results indicated an increase in the permeability and instability of the mitochondrial membrane.Mitochondrial membrane potential(MMP)examination after IR further confirmed the participation of these molecules(Figure 5 c-d);a decrease in MMP(IR induced)and membrane stability(CTSH KD induced)made it easier for molecules to be released from the mitochondria after IR,thus promoting the apoptotic process.The above results suggested an explanation responsible for the increased apoptotic flux;CTSH knockdown promoted the release of some proapoptotic factors after IR through modulating the permeability and stability of the mitochondria membrane(Figure 5 e).6.CTSH and targets were correlated with tumorigenesis and poor prognosisThen,to verify the potential of CTSH in clinical application,a series of bioinformatic analyses were performed using well-known databases.For the m RNA level,CTSH showed significantly higher expression in HCC than non-tumor samples(Figure 6 a)(derived from GEPIA).In addition,in a clinical cohort of 370 HCC patients,XVI using a validated m RNA signature,higher CTSH signaling was associated with poor prognosis(p-value = 0.025)(Figure 6 b)(derived from Kaplan–Meier plotter).A survival Kaplan–Meier(KM)analysis of 365 patients showed similar results in the CTSH protein level(Figure 6 c)(from The Human Protein Atlas).A multi-gene expression comparison of CTSH targets,especially those glycolysis-related genes,showed homogeneous trends related to tumorigenesis(Figure 6 d)(from TNMplot).Furthermore,by combining CTSH expression and its downstream target expressions(i.e.,CTSH combined with PFKL,PKM,LDHA,HK2;and low IDH,MPC1,AIFM1,and HTRA2 combined with high CTSH expression),we found a remarkably significant facilitation of tumorigenesis and poor prognosis in HCC(Figure 6 e)(Kaplan–Meier plotter).Then,to verify the universality of the above findings,profiles of CTSH expression were drawn out using two different databases(Figure 6 f;Supplemental Figure 4 A)(TNMplot and GEPIA).Among them,human malignancies were selected because of the high CTSH tumor expression.Remarkably,CTSH was related with poor prognosis in all these cancers(Figure 6 G).Furthermore,survival KM plotting showed consistent facilitations of poor prognosis in malignancies such as cervical squamouscell carcinoma,esophageal carcinoma,and pancreatic adenocarcinoma(Figure 6 H-I;Supplemental Figure 4 B-C)(Kaplan–Meier plotter).However,in other low-grade malignancies,this relationship seems not notable or even reversed(Supplemental Figure 4 D).Taken together,these findings implied significant therapeutic and diagnostic potential for clinical use.Conclusion:1.Large segmentation radiotherapy inhibits tumor growth and induces apoptosis,and this phenomenon has been verified in human hepatocellular carcinoma cells.However,different cells have different radiation sensitivity,and CTSH plays a potentially important role in this process.2.Inhibition of CTSH can interfere with the tumour-dependent aerobic glycolysis and its downstream metabolic process,and promote its energy metabolism to the direction of aerobic respiration.3.The enhanced aerobic respiration pathway can increase the oxidative stress level of cells and promote apoptosis by increasing downstream products.Meanwhile,the upregulation of AIF in the aerobic respiration chain and the upregulation of HTRA2 and DIABLO can promote apoptosis through two mitochondrial pathways,Caspase-independent and dependent(inhibition of IAP protein family).4.The above promotion of apoptosis process is the result of synergistic action with ionizing radiation after increasing permeability of mitochondrial membrane and destroying its structural stability.5.The combined effect of CTSH and its downstream regulatory genes may provide great applications for the diagnosis and treatment of liver cancer... |
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