| Tumor recurrence and therapeutic resistance are the main leading causes of treatment failure.Chemotherapy and immunotherapy are the clinical therapeutic strategies for the management of recurrent and advanced cancers.However,the generation of drug resistance,or the generation of strong toxic and side effects to chemotherapeutic drugs are important reasons to limit the effect of chemotherapy.Multidrug resistance(MDR)is a major problem in clinical treatment,which often leads to the failure of final chemotherapy.The mechanism of MDR is complex,including the high expression of ABC transporters,the change of anti-apoptosis mechanism,and the metabolic reprogramming.There have been much reports on the mechanism of drug resistance,but there are few effective strategies for clinical treatment.Therefore,it is urgent to find new therapeutic targets and reverse drug resistance strategies.We first assessed changes in gene expression in docetaxel(Doc)-induced multidrug-resistant PC3/Doc prostate cancer cells.The expression profiles of genes related to metabolism in lysosomal and mitochondrial pathways were particularly significantly different in resistant cells compared to control cells.According to Gene Ontology(GO)analysis,changes in the signatures of organelle-related genes were most evident in lysosomes,as indicated by the highest ratio of lysosomal GO diffgenes/GO genes among organelle-related genes.Therefore,this paper will focus on the role of lysosomes in multidrug resistance,the target screening of lysosomotropic agent RDN,and the mechanism of reversing multidrug resistance.In this paper,we analyzed the key cancer driver genes and new mechanisms of drug resistance by spectrum chip,TCGA data platform and proteomics.We screen new natural small molecule compounds to reverse the drug resistance according to new mechanism and potential,new drug resistance targets.It provides a new theoretical basis for clinical tumor treatment and overcoming drug resistance.Part I.TFE3-mediated lysosomal reprogramming is involved in multidrug resistance.i.Metabolic reprogramming occurs in drug-resistant cells,and lysosome changes are the most significant.1、The update rate and number of lysosomes in PC3/Doc resistant cells are much higher than PC3 cells.According to GO analysis,changes in the signatures of organelle-related genes were most evident in lysosomes.as indicated by the highest ratio of lysosomal GO diff-genes/GO genes among organelle-related genes.Compared to PC3 cells.The numbers of autophagosomes and lysosomes increased significantly in PC3/Doc cells by transmission electron microscopy and the fluorescent intensities of Lyso-Tracker.We also detected the lysosomal related proteins(lysosomal-associated membrane protein 1,LAMP1;lysosomal-associated membrane protein 2,LAMP2;Mucolipin,MCOLN1;vacuole protein sorting 18,VPS 18;cathepsin B,CTSB;cathepsin D,CTSD;cathepsin L,CTSL;ATPase,H+transporting,lysosomal 50/57kDa.V1 subunit H,ATP6V1H)by qPCR and Western blotting.The results showed that lysosomal related proteins(including mitochondria and autophagy)were generally elevated.2、The lysosomal activity was enhanced in PC3/Doc cells.The shift to lysosomal metabolism in the resistant cells might be ascribed to this increase in gene transcription,which in turn led to enhanced lysosomal activity,as indicated by the increase in acidic sphingomyelinase(ASM)activity.3、Different clinical chemotherapeutic drugs also can promote lysosomal metabolism.These changes in lysosomes were further corroborated in several other drug-resistant cells(Doc-resistant cells(RM1/Doc),Taxol-resistant H460 cells(H460/Tax),and vinblastine-resistant cells(KB/VCR)),and increased numbers of basal lysosomes were consistently observed in all resistant cell lines compared to the number of basal lysosomes in their paired cell lines.We constructed drug-resistant animal models by challenge with first-line clinical chemotherapeutics to confirm the metabolic shift accompanied by multidrug resistance in vivo.Consistent with the observations in cultured cells,an increase in the expression of lysosomal-associated genes was clearly shown in tumors treated with Doc,VCR,Dox,and VP 16,although this response was varied to some extent.Therefore,lysosome activation is an outcome of treatment with various antitumor agents that contributes to the development of drug resistance regardless of the structures and targets of these antitumor agents.4、The expression of four representative lysosomal genes were correlated with poor treatment outcomes.We next analyzed publicly available data in TCGA database on the expression of four representative genes in the autophagic/lysosomal pathway in patients to validate the role of the lysosomal metabolic pathway in the efficacy of anticancer therapy:lysosomal-associated membrane protein 2(LAMP2),cathepsin L(CTSL,a lysosomal enzyme),ATP6V1H(a subunit of the peripheral V1 complex of the vacuolar ATPase),and VPS18(a central member of the VPS-C core complex).Indeed,LAMP2,CTSL,ATP6V1H,and VPS18 expression was negatively correlated with the survival of bladder cancer,lung cancer,breast cancer,and colon cancer patient cohorts,respectively.Notably,none of the four genes was positively correlated with the survival of patients in all the listed cancers.Thus,the lysosome is activated prominently in drug-resistant cells and correlated with poor treatment outcomes.ii.Therapy-activated TFE3 acts as a critical regulator to control the expression of most lysosomal related genes and lysosomal biogenesis.The microphthalmia-transcription factor E(MiT/TFE)family members,especially TFEB and TFE3.act as the most important regulators to control the lysosomal biogenesis.Relevant studies have confirmed that TFEB and TFE3 control lysosomal biosynthesis by regulating the expression of most lysosomal related genes.Usually,TFEB and TFE3 phosphorylation remains in the cytoplasm,and after dephosphorylation,it relocates to the nucleus and promotes the expression of lysosomal related genes.We first determined whether the biogenesis of lysosome in drug-resistant cells was depended on TFEB or TFE3.1、Therapy-activated TFE3 acts as a critical regulator to control lysosomal biogenesis.1)Docetaxel activates TFEB,TFE3 and control lysosomal biogenesis.Since the number and activity of lysosomes increased significantly after chemotherapy treatment,we first analyzed whether Doc could activate TFEB and TFE3 while exerting anti-tumor effects and lysosome metabolism.Compared to parental cells,either TFE3 or TFEB was present in its active form(decreased phosphorylation status)in PC3/Doc cells.The depletion of TFE3 or TFEB significantly suppressed lysosomes and their associated gene expression in resistant cells.Additionally,the presence of nuclear TFEB and TFE3(activated transcription)was examined after Doc or Dox treatment.Both drugs clearly promoted the accumulation of TFE3 and TFEB in the nucleus,and the activation of TFE3 was more noticeable than that of TFEB after prolonged treatment.Therefore,both transcription factors participate in controlling lysosomal gene expression in resistant cells,and TFE3 was selected for further mechanistic study.2)Multiple chemotherapeutic drugs induced drug resistance is accompanied by TFE3 activation.We also found noticeable activation of TFE3 in vivo with Doc、Dox、VCR and VP 16 treatment.This means that the activation of TFE3 is prevalent during drug resistance.2、Doc facilitates the nuclear translocation of TFE3 by calcineurin.A study was designed to identify the regulator that activates TFEB after chemotherapeutic treatment.Since reactive oxygen species(ROS),which are often induced by chemotherapeutic agents and have been implicated in TFEB activation(decreased phosphorylation)were reduced in resistant cells,the possibility that the activation of TFE3 and TFEB is ROS-independent was ruled out.We next analyzed changes in several kinases responsible for TFE3/TFEB phosphorylation.The activation of mTORC1 and p-70S6K was evident in resistant cells,while phosphor-AKT and phosphor-GSK3β levels remained unchanged.These data suggested that phosphatase,instead of the kinase,contributes to the activation of TFE3 through dephosphorylation.We therefore compared the activity of calcineurin,a phosphatase involved in the regulation of TFE3,in PC3 and PC3/Doc cells.The calcineurin expression and activity were significantly upregulated in resistant PC3/Doc cells.FK520,an inhibitor of calcineurin,could partially reverse the nucleation of TFE3,supporting the regulatory effect of calcineurin on TFE3 phosphorylation.Therefore,activation of TFE3,which was induced at least in part,by calcineurin in response to chemotherapeutics,controls lysosome biogenesis.iii.TFE3 promotes lysosomal MRP2-mediated drug sequestration.1、TFE3 contribute to drug resistance.We further explored whether TFE3-driven changes in lysosome metabolism contribute to drug resistance.TFE3 downregulation remarkably restored the sensitivity of PC3/Doc cells to either Doc or Dox.and the overexpression of TFE3 significantly increased the tolerance of PC3 cells to Doc and Dox.A similar result was observed following TFEB downregulation,but this effect was to a lesser extent,indicating that the activated TFE3/TFEB axis in lysosomes is involved in conferring drug resistance.2、TFE3-driven changes in lysosome metabolism contribute to drug resistance.Since TFE3 acts as a critical regulator to contribute to drug resistance,and drug pump protein is an important mechanism to mediate multi-drug resistance independent of drug structure,we first analyzed whether TFE3 is involved in drug resistance through changing drug storage capacity.We tested whether TFE3 affects drug accumulation in cells by modulating transporters that pump drugs.Rhodamine B and Dox,two indicators used to detect drug efflux,were significantly reduced in resistant cells.However,regardless of whether TFE3 was downregulated or overexpressed,the drug storage capacity in the cells remained unchanged,suggesting that TFE3 affects lysosomes rather than cell membrane transporters.These data thus demonstrated that TFE3 triggers enhanced drug trapping in lysosomes,resulting in lysosome-mediated resistance.3、TFE3 drives lysosomal biogenesis to drug-resistance by increasing the distribution of Doc and Dox in lysosomes.To determine if lysosomal sequestration,a main mechanism of drug resistance,enforces Doc sequestration in lysosomes,concentrations of Doc in lysosomes were measured by high-performance liquid chromatography(HPLC).Typical markers served as indicators for every organelle fraction.There was significantly less Doc and Dox in the cytoplasm of resistant cells compared to parental cells,whereas high levels of trapped Doc and Dox were detected in the lysosomes of resistant cells,indicating that drug trapping in lysosome causes off-target effects on their sites of activity,thereby reducing their cytotoxic effects.4、TFE3 promotes lysosomal MRP2-mediated drug sequestration.1)MRP2 mediated drug sequestration in PC3/Doc cellsWe next tested whether ABC family transporters,which are located in the lysosomal membrane and required for drug sequestration,contribute to facilitating Doc accumulation in lysosomes.Gene expression profiles revealed that the levels of most ABC superfamily efflux transporters were increased in drug-resistant cells,and some of these increases were verified by qPCR.As multidrug resistance-associated protein 2(MRP2)was significantly increased in PC3/Doc cells,but P-glycoprotein(P-gp)was absent in PC3 and PC3/Doc cells although P-gp gene(ABCB1)expression was high.We examined the involvement of MRP2 in lysosome-mediated drug resistance.Downregulation of MRP2 markedly restored the sensitivity of resistant cells to chemotherapeutic drugs.2)MRP2 was present in the cytoplasm and shown to colocalize with lysosome.Importantly,in addition to its localization in the cell membrane,a significant amount of MRP2 was present in the cytoplasm and shown to colocalize with LAMP2,suggesting that MRP2 contributes to lysosomal drug sequestration.3)The association of TFE3 and MRP2.MRP2 is a proton pump transmembrane protein and can be located in the lysosome membrane,while the most significant enrichment of TFE3 target genes is lysosomal pump protein.We further analyzed whether TFE3 promotes lysosomal mediated drug resistance by regulating the expression and localization of MRP2 to complete drug accumulation in lysosomes.We therefore examined the association of TFE3 and MRP2.TFE3 depletion inhibited MRP2 transcription,and Doc-and Dox-induced elevated MRP2 levels were also noticeably decreased in cells lacking TFE3.In contrast,the ectopic expression of TFE3 induced MRP2 expression and,in particular,facilitated MRP2 transcription in the presence of Doc and Dox.Importantly,the ectopic expression of TFE3 stimulated MRP2 localization in lysosomes and lysosomal drug trapping.Thus,TFE3-stimulated MRP2 expression and localization in lysosomes promotes lysosomal drug trapping and resistance.Part II.Targeting of VPS18 by the lysosomotropic agent RDN reverses therapy-induced drug resistance.In part I,it was clear that most chemotherapy drugs can activate TFEB or TFE3,which leads to multidrug resistance in tumors mediated by lysosomes.Based on the preliminary screening and identification of potential dibenzyl compounds,we further screened and identified small molecule compounds that could regulate or target the"TFEB/TFE3-lysosome-multidrug resistance" pathway.Riccardin D-N(RDN),an aminomethyl derivative of dibenzyl compound Riccardin D,is a cationic compound,which can target lysosomes,cause lysosome permeability and mediate cell death,but its mechanism and target are unknown.Firstly,we analyzed the effect of RDN on lysosomes and its efficacy in reversing drug resistance.Secondly,the mechanism of RDN causing lysosome permeability and inducing DNA damage was determined.Finally,to define the molecular target of RDN in the lysosome.a chemical probe,RDN-biotin,was designed to allow streptavidin affinity purification of RDN interactors.i.Therapy-induced drug-resistant cells are vulnerable to lysosome-targeting agents1.Therapy-induced drug-resistant cells are vulnerable to lysosome-targeting agents.To explore whether predominant lysosome-dependent metabolism is critical for the survival of cancer cells with acquired resistance and,if so,whether the resistant cells exhibit enhanced sensitivity to lysosome-targeting agents,we used the small molecule RDN,which acts selectively towards lysosomes.Compared to parental cells(IC50=6.12±0.12(μM).the resistant PC3/Doc cells were more sensitive to RDN treatment(IC50=2.15±0.03 μM),while Doc was less effective.Similar to RDN,another two agents that typically target lysosomes,salinomycin(SAL)and hydroxychloroquine(HCQ),inhibited resistant cells to a greater extent than paired cells and perturbed lysosomes as well.2.Drugs targeting the same organelle have obvious synergistic effects.To further highlight the importance of lysosomes in the survival of therapy-induced resistant cells and overcome potential off-target effects and toxicity often caused by treatment with a single agent at relatively high doses,we evaluated the efficacy of combination treatment regimens with the combination index(Cl),which reflects the synergistic effect of combined drugs in cultured cells.Screening assays revealed that the combination of alkaline agents,in particular,SAL and RDN,predominantly augmented the response of resistant cells and that cotreatment induced extremely low CI values,indicating a strong synergistic effect.At the same time,we found that the mitochondrial target drugs in particular,Syr and Met,Syr and BPTES also have a significant synergistic effect.The effect of Syr combined with Met has been reported in a paper of Science in 2016.3.The potential antitumor efficacy of SAL and RDN in vivo.Intrigued by the significant synergistic effect of SAL and RDN,we next investigated their potential antitumor efficacy in PC3/Doc-Luc xenografts.While SAL(10 mg/kg,SAL-H)marginally reduced tumor growth,four mice treated with SAL-H failed to complete the study due to SAL toxicity;however,a low dose of SAL(1 mg/kg,SAL-L)had limited activity against tumor growth.RDN(20 mg/kg,RDN-H),however,significantly suppressed tumor growth without generic toxicity,as evidenced by the unchanged mouse body weights and aspartate transaminase(AST),glutamic-pyruvic transaminase(ALT),blood urea nitrogen(BUN),and creatinine(CREA)levels,which indicate liver and kidney function.Importantly,RDN(10 mg/kg,RDN-L)in combination with SAL-L substantially suppressed drug-resistant tumor growth and had no detectable toxicity at effective low doses.Conversely,the treatment of drug-resistant tumors with Doc was ineffective.These results suggest that a combinatorial strategy more potently blocks the proliferation of resistant cells by simultaneously targeting adaptive resistance mechanisms.Thus,therapy-resistant cell survival is predominantly lysosome-dependent,indicating metabolic vulnerability to lysosome-targeting agents,particularly combinatorial treatment with these agents.ii.Suppression of BRCA1 is essential for conferring CTSB-induced DNA damage in response to RD-N1.RDN causes lysosomal membrane permeabilization and blocks the fusion of lysosomes and autophagosomes.Having demonstrated the importance of therapy-activated lysosomes in cancer cell survival and drug resistance,together with the specific lysosomal inhibitory activity of RDN,we set out to determine the target of RDN in resistant cells.As RDN causes lysosomal membrane permeabilization,we first examined changes in ASM,a key component of the lysosomal lipid membrane.ASM activity was significantly reduced in resistant cells exposed to RDN,supporting the activity of RDN in lysosomes.As shown by analysis of a pH-sensitive LC3 reporter plasmid containing both GFP and RFP fusion tags,autophagosomes appeared yellow due to overlapping red and green fluorescence in a less acidic pH upon RDN treatment,which together with results indicating that RDN causes the accumulation of LC3 and P62,indicated that RDN might act on membrane proteins.2.RD-N induces lysosomal CTSB translocation to the nucleus.Our previous studies have shown that inhibition of CTSB by inhibitor CA074Me blocked RD-N-induced cell death.But the underlying molecular mechanisms remain unknown.The importance of CTSB in RD-N-induced apoptosis was assessed.Immunofluorescence staining supported the observations that,in contrast to the untreated cells where CTSB was excluded from the nucleus and exhibited the typical lysosome pattern.3.RD-N exacerbate DNA damage by inducing lysosomal CTSB translocation to the nucleus and degrading BRCA1.Western blotting confirmed that forced expression of CTSB markedly abolished p-BRCA1 in the nucleus and enhanced yH2AX foci in RDN-treated cells.To determine whether the effect of CTSB on BRCA1 relies on its proteolytic activity,we constructed a mutant CTSB expression plasmid lacking enzyme domain,resulting in an enzyme-inactivated product.Expression of enzyme-negative CTSB(ACTSB)predominantly reduced its catalytic activity as evidenced by losing the active bands,importantly,the level of phosphor-BRCA1 was restored in cells when expression of ACTSB in the presence of RDN.4.The nuclear localization of CTSB plays a role in the degradation of BRCA1.To further determine whether nuclear localization of CTSB plays a role in the degradation of BRCA1,we constructed a mutant CTSB expression plasmid lacking nuclear localization signal(CTSB-ANLS).Expression of CTSB-ANLS predominantly reduced its nuclear localization upon treatment with RDN,importantly,the level of phosphor-BRCA1 was restored in cells when expression of CTSB-ANLS in the presence of RDN.iii.The targeting of VPS18 by RDN interrupts the lysosomal membrane1.The targeting of VPS 18 by RDN.1)RDN-biotin,a chemical probe.To define the molecular target of RDN in the lysosome,a chemical probe,RDN-biotin,was designed to allow streptavidin affinity purification of RDN interactors.The activity of biotin-conjugated RDN(RDN-Bio)was evaluated prior to identifying proteins/enzymes that interact with the probe.RDN-Bio,to some extent,had a more potent inhibitory effect on cell survival than biotin alone.Additionally,the colocalization of RDN-biotin with LAMP2 in the lysosome was clearly shown by immunofluorescence staining.2)The targeting of VPS18 by RDN.After the incubation of PC3/Doc cell lysates with RDN-Bio or biotin alone,a specific band in precipitated RDN-Bio-streptavidin complexes was subjected to proteomic analysis.Putative RDN-biotin-interacting partners identified by mass spectrometry were VPS 18,ABCF1,HSPH1,and HSPA4L(data not shown).Considering the observed dependence of resistance on the lysosome,we focused our attention on vacuole protein sorting 18(VPS18),a central member of the VPS-C core complex that plays a pivotal role in lysosomal maturation.To test whether the interaction between RDN and VPS 18 is specific,PC3/Doc cell lysates were incubated with RDN-Bio or biotin control,and the immunoprecipitated proteins were probed using a specific antibody against VPS 18.VPS 18 was shown to coprecipitate with RDN-Bio but not control biotin.Furthermore,the interaction between VPS 18 and RDN disrupted VPS 18 function,as evidenced by the loss of the association between VPS 18 and its VPS-C partner,VPS 16,suggesting that RDN impairs the interaction between VPS 18 and the VPS-C complex.3)RDN binds to the RING domain of VPS 18,leading to disruption of the VPS-C core complex and lysosomal function.To determine the domain of VPS 18 required for RDN targeting,we generated Flag-tagged deletion mutants of human VPS 18 lacking three domains in the C-terminus(VPSA1),two domains in the C-terminus(VPSA2),one domain in the C-terminus(VPSA3),the RING domain(VPSA4),or the RING domain plus the CC domain(VPSA5).Whereas full-length VPS 18 strongly bound to RDN and VPS 18 containing the RING domain showed some residual binding activity,the truncated VPSA1,VPSA2,and VPSA3 mutants showed almost no association with RDN,indicating that the RING domain is required for the interaction of VPS 18 with RDN.Additionally,as the RING domain of VPS 18 is essential for recruiting VPS 18-binding partners,including VPS 16,it is reasonable that VPS 18 is unable to interact with VPS 16 in cells pretreated with RDN.2.Overexpression of lysosomal VPS 18 correlates with poor prognosis and chemoresistance.1)Overexpression of lysosomal VPS18 in several human tumors.Few studies have reported the role of VPS18 in diseases.In order to clarify whether VPS 18 has clinical significance and can be used as a molecular target for tumor therapy,we collected cancer tissues and adjacent tissues(prostate cancer,bladder cancer,lung cancer and colorectal cancer)of patients with different tumors,and analyzed its expression by qPCR and immunohistochemistry.2)VPS 18,but not other VPS proteins,is correlated with poor prognosis in several human tumors.Given that Doc transcriptionally induces VPS 18 expression,which is essential for autophagosome-lysosome fusion and binding to RDN,we were prompted to determine if VPS 18 is a significant pathological effector.As VPS 18,but not other VPS proteins,is correlated with poor prognosis in several human tumors.3)VPS 18 expression was elevated in drug-resistant cells and involved in drug resistance.As a target of RDN and highly expressed in a variety of common malignant tumors,we further analyzed whether VPS 18 was related to drug resistance.VPS 18 silencing significantly inhibited cell proliferation and increased the sensitivity of resistant cells to Doc and Dox,while the overexpression of VPS 18 increased the tolerance of PC-3 cells to chemical agents,indicating the involvement of VPS 18 in drug resistance.Therefore,a marked increase in VPS 18 in tumors correlates with poor prognosis and drug sensitivity3.TFE3 can transcriptionally regulate the expression of VPS 18.1)As a key component of VPS-C core complex,whether the high expression of VPS18 in tumor and drug-resistant cells is related to TFE3.The results of qPCR showed that VPS18 was transcriptional regulated by TFE3,and overexpression of TFE3 could significantly up-regulate the expression of VPS 18,indicating that VPS 18 was the downstream regulatory gene of TFE3.2)VPS 18 affects the maturation of lysosomes.Lyso-tracker staining showed that downregulation of VPS 18 resulted in a decrease in lysosomes.Western blotting results showed that the down-regulation of VPS 18 significantly reduced the active forms of LAMP2 and CTSB,but did not affect the expression of RAB5.The staining results of RFP-GFP-LC3 double fluorescent reporter gene showed that the down-regulated VPS 18 could block the formation of rapamycin induced autophagosomes.Therefore,VPS 18 is related to the maturation and permeability of lysosomes.Part Ⅲ.Inactivation of TFEB by Marchantin M alleviates the chemotherapy-driven pro-tumorigenic senescent secretionIn part I,it was clear that most chemotherapy drugs can activate TFEB or TFE3,which leads to multidrug resistance in tumors mediated by lysosomes.Based on the preliminary screening and identification of potential dibenzyl compounds,we further screened and identified small molecule compounds that could regulate or target the"TFEB/TFE3-lysosome-multidrug resistance" pathway.In part II,we have identified that targeting of VPS 18 by the lysosomotropic agent RDN reverses therapy-induced drug resistance through interrupting TFE3-mediated lysosome biogenesis.At the same time,during the screening of dibenzyl compounds,we were surprised to find that Marchantin M(Mar-M)can significantly reduce the expression of TFEB,and a low concentration of Mar-M can induce senescence in drug resistant cells.Another important hallmark feature of senescence is the secretion of numerous inflammatory cytokines,growth factors and proteases,which is termed senescence-associated secretory phenotype(SASP).The SASP is physiologically beneficial for tissue repair,wound healing.However,the secretion of inflammatory cytokines and matrix metalloproteinase in SASP influences neighboring cells and microenvironment by paracrine activities,resulting in acceleration of aging and onset the secondary cancers and some of the side effects of chemotherapy.Given the fact that senescent cells act as a double-edged sword in human cancers,pro-senescent and anti-senescent therapies are actively being explored.We have investigated the novel macrocyclic bisbibenzyl-based class of agents that were isolated from liverwort plants,as bioactive compounds to exert antitumor and anti-inflammatory activities.The present study went a further step to test the effect of Marchantin M(Mar-M)on the SASP.i.Mar-M triggered inactivation of TFEB and induced cell death and senescence1.Marchantin M(Mar-M)can significantly reduce the expression of TFEB,and a low concentration of Mar-M can induce senescence in drug resistant cells.According to previous studies,most drugs activate TFEB,TFE3 and control lysosomal biogenesis.But,during our screening of dibenzyl compounds,we were surprised to find that Marchantin M can significantly reduce the expression of TFEB,and a low concentration of Mar-M can induce senescence in drug resistant cells.ii.Mar-M-mediated proteasome inhibition is important for senescence induction1.Low concentration of Mar-M mainly causes cell cycle arrest through the p21 signaling pathway.Cycle arrest is one of the characteristics of cell senescence.The time-dependent induction of P21CIP1 and P27KIP.cell cycle inhibitors associated with senescence,was also evident in Mar-M-treated cells.Down-regulation of P21CIP1 but not P27KIP can reverse cell senescence induced by Mar-M.2.Mar-M-mediated proteasome inhibition is important for senescence induction.To gain insight into potential mechanisms required for regulation of Mar-M induced senescence,proteasome activity was measured to determine the involvement in Mar-M-induced senescence,because Mar-M has been shown to exert antitumor activity by inhibition of proteasome activity.Both chymotrypsin-like(CT-like)and peptidylglutamyl hydrolyzing(PGPH)activities were significantly decreased in a time dependent manner upon Mar-M treatment,while trypsin-like activity remained unchanged.To validate the role of the proteasome activity in Mar-M induced senescence,ectopic expression of β5 and β7,two major subunits of proteasome,resulted in enhanced proteasome activity.The enhancement of proteasome activity was associated with increased cell proliferation in response to Mar-M.Also,induction of γ-H2AX by Mar-M was decreased in cells expressing of β5 and β7 when compared to the Mar-M treatment alone,which was associated with reversal of the senescence phenotype induced by Mar-M.These data indicated that senescence induction by Mar-M was ascribed to,at least in part,proteasome inhibition.iii.Mar-M-triggered inactivation of TFEB contributes to suppression of SASP1.Mar-M suppress senescent cells expressing SASP.Screening assays have identified several candidate bisbibenzyls as potential anti-inflammatory and antitumor agents.We therefore aimed to analyze the effect of Mar-M on major SASP components of senescent PC3/Doc cells using antibody arrays.Mar-M,to some extent,reduced the secretion of several pro-inflammatory proteins including IL-la,IL-1β,IL-6,and TNF-α,associated with the decrease in IL-8 and IL-13.However,Dox treatment resulted in significant elevation in IL-1α,IL-1β,and IL-6 in SASP.To verify the changes in SASP upon Mar-M treatment,we assessed the cytokine transcription by quantitative PCR assays.Consistent with the observations in antibody arrays,Dox significantly stimulated gene expression related to inflammation,however,the corresponding mRNA levels of IL-1α,IL-1β,IL-6,IL-8,IL-13 and TNF-a were declined in response to Mar-M2.Mar-M-triggered inactivation of TFEB contributes to suppression of SASP.Transcription factor TFEB and TFE3 are recently emerged as modulators for IL-6 expression.We are prompted to examine the involvement of TFEB and TFE3 in the regulation of SASP in senescent PC3/Doc cells,because activated TFEB and TFE3(in the nucleus)was pronounced in PC3/Doc cells when compared to that of PC3 cells.The results indicated that a decreased TFEB,rather than TFE3,was noticeable in Mar-M-treated cells.Immunofluorescence analysis confirmed the reduction in TFEB nuclear translocation in Mar-M-induced senescent cells.To validate the importance of TFEB in regulation of inflammatory factors,the changes of SASP components were examined in cells overexpressing or knockdown of TFEB.Knockdown TFEB alone could partly abolish the SASP in PC3/Doc cells.By contrast ectopic expression of TFEB resulted in enhancement of IL-1α,IL-1β,and IL-6,leading to predominant restoration of SASP that were inhibited by Mar-M.Therefore,Mar-M suppressed the SASP components through inactivation of TFEB.3.Mar-M exerts antitumor efficacy with prolonged survival and inhibited the paracrine effect of SASP in tumor-bearing homograft mice.We next evaluated whether Mar-M could alleviate tumor burden due to its ability in the induction of senescence and decrease of SASP using RM1/Doc-Luc homograft mice.Tumor-bearing RM1/Doc homograft mice were pretreated with Mar-M or Dox for 10 days,the mice were subsequentially introduced RM1/Doc-luciferase(RM1/Doc-Luc)cells into the other side of each mouse to examine the paracrine effect of SASP released from RMl/Doc tumors.The paracrine effect of SASP was evaluated by measuring changes of bioluminescent luciferase in tumors arising from RMl/Doc-Luc.1)Mar-M induced RM1/Doc tumor senescent in vivo.We first moved to examine the effect of Mar-M on senescence induction in RM1/Doc tumor samples.Indeed,Mar-M induced RM1/Doc tumor senescent as evidenced by increased SA-β-Gal staining,consistent with results in cultured cells.It appeared that Dox exerted more potential ability on cellular senescence induction.2)Mar-M inhibited the paracrine effect of SASP in tumor-bearing homograft mice Analysis of typical markers of SASP verified that Mar-M markedly inhibited the expressions of IL-1α,IL-1β,and IL-6,whereas Dox significantly stimulated the expression of these factors,in agreement with the observations in cultured cells.The fluorescence intensity of RM1/Doc-Luc tumors in mice that were pre-treated with Mar-M was comparable to the control,while tumor growth was noticeably accelerated in Dox-pretreated mice,indicating that SASP released from RM1/Doc tumors,which were pretreated with Mar-M,had little p... |
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