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Qiqiharof ChineseReceived 1 November 2013hepatic stellate cells (HSCs) phenotype by both proliferation and inhibition of apoptosis. Thus, the induc-ing hepatic fibrogenesis (Li et al., 2013). Increasing evidence sug-gests that hepatic fibrosis is reversible (Rockey, 2013), and theelimination of activated HSCs through cell death has been pro-posed as a therapeutic anti-fibrogenic strategy (Lim et al., 2013).One of the intracellular signaling elements that have recentlyattracted attention as a potential modulator of cell death inet al., 2000).in preventingSeveral indepen-to suggestphosphatidylinositol 3 kinase (PI3K) pathway has been implicatedin the enhancement of NF-jB-dependent transcription (Avni2012; Rasul et al., 2012). The importance of PI3K signaling inhas been established as blocking PI3K activity, using either phar-macological or genetic approaches, inhibits HSCs proliferation,although unanswered questions remain (Son et al., 2009).Curcumol (for its structure, see Fig. 1a) is one of the major com-ponents of Curcuma phaeocaulis Val. with the structure of a guai-ane-type sesquiterpenoid hemiketal. It exhibits characteristicssuch as anti-inflammatory, anti-hepatic fibrosis (Jiang et al.,Corresponding author. Tel./fax: +86 452 2663373.E-mail address: (Y. Niu).1Gang Chen and Yinghang Wang contributed equally to this work.European Journal of Pharmaceutical Sciences 65 (2014) 2128Contents lists availableEuropean Journal of Pharmelagents, there is no efficacious treatment for live fibrosis due to apoor understanding of the complex pathological process (Liuet al., 2011). The activation of hepatic stellate cells (HSCs) inresponse to liver injury is considered a key cellular event underly-it is not required for the process of activation (LangIn contrast, active NF-jB plays an important roleapoptosis of activated HSCs (Kong et al., 2013).dent lines of investigation have now converged/10.1016/j.ejps.2014.09.0010928-0987/C211 2014 Elsevier B.V. All rights reserved.thatet al.,HSCs1. IntroductionHepatic fibrosis is caused by a wound-healing response tochronic liver injuries, such as persistent viral infections (Aksuet al., 2012), overuse of alcohol (for review, see reference Purohitand Brenner (2006), and drug intoxication (for review, see refer-ence Begriche et al. (2011). Despite efforts to develop antifibroticactivated HSCs is the nuclear factor-jB (NF-jB) Byun et al., 2013.NF-jB ordinarily is retained in the cytoplasm in an inactive formthrough association with one of the nuclear factor-jB inhibitorprotein (IjB) inhibitory proteins. Degradation of IjBa enhancenuclear NF-jB DNA binding activity and transcriptional activationof its target genes, including Bcl-2 family members (Moss et al.,2012). NF-jB activity is increased in cultured activated HSCs butReceived in revised form 15 July 2014Accepted 1 September 2014Available online 9 September 2014Chemical compounds studied in this article:Curcumol (PubChem CID: 160771)SP600125 (PubChem CID: 8515)PD 98059 (PubChem CID: 4713)SB-203580 (PubChem CID: 176155)Keywords:CurcumolApoptosisHepatic fibrosisNuclear factor-jBBcl-2tion of activated HSCs apoptosis has been proposed as an antifibrotic treatment strategy. Curcumol haspro-apoptotic activity in a number of cancer cell types. The aim of this study is to test the hypothesis thatthe interruption of the phosphatidylinositol 3 kinase (PI3K)/nuclear factor-jB (NF-jB) signaling pathwayby curcumol might induce apoptosis of activated HSCs. Our results indicated that curcumol-inducedgrowth inhibition correlated with apoptosis induction as evidenced by Annexin V staining, and cleavageof caspase-3 and poly (ADP-ribose) polymerase (PARP) in HSC-T6. Importantly, we show that theapoptotic effect of curcumol was specific to the activated HSCs (HSC-T6). Suppression of the NF-jBtranslocation via inhibition of IjB-a phosphorylation by the curcumol led to the inhibition of expressionof NF-jB-regulated gene, e.g. Bcl-xL and Bcl-2, in a PI3K-dependent manner, which is upstream of NF-jBactivation. Also, curcumol-mediated apoptosis of HSC-T6 were reversed by LY294002 and Bay 11-7082.Taken together, our findings perfectly support the hypothesis and demonstrate that the inhibition ofPI3K/NF-jB pathway by curcumol lead to HSC-T6 apoptosis. Thus, our study indicates that curcumol isa potential candidate for further preclinical study aimed at the treatment of liver fibrosis.C211 2014 Elsevier B.V. All rights reserved.article infoArticle history:abstractThe major feature in the molecular pathogenesis of hepatic fibrosis requires maintenance of the activatedCurcumol induces HSC-T6 cell death throughInvolvement of PI3K and NF-jB pathwaysGang Chena,1, Yinghang Wangb,1, Meiqian Lic, TianjiaoaThe Institute of Medicine, Qiqihar Medical University, 333 BuKui Street, JianHua District,bRheumatoid Immunology Clinic, The First Affiliated Hospital to Changchun, UniversitycSchool of Nursing, Qiqihar Medical University, Qiqihar 161006, Chinajournal homepage: www.suppression of Bcl-2:Xua, Xiaoli Wanga, Bo Honga, Yingcai Niua,161006, ChinaMedicine, Changchun 130117, Chinaat ScienceDirectaceutical S/locate/ejps22 G. Chen et al./European Journal of Pharmaceutical2005), and importantly, as an anti-proliferative compound induc-ing a loss in viability in a number of tumor cell lines (Zhanget al., 2011). However,the mechanisms bywhich curcumolamelio-rates hepatic fibrosis are not well characterized. Studies from sev-eral laboratorieshave shownthat the eliminationof activated HSCsby apoptosis might be an important mechanism of terminating andpotentially reversing liver fibrosis (Tashiro et al., 2013). Given theanti-proliferative and anti-hepatic fibrosis properties of curcumol,we hypothesized that curcumol could exhibit therapeutic potentialin the context of liver fibrosis by inducing apoptosis of activatedmeasured at 490 nm using a microplate reader (Safire2, TecanFig. 1. Different susceptibility to curcumol-mediated death in activated HSCs (HSC-T6) and hepatocytes (BRL-3A). (a) Chemical structure of curcumol. (b) Cells, afterserum-starvation for 24 h, were exposed to curcumol with indicated concentrationsfor 48 h (b and c) or at 300lM for the indicated times (d), and then assayed for cellviability using the MTS assay. Data represent means S.D. of three separateexperiments.*p 0.05 vs. cell with no treatment.Group Ltd., Maennedorf, Switzerland), and results were expressedas percentage of Triton X-100-induced LDH release.2.4. Cellular caspase-3 activity assayCellular caspase-3 activity was measured with a commerciallyavailable Colorimetric Assay Kit (Beyotime Biotechnology, Haimen,China) according to the manufacturers protocol. In brief, aftertreatment, the cells were harvested in cell lysis buffer (25 mMHEPES, pH 7.4, 5 mM CHAPS, and 5 mM DTT), and incubated withAc-DEVD-pNA, a colorimetric substrate for caspase-3, for 1 h atHSCs. Indeed, Jiang et al. give the first evidence that this hypothesisholds true (Jiang et al., 2005).Our preliminary studies revealed that curcumol selectivelyinduces cell death in culture-activated primary rat HSCs, but notin quiescent HSCs (our unpublished observations). This led us toquestion whether curcumol is capable of inducing activated HSCsapoptosis and, if so, whether the NF-jB signaling pathway wasinvolved in curcumol-mediated apoptosis of activated HSCsin vitro. To answer the question, we determine the apoptosis-inducing potential of curcumol and to characterize involved signaltransduction pathways in HSC-T6.2. Material and methods2.1. Cell culture and treatmentHSC-T6 cells, a rat immortalized HSC line, was provided byCancer Institute and Hospital, Chinese Academy of MedicalSciences (Beijing, China), and the BRL-3A (rat liver cell line) werepurchased from Shanghai Institute of Cell Biology, Chinese Acad-emy of Sciences (Shanghai, China). BRL-3A were maintained inPRMI-1640 medium supplemented with 10% fetal calf serum(FCS) and antibiotics, and HSCs were grown in Dulbeccos modifiedEagles medium (DMEM) supplemented with 10% fetal calf serumand antibiotics. Before the experiments the medium was changedto serum-free, which rendered cells more sensitive to treatment.Cells were subsequently treated with and without curcumol(National Institute for Food and Drug Control, Beijing, China) atthe indicated concentrations for the indicated times. In someexperiments, cells were pretreated with LY294002, a PI3Ks specificinhibitor, or/and Bay 11-7082, an irreversible inhibitor of IjBaphosphorylation, for 1 h prior to the addition of curcumol.2.2. Assessment of cell viabilityA MTS/PMS reagent based CellTiter 96C210Aqueous Non-Radioac-tive Cell Proliferation Assay kit (Promega, Madison, WI, USA) wasused to quantify proliferation of HSC-T6 and BRL-3A followingthe manufacturers instructions. Reagents were added directly inthe incubation media and incubated at 37C176C for 2 h. Absorbancewas recorded at 490 nm with a microplate reader (Safire2, TecanGroup Ltd., Maennedorf, Switzerland). The data are expressed asmean percent viable cells compared with respective controlcultures.2.3. Lactate dehydrogenase (LDH) leakage assaysLDH released into the culture supernatants was measured bycytotoxicity detection kit (Genmed, Westbury, NY) following themanufacturers instructions. The absorbance of the samples wasSciences 65 (2014) 212837C176C. pNA absorbance was quantified using a microplate reader(Safire2, Tecan Group Ltd., Maennedorf, Switzerland) at a 405-nmwavelength. Enzyme activity were calculated based on the300lM for 48 h was used in subsequent experiments. Importantly,LY294002 or Bay 11-7082 alone did not cause LDH release fromHSC-T6.3.2. Curcumol induces apoptosis of HSC-T6Given that no effect of curcumol on LDH activity in HSC-T6could be observed, next we examined whether the curcumol-induced apoptosis of HSC-T6 is attributable to HSC-T6 cell death.Cell apoptosis was quantified by staining cell with Annexin-V-FITC/PI. As shown in Fig. 2a and b, compared with control, curcu-mol caused significant increases in apoptosis of HSC-T6. BlockingPI3K or/and NF-jB by LY294002 or/and Bay 11-7082 significantlyreduced the apoptosis-inducing effect of curcumol on HSC-T6 butdid not abolish it completely, suggesting that apoptosis-stimulat-ing effect is at least in part regulated by PI3K and NF-jB pathways.standard curve run under the same conditions, and represented asthe units/mg of protein.2.5. Fluorescence activated cell sorting (FACS) analysisAfter treatment, cells were stained with FITC-AnnexinV andpropidium iodide (PI) provided in the Annexin V-FITC ApoptosisDetection Kit I (Becton Dickinson, San Jose, CA) according to man-ufacturers protocol. The population of Annexin V-positive cellswas evaluated by FACS Calibur flow cytometer (BD Biosciences,San Jose, CA) and FlowJo software (Tree Star, San Carlos, CA).2.6. The activity of NF-jBActivation of NF-jB was estimated using enzyme-linked immu-nosorbent assay (ELISA)-based TransAM NF-jB p65 Assay Kit(Active Motif, Carlsbad, CA, USA) according to the manufacturersprotocol. Nuclear extracts from cells were prepared using Nuclearand Cytoplasmic Protein Extraction kit (Beyotime Biotechnology,Haimen, China). Oligonucleotides containing the NF-jB consensusbinding site (50-GGGACTTCC-30) were immobilized on a 96-wellplate. The active forms of NF-jB in the nuclear extracts werebound to the oligonucleotides on the plate and detectedcolorimetrically by a microplate reader (Safire2, Tecan GroupLtd., Maennedorf, Switzerland) at 450-nm wavelength.2.7. Western blotting analysisWhole cell lysate, cytosolic extract, and nuclear extract wereseparated by 10% SDSpolyacrylamide gel electrophoresis andelectrotransferred to nitrocellulose membrane as previouslydescribed. The membrane was blocked overnight at 4 C176C in PBS-0.1% Tween 205% skim milk powder and incubated for 1 h atroom temperature with antibodies against cleaved caspase-3,Bcl-2, Bcl-xL, Bax, NF-jB p65, p-Akt, and p-ERK1/2, which wereobtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), aswell as antibodies to cleaved poly (ADP-ribose) polymerase (PARP),p-IjB, p- c-Jun N-terminal kinases (JNK), and p-p38 mitogen-activated protein kinase (MAPK), which were obtained from CellSignaling Technology (Beverly, MA, USA). After incubation withappropriate secondary antibody (Medical Biological LaboratoryCo., Nagoya, Japan), the bands were visualized by an enhancedchemiluminescence (ECL) system (Amersham Biosciences, Piscata-way, NJ, USA) and Kodak XBT-1 film. Blots were reprobed withantibodies against glyceraldehyde 3-phosphate dehydrogenase(GAPDH), Lamin B (Santa Cruz, CA, USA), and corresponding totalprotein to demonstrate equal loading. Densitometry analysis wasperformed by using AlphaEaseFCSoftware (Witec, Littau,Switzerland). The variation in the density was expressed as foldchanges compared to the control in the blot.2.8. Real-time polymerase chain reaction (PCR)Total RNA was isolated from HSC-T6 using the TRIzol method(Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performedusing SuperScript II (Invitrogen Corp., Carlsbad, CA). Gene expres-sion levels were measured by real-time PCR on an ABI7300machine (Applied Biosystems, CA) with iTaq SYBRC210Green Super-mix with ROX (Bio-Rad Laboratories, Hercules, CA, USA), and theprimers used in this study are listed in Table 1. Threshold cycle(Ct) data were collected using the Sequence Detection Softwareversion 1.2.3 (Applied Biosystems, CA). The relative expression lev-G. Chen et al./European Journal of Pharmaceuticalels of the genes of interest were calculated using theDDCt method,normalizing for the expression of housekeeping gene GAPDH andrelated to the control treatment.the inhibitive effect of curcumol was specific to HSC-T6. In con-trast, rat hepatocytes BRL-3A showed only minor death in responseto 1000lM curcumol (Fig. 1d), suggesting that curcumol may be aselective inducer of HSCs death. However, a slight but not signifi-cant alteration of LDH activity due to curcumol could also beobserved in both HSC-T6 and BRL-3A (Fig. 1e). Given that inhibit-ing Akt or NF-jB does not affect cell survival, LDH release wasdetermined in the extracellular medium to examine if LY294002or Bay 11-7082 causes necrosis of HSC-T6. Results revealed that2.9. Statistical analysisAll data represent the mean of at least three independentexperiments standard deviation (S.D.), if not otherwise stated.Statistical analysis was performed by one-way analysis of variance(ANOVA)followed byStudentNewmanKeuls(SNK) tests formul-tiple comparisons between treatment groups using SPSS 13.0 soft-ware (SPSS Inc., Chicago, IL). P values of 0.05 were considered tobe statistically significant.3. Results3.1. Curcumol induces cell death in activated HSCs (HSC-T6) but not inhepatocytes (BRL-3A)To determine potential antifibrogenic effects of curcumol, weinvestigated whether curcumol mediated cell death in activatedHSCs. The MTS assay for cell viability indicated that curcumolinduces death of activated HSCs reached a plateau at a concentra-tion of 300 lM for 48 h in HSCs. Higher concentrations and longerduration of curcumol yielded no further decrease in the cell viabil-ity. Given this result, the treatment of HSC-T6 with curcumol atTable 1Primers used for real-time PCR detection of gene expression.Group Accession no.*Direction SequenceBcl-xL U72350.1 Forward CAGCTTCATATAACCCCAGGGACReverse GCTCTAGGTGGTCATTCAGGTAGGBcl-2 NM_016993 Forward TGAACCGGCATCTGCACACReverse CGTCTTCAGAGACAGCCAGGAGBax NM_017059 Forward AGACACCTGAGCTGACCTTGGAGReverse GTTGAAGTTGCCATCAGCAAACAGAPDH NM_017008 Forward GACAACTTTGGCATCGTGGAReverse ATGCAGGGATGATGTTCTGG*Note: Genbank accession number of cDNA and corresponding gene, available at/.Sciences 65 (2014) 2128 23Furthermore, the LY294002 alone had no effect on apoptosis ofHSC-T6. Complementary data from cellular caspase-3 activityassay confirmed apoptosis obtained by Annexin-V-FITC/PI stain24 G. Chen et al./European Journal of Pharmaceuticalassay presented above (Fig. 2c). Western blot and densitometryanalysis for cleaved caspase-3 levels and PARP cleavage, anothermarker of caspase-dependent apoptosis, also corroborated thesefindings (Fig. 2d and e).3.3. Curcumol suppress anti-apoptotic Bcl-2 and Bcl-xL expression inHSC-T6, but not BaxGiven that the members of the Bcl-2 family play a critical role inHSCs survival, we, therefore, further hypothesized that regulationof the Bcl-2 family members by curcumol might be mainly causeof HSC-T6 apoptosis. As shown in Fig. 3ac, curcumol significantlyreduced, as expected, expression of Bcl-2 and Bcl-xL at both levelsof protein and mRNA in HSC-T6, while no significant differencewas observed for Bax. Inhibiting PI3K or/and NF-jB by LY294002or/and Bay 11-7082 only partially canceled the downregulationof Bcl-2 and Bcl-xL by curcumol in HSC-T6.Fig. 2. Curcumol induces apoptosis of HSC-T6. HSC-T6, after serum-starvation for 24 h, wasLY294002 and/or NFjB inhibitor BAY 11-7082. (a) Cells were stained for FITC-Annexin Vin the histograms (b). (c) The activity of caspase-3 proteases was measured using Ac-DEVD-pNAcleaved PARP were analyzed by Western blot with antibodies against cleaved caspase-3antibodies against GAPDH to verify protein levels, are reported in the histograms. (e)densitometry and normalized to GAPDH. Data represent means S.D. of three separateSciences 65 (2014) 21283.4. Curcumol represses NF-jB activation in HSC-T6Since Bcl-2 family member are main targets of NF-jB, which iscentral to regulating apoptosis and cell survival in HSCs, next wetested if curcumol suppress NF-jB activation in HSC-T6. We assessthe effects of curcumol on nuclear translocation of NF-jB. As dem-onstrated in Fig. 4ac, curcumol significantly decreased nuclearNF-jB p65 protein expression, whereas increased cytoplasmicNF-jB p65 protein in HSC-T6. Phospho-IjBa was dec

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