Identification Of C0818,a Novel Curcumin Derivative As Antitumor And Senolytic Agent Through Disruption Of Hsp90 Function In Human Hepatocellular Carcinoma Cells

BackgroundCancer,a category of diseases characterized by uncontrolled cell proliferation,is the leading cause of death worldwide.Hepatocellular carcinoma（HCC）is one of the most popular cancers around the world.While the majority of HCC cases are reported in the developing countries of Asia and Africa,there has been an alarming rise in HCC cases in Western Europe and United States.HCC is caused by chronic liver disorders,viral hepatitis,alcoholism,and dietary carcinogens such as aflatoxins and nitrosoamines.Cellular senescence can play a role in the onset and progression of liver diseases like liver cancer.The accumulation of senescent hepatocytes in large numbers due to the lack of immune clearance of senescent cells in advanced chronic liver diseases results in a senescence associated secretory phenotype（SASP）rich microenvironment,which may induce tumorigenesis in neighbouring DNA-damaged,pre-senescent hepatocytes.The most popular treatments are liver transplantation and surgical resection.The most commonly used chemotherapeutics（sorafenib,doxorubicin,and others）do not provide a six-month increase in the average survival time,and chemotherapeutic therapy has been linked to a rise in the number of senescent cells.Thus,there is a vital need to examine and evaluate alternative chemopreventive and therapeutic methods that may be successful in the management of liver cancer and selectively trigger senescent cell removal,as well as relieve several age-related phenotypes known as senolytics.Since cancer is a multifactor disease,it may necessitate the use of drugs that target a variety of intracellular components.As a result of extensive studies in recent years,various plant derivatives,involving polyphenols,have been demonstrated to have anticancer properties.Bioactive molecules such as curcumin,resveratrol,caffeine and flavonoids have been reported in various studies to have potential therapeutic efficacy in tumor suppression.Thus,emphasis should be given on the determination of possible molecular mechanisms behind anticancer activity of these polyphenols.This will also support the opinion that polyphenols could be useful in cancer treatment and prevention.Curcumin,the main polyphenolic curcuminoid in the turmeric rhizome Curcuma longa has long been utilized to treat a wide range of chronic diseases,including cancer and neurodegenerative disorders.Curcumin has been shown in several studies over the last few decades to be a potent anti-inflammatory agent with promising therapeutic potential against a variety of cancers.It prevents the proliferation and metastasis of human tumors by regulating transcription factors,growth factors,inflammatory cytokines,protein kinases,and other enzymes.It induces apoptotic cell death as well as cell cycle arrest,which inhibits the proliferation of cancer cells.In addition,curcumin and its analogs have some anti-aging properties involving;alleviate ageing symptoms and delaying the onest of age-related diseases in which cellular senescence is directly involved.However,the mechanisms of their action are still unknown.Despite these promising findings,new analogs of curcumin with improved therapeutic properties are needed to fulfill therapeutic needs.3,5-（E）-bis（3-methoxy-4-hydroxybenzal）-4-piperidinone hydrochloride（C0818）is a novel analogue of curcumin that is more potent than curcumin.Heat shock proteins（HSP）are overexpressed in cancer cells and establish a favourable environment for tumor development.Heat shock protein 90（Hsp90）is a molecular chaperone implicated in the maturation of various oncoproteins that are essential in tumor growth.Cancer cells are highly dependent on Hsp90 chaperone function for survival and proliferation in this regard.Furthermore,both senescent and cancer cells have been shown to use pro-survival pathways,which can at least in part be attributed to HSP90 and its stabilizing effect on its client proteins.Thus,Hsp90 has been considered as a promising target for the treatment of different types of cancer via targeting of different signaling pathways.Besides,the Hsp90 inhibitors demonstrated senolytic effect in addition to their original activity as antitumor agents.However,the clinical significance of Hsp90 and the mechanisms regulating the tumor-promoting effects of Hsp90 in HCC,the role of Hsp90 in the development of senescence and the benefit of developing Hsp90 inhibitors as senolytic agents in senescent HCC cells remain unobvious.This study is going to investigate the correlation between suppressing the growth of human hepatocellular carcinoma cells in vitro and Hsp90,and to evaluate the effect of C0818,a Hsp90 inhibitor through determination the mechanism by it C0818 induces the degradation of Hsp90 client proteins in HCC cells and determination its effect on the energy metabolism in cancer cells which mediated by targeting mitochondria.In addition,Identification of C0818 as a new senolytic agent targeting adriamycin-induced senescent HCC cells through its ability to kill senescent cells or inhibit the senescence-associated secretory phenotype（SASP）which mediated through its ability to inhibit Hsp90 and autophagy in senescent cancer cells.MethodsHuman hepatocellular carcinoma Hep G2 and Sk-Hep-1 cell lines were used.Cellular senescence was induced in Hep G2 and Sk-Hep-1cell lines by treating cells with Adriamycin（ADR）at 0.125μg/m L,ADR was washed out after 48 hours and cells were incubated in normal medium for five additional days.Cell viability was assessed using MTT,colony formation and Ed U incorporation assays.Apoptosis,cell cycle analysis,loss of mitochondrial membrane potential（MMP）and quantification of ROS production were determined using flow cytometry.The expression of Hsp90 client proteins,the proteins involved in apoptosis,proteins regulate cell cycle,proteins regulate oxidative stress,proteins regulate energy metabolism and mitochondrial mass,proteins of molecular senescence markers and the DNA damage marker,autophagy associated proteins and the proteins regulate SASP modulation were determined using Western blotting.To further validate the essential role of Hsp90 in hepatocellular carcinoma cells survival and induction of senescence,we transfected si RNAs targeting Hsp90 into Hep G2 and Sk-Hep-1 cell lines.To confirm the significance of caspase activation in C0818 induced cell death,Hep G2 and Sk-Hep-1 were pretreated with the general caspase inhibitor z-VAD-fmk.To investigate the molecular mechanism underlying C0818 induced Hsp90 client proteins depletion,cycloheximide,an inhibitor of protein synthesis,MG132 a proteasome inhibitor and NH4Cl a lysosome inhibitor were used in the presence or absence of C0818.Immunoprecipitation（IP）experiment was carried out to investigate the effects of C0818 on the chaperone functions of Hsp90.Quantitative real-time PCR was used to test the m RNA expression levels of Hsp90 clients Raf-1 and Akt and the markers of senescence P21,P16 and IL6 using GAPDH as control.To further analyze the role of ROS,the ROS scavenger N-Acetyl-L-cysteine（NAC）was used to inhibit the accumulation of intracellular ROS.The levels of cellular ATP were measured using ATP assay kit.To quantify senescence,the lysosomalβ-galactosidase enzyme（SA-β-Gal）activity was measured using the colorimetric substrate X-gal.ELISA was used to quantify the levels of SASP.To clarify the modulation of C0818 on autophagy flux,senescent HCC cells were treated with rapamycin,a well-known autophagy inducer and lysosomotropic agents chloroquine（CQ）and bafilomycin A1（Baf）.ResultsThe IC₅₀values of C0818 and curcumin at 48 h were 2.1μmol/L and were 19.3μmol/L,1.9μmol/L and 19μmol/L for Hep G2 and Sk-Hep-1 cells,respectively.The inhibition in colony formation by C0818 was observed in both Hep G2 and Sk-Hep-1 cells in a dose dependent manner and a nearly complete inhibition in the colony formation was observed at the highest dose of C0818 in both cell lines.It was found that C0818induced an increase in both early and late apoptotic cells percentage in a dose-dependent manner in both Hep G2 and Sk-Hep-1 cells.Click-i T EDU proliferation assay showed that C0818 significantly inhibited DNA synthesis in both Hep G2 and Sk-Hep-1cells in a dose dependent manner.C0818 induced an obvious G2/M phase arrest in a dose dependent manner in both Hep G2 and Sk-Hep-1 cells.The increase in the cells percentage in G2/M phase was consistent with the reduction in the percentage of cells in both the S and G0/G1 phases.The expression levels of Cdc2,P-Cdc2（Thr161）,Cyclin B1 and Cdc25c were down-regulated,and P21 was up-regulated by C0818.Western blot analysis showed that C0818 induced an increase in cleavage of caspase-3,-7,-8,and-9 and PARP in a dose and time dependent manner in both Hep G2 and Sk-Hep-1 cells.The cleavage of PARP and caspase-3 induced by C0818 was significantly inhibited by z-VAD-fmk.In addition,C0818 induced cell death and apoptosis were prevented by z-VAD-fmk,suggesting that C0818 induced apoptosis in HCC cells is caspase-dependent.The knockdown of Hsp90 significantly reduced the viability of HCC cells.C0818 was found to reduce the levels of RAS,C-Raf,P-C-Raf,ERK,P-ERK,MEK,P-MEK,PI3K,AKT,P-AKT,mTOR and P-mTOR in a dose and time-dependent manner in both Hep G2 and Sk-Hep-1 cells.It was found that C0818 degraded C-Raf much faster when new protein synthesis was inhibited by cycloheximide（CHX）in Hep G2 and Sk-Hep-1cells,suggesting that C0818 induced C-Raf degradation instead of inhibition of the protein synthesis.It was found that treatment with the proteasome inhibitor MG132completely blocked C0818 induced degradation of C-Raf and AKT.In addition,degradation was not affected by the treatment with the lysosome inhibitor NH4Cl in both cells,indicating that C0818 induced the degradation of Hsp90 clients by the proteasome rather than the lysosome.Besides,after down-regulation of the expression of Hsp90 using si RNA,C0818 was unable to promote C-Raf and AKT proteins degradation,suggesting that Hsp90 is a direct target of C0818.Immunoprecipitation（IP）experiment showed that C0818 inhibited the binding of C-Raf and AKT with Hsp90 and induced their degradation by the proteasome.In addition,C0818 had no obvious effect on the transcription of Hsp90 client proteins such as C-Raf and AKT.It was found that C0818 could up-regulate the Bax/Bcl-2 ratio in a dose and time dependent manner in both Hep G2 and Sk-Hep-1 cells and induced a significant reduction in MMP in a dose-dependent manner in both cell lines.After C0818 treatment for 48 h,flow cytometry analysis revealed a significant increase in ROS production in a dose dependent manner in both cell lines.C0818 down-regulated the level of PINK1 and release of mitochondrial cytochrome c into the cytosol of HCC cells in a dose dependent manner.The results of annexin V–APC/PI staining showed that the percentage of apoptotic cells was significantly attenuated by pre-treatment with NAC.Besides,the change in the expression levels of Bax,Bcl2,cleaved PARP and cleaved caspase 9 was partially reversed by pre-treatment with NAC.C0818-induced cell growth inhibition was markedly decreased by pre-treatment with NAC,suggesting that C0818 induced ROS-dependent cytotoxicity in HCC cells.C0818 was found to reduce the levels of AMPK,P-AMPK and increased the level of TOMM20 in a dose and time-dependent manner in both Hep G2 and Sk-Hep-1 cells.C0818 induced a significant reduction in the ATP levels after 24 h and a nearly complete depletion of ATP was seen after 48 h of C0818 treatment,suggesting that C0818 induced mitochondrial dysfunction in HCC cells.Cell senescence was confirmed via measuring markers of senescence including increased expression of the cell cycle inhibitors p21 ^Cip1/Waf1,p16^Ink4a,P53,as well as increased expression of the DNA damage markerγ-H2AX in ADR treated Hep G2 and Sk-Hep-1 cells.C0818 showed a significant senolytic activity in ADR-induced senescent HCC cells.As confirmed by SA-β-Gal staining,C0818 induced a significant reduction in the density and number of stained cells in both Hep G2 and Sk-Hep-1senescent cells in a concentration-dependent manner.P16^INK4aand p21^Cip1/Waf1were significantly down-regulated and the m RNA levels of P16^INK4aand p21^Cip1/Waf1were decreased in both senescent Hep G2 and Sk-Hep-1 cells after the treatment with C0818in a concentration-dependent manner.C0818 treatment increased the number of Annexin-V-positive cells in senescent cells.Western blot analysis showed elevated levels of activated caspase-7,caspase-8 and degradation of poly（ADP-ribose）polymerase（PARP）in senescent cells.C0818 induced apoptosis in senescent cells was significantly reduced by the pan-caspase inhibitor z-VAD-fmk.The cleavage of PARP and caspase-8 induced by C0818 was significantly inhibited by z-VAD-fmk in senescent cells.A reduction in mitochondrial membrane potential and down regulation of Bcl-2 was observed upon C0818 treatment in senescent cells in a concentration-dependent manner,suggesting that C0818 induced caspase-dependent apoptosis in senescent HCC cells through the mitochondrial-mediated pathway.AKT and its activated form P-AKT（Ser473）were upregulated in ADR treated cells comparing to control HCC cells.C0818 was able to reduce the levels of AKT and P-AKT in senescent cells.The knockdown of HSP90 induced the apoptosis and decreasing the levels of AKT and P-AKT in senescent cells,suggesting that C0818 induced apoptosis in senescent HCC cells is Hsp90 dependent.C0818 significantly increased LC3II accumulation,increased the level of p62 and decreased the levels of Beclin1 and ATG5in senescent cells in a concentration-dependent manner.Autophagy inducer rapamycin reduced the expression of P62,while,C0818 increased the expression of P62 consistent with the autophagy inhibitors chloroquine and bafilomycine.All tested drugs increased LC3II accumulation.Besides,the accumulation of P62 was downregulated when cells were treated with C0818 combined with rapamycin compared with C0818 alone,suggesting that C0818 inhibits autophagy at the late stage in senescent HCC cells.C0818 triggered the upregulation of p62;whereas 17-AAG decreased the expression level of p62.Transfection of senescent cells with Hsp90-target si RNA decreased Hsp90expression.After knocking down of Hsp90,C0818 still effectively prompted p62upregulation and LC3II accumulation,indicating that inhibition of autophagy by C0818is independent of Hsp90.Analysis of a typical marker of SASP using ELISA and quantitative real-time PCR showed that C0818 significantly inhibited the expressions IL-6.C0818 significantly down regulated the expression levels NF-kB and its phosphorylated form in senescent cells.In addition,C0818 not only inhibits NF-kB activation,but also triggers the downregulation of the expression levels of MAPK associated proteins P38 and Erk.Besides,C0818 induced the down regulation of mTOR and its phosphorylated form in a dose dependent manner in senescent cells,suggesting that C0818 modulates SASP in senescent HCC cells by inhibiting NF-κB,P38,ERK and mTOR signaling pathways.ConclusionC0818 induces apoptosis and G2/M phase arrest by inhibition of PI3K/AKT and RAF/MEK/ERK signaling pathways in HCC cells.C0818 promotes the degradation of Hsp90 client proteins including C-Raf,P-C-Raf,Erk,P-ERK,MEK,P-MEK,AKT,P-AKT and RAS in a proteasome dependent path way.Additionally,C0818 potentiates both the mitochondrial and death receptor pathways of apoptosis and promotes Bax up-regulation,Bcl2 down-regulation,activation of caspase-8 and cleavage of caspase-3,-7,-9 and PARP.C0818 triggered mitochondrial dysfunction including,the loss of MMP,excessive ROS production and disruption of the energy supply are important in inhibition the growth and proliferation of HCC cells after C0818 treatment.This study was the first to show that C0818 is a novel senolytic agent against adriamycin-induced senescent HCC cells through inhibition of Hsp90 and blocking of autophagy.In addition,C0818 can be a suppressor of the SASP via the inhibition of NF-κB,P38,ERK and mTOR signaling pathways.Our data indicate that C0818 is a promising Hsp90-targeted therapy in the treatment of HCC and the elimination of senescent HCC cells and inhibition of HCC progression.