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Induction of apoptosis by streptochlorin isolated from Streptomyces sp. in human leukemic U937 cells

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Induction of apoptosis by streptochlorin isolated from Streptomyces sp. in human leukemic U937 cells
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  Induction of apoptosis by streptochlorin isolated from  Streptomyces  sp.in human leukemic U937 cells Cheol Park a , Hee Jae Shin b , Gi-Young Kim c , Taeg Kyu Kwon d , Taek-Jeong Nam e , Se-Kwon Kim f  , Jaehun Cheong g , Il-Whan Choi h, * , Yung Hyun Choi a, * a Department of Biochemistry, Dongeui University College of Oriental Medicine, Department of Biomaterial Control (BK21 Program), Dongeui University Graduate School,Yangjung-dong San 45, Busanjin-gu, Busan 614-052, South Korea b Marine Natural Product Chemistry Laboratory, Ocean Research and Development Institute, Ansan 425-600, South Korea c Faculty of Applied Marine Science, Cheju National University, Jeju 690-756, South Korea d Department of Immunology, School of Medicine, Keimyung University, Taegu 700-712, South Korea e Department of Food and Life Science, Pukyong National University, Busan 608-737, South Korea f  Department of Chemistry and Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, South Korea g Department of Molecular Biology, Pusan National University, Busan 609-735, South Korea h Department of Microbiology, College of Medicine and Center for Viral Disease Research, Inje University, Busan 614-735, South Korea a r t i c l e i n f o  Article history: Received 24 March 2008Accepted 23 June 2008Available online 1 July 2008 Keywords: StreptochlorinU937ApoptosisBcl-2Caspase-3. a b s t r a c t Streptochlorin is a small molecule that produced by marine  Streptomyces  sp. that is known to have anti-angiogenicandanti-cancerproperties. However, themechanismbywhichstreptochlorinfunctions isnotwell understood. In this study, we investigated the pro-apoptotic effect of streptochlorin in human leu-kemic U937 cells. Streptochlorin treatment resulted in concentration- and time-dependent growth inhi-bitionbyinducingapoptosis.Theincreaseinapoptosisthatwasinducedbystreptochlorinwascorrelatedwith down-regulation of anti-apoptotic Bcl-2 expression, up-regulation of pro-apoptotic Bax and FasL, adecrease in the mitochondrial membrane potential (MMP), activation of caspases and degradation of poly-(ADP-ribose)polymerase and phospholipase C- c 1 protein. In addition, the cytotoxic effects andapoptotic characteristics induced by streptochlorin were significantly inhibited by z-DEVD-fmk, a cas-pase-3 inhibitor, which demonstrates the important role that caspase-3 played in the process. Further-more, Bcl-2 overexpression significantly reversed the streptochlorin-induced growth inhibitory effectsvia inhibition of the MMP collapse and caspases activation and effectively attenuated the apoptoticresponse to streptochlorin. However, the elevated levels of FasL expression induced by streptochlorinwerenotreducedbyBcl-2overexpression.Takentogether,thesefindingsdemonstratethatthepro-apop-totic effect of streptochlorin is mediated through activation of caspases and mitochondria in U937 cells.   2008 Elsevier Ltd. All rights reserved. 1. Introduction Apoptosis is the active process of programmed cell death thatoccurs during many important physiological conditions, such asembryonic development and tissue remodeling. However, mostcancer cells block apoptosis, which allows them to survive despiteundergoing genetic and morphologic transformations. Apoptoticcells are characterized by several unique features, including cellshrinkage, chromatin condensation, DNA fragmentation, cell sur-face expression of phosphatidylserine, and membrane blebbing(Okada and Mak, 2004; Jin and el-Deiry, 2005; Han et al., 2008).A central component of the apoptotic machinery is a family of cy-stein-containing aspartate-specific proteases, termed caspase.Caspases are present in cells as inactive proenzymes, with the ac-tivetetramerbeingformedasaresultofremovaloftheprodomainand cleavage between the large and small subunits (Hengartner,2000; Suda et al., 1993). Caspase activity is either directly or indi-rectly responsible for the cleavage of several intracellular proteinsthatarecharacteristicallyproteolysedduringapoptosis.Ithasbeenreported that two major pathways, the Fas/tumor necrosis factor(TNF) death receptor (extrinsic) pathway and the mitochondria-dependent (intrinsic) pathway, lead to activation of caspases and 0887-2333/$ - see front matter    2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.tiv.2008.06.010  Abbreviations:  DAPI, 4,6-diamidino-2-phenylindile; DEVD, Asp-Glu-Val-Asp;DMSO, dimethyl sulfoxide; ECL, enhanced chemiluminescence; EtBr, ethidiumbromide; FasL, Fas ligand; FBS, fetal bovine serum; IETD, Ile-Glu-Thr-Asp; JC-1, 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethyl-imidacarbocyanine iodide; LEHD, Leu-Glu-His-Asp; MMP,mitochondrialmembranepotential;PARP,poly-(ADP-ribose)polymerase;PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PI, propidiumiodide; PLC, phospholipase C; pNA,  p -nitroaniline; TNF, tumor necrosis factor. *  Correspondingauthors.Tel.:+82518507413;fax:+82518534036(Y.H.Choi),tel.: +82 51 890 6461; fax: +82 51 891 6004 (I.W. Choi). E-mail addresses:  cihima@inje.ac.kr (I.-W. Choi), choiyh@deu.ac.kr (Y.H. Choi). Toxicology in Vitro 22 (2008) 1573–1581 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit  consequent apoptosis in mammalian cells (Fulda and Debatin,2006; Wajant et al., 2005). The Fas/TNF death receptor pathwayis initiated by binding of a ligand to the Fas/TNF death receptoron the cell surface, which then activates caspase-8 and apoptoticcell death. However, changes in the mitochondrial integrity in re-sponse to a broad range of physical and chemical stimuli can trig-ger the intrinsic pathway of apoptosis (Chowdhury et al., 2006;Fulda and Debatin, 2006). It is believed that the release of cyto-chrome  c   from the mitochondria into the cytosol is a key eventin the intrinsic pathway (Mow et al., 2001; Mohamad et al.,2005). Once in the cytosol, cytochrome  c   can activate caspase-9,which in turn cleaves and activates the executioner, caspase-3.After activation, several specific substrates for caspase-3 includingpoly-(ADP-ribose)polymerase (PARP) and phospholipase C (PLC)- c 1 are cleaved, which eventually leads to apoptosis (Lazebnik etal., 1994; Bae et al., 2000). Consistent with the role that the mito-chondria plays in the control of cell death, survival or apoptoticfactors such as the Bcl-2 family act on the organelle to prevent orfacilitate the release of apoptogenic factors (Yin, 2000; Zinkel etal., 2006; Jeong and Seol, 2008). Many recent studies have shownthat many chemopreventive and/or chemotherapeutic agents cancause tumor cell death via the induction of apoptosis, which isthepreferredmethodof managingcancer. Therefore,theinductionof apoptotic cell death is an important mechanismin the anti-can-cer properties of many drugs.Recent studies have shown that marine microorganisms are anovel and rich source of bioactive compounds due to their poten-tial pharmacological activities. However, it is believed that mole-cules produced by those microorganisms have therapeuticproperties that have not yet been discovered (Schweder et al.,2005; Singh and Pelaez, 2008). In addition, studies evaluating thecomponents of marine microorganisms have shown that manyare not general cytotoxic agents, but are instead targeted towardsspecificcellular or biochemical events andmay therefore beusefulas anti-microbial, anti-cancer or anti-inflammatory agents (Pro-ksch et al., 2002; Singh and Pelaez, 2008). We previously screenedmarine natural products for the ability to induce apoptosis in can-cer cells and found that streptochlorin isolated from  Streptomyces sp. exhibited selective cytotoxicity against several cancer cell lines(Choi et al., 2007; Shin et al., 2007). However, the mechanism bywhichstreptochlorinexertsitsfunctionisnotyetfullyunderstood.Therefore, we used the human leukemic U937 cell line as a modelsystemtoinvestigatetheeffectsof streptochlorin-inducedapopto-sis. We found that streptochlorin-induced apoptosis is accompa-nied by modulation of the Fas/Fas ligand (FasL) system, Bcl-2family members, mitochondrial dysfunction and activation of caspases. In addition, blocking of caspase-3 activation and Bcl-2overexpression reduced streptochlorin-induced apoptosis inU937 cells. 2. Materials and methods  2.1. Cell culture and viability assay U937 cells were obtained from the American type culture col-lection (Rockville, MD), and cultured in RPMI 1640 medium sup-plemented with 10% heat-inactivated fetal bovine serum (FBS),2mM glutamine, 100U/mL penicillin, and 100 l g/mL streptomy-cin (Gibco BRL, Gaithersburg, MD) at 37   and 5% CO 2 . The Bcl-2overexpressing U937 (U937/Bcl-2) cells were a generous gift fromDr. T.K. Kwon (Department of Immunology, Keimyung UniversitySchool of Medicine, Taegu, Korea) and maintained in a mediumcontaining 0.7 l g/mL geneticin (G418 sulfate). Streptochlorin iso-lated from  Streptomyces  sp. (strain 04DH110) was prepared as de-scribed previously (Shin et al., 2007) and dissolved in dimethylsulfoxide (DMSO) as a stock solution at 1mg/mL concentration,andstoredinaliquots at  20  C. Cell numberandits viabilityweredetermined by tryphan blue exclusion assay and MTT assay,respectively.  Table 1 Gene-specific primers for RT-PCR  Name Sequence of primersTRAIL Sence 5 0 -ATG GCT ATG ATG GAG TCC AG-3 0 Antisence 5 0 -TTG TCC TGC ATC TGC TTC AGC-3 0 DR4 Sence 5 0 -CAG AAC GTC CTG GAG CCT GTA AC-3 0 Antisence 5 0 -ATG TCC ATT GCC TGA TTC TTT GTG-3 0 DR5 Sence 5 0 -GGG AAG AAG ATT CTC CTG AGA TGT G-3 0 Antisence 5 0 -ACA TTG TCC TCA GCC CCA GGT CG-3 0 Fas Sence 5 0 -TCT AAC TTG GGG TGG CTT TGT CTT C-3 0 Antisence 5 0 -GTG TCA TAC GCT TTC TTT CCA T-3 0 FasL Sence 5 0 -GGA TTG GGC CTG GGG ATG TTT CA-3 0 Antisence 5 0 -AGC CCA GTT TCA TTG ATC ACA AGG-3 0 Bax Sence 5 0 -ATG-GAC-GGG-TCC-GGG-GAG-3 0 Antisence 5 0 -TGG-AAG-AAG-ATG-GGC-TGA-3 0 Bcl-2 Sence 5 0 -CAG-CTG-CAC-CTG-ACG-3 0 Antisence 5 0 -GCT-GGG-TAG-GTG-CAT-3 0 Bcl-xL Sence 5 0 -CAG CTG CAC CTG ACG-3 0 Antisence 5 0 -GCT GGG TAG GTG CAT-3 0 GAPDH Sence 5 0 -CGG-AGT-CAA-CGG-ATT-TGG-TCG-TAT-3 0 Antisence 5 0 -AGC-CTT-CTC-CAT-GGT-GGT-GAA-GAC-3 0 Fig. 1.  Growth inhibition in response to streptochlorin treatment of U937 cells.U937cellswereplatedataconcentrationof1  10 5 cellsper60-mmplateandthenincubated for 24h, after which they were treated with various concentrations of streptochlorin for the indicated times. (A) Cell number and (B) viability weredetermined by hemocytometer counts of tryphan blue-excluding cells and an MTTassay,respectively.Eachtimepointrepresentsthemeans±SDofthreeindependentexperiments. The significance was determined by a Student’s  t  -test (*  p  <0.05 vs.vehicle control).1574  C. Park et al./Toxicology in Vitro 22 (2008) 1573–1581   2.2. Nuclear staining with DAPI  AftertreatingU937cellswithstreptochlorin,thecellswerehar-vested, washed in ice-cold phosphate-buffered saline (PBS) andfixed with 3.7% paraformaldehyde (Sigma Chemical Co., St. Louis,MO) in PBS for 10min at room temperature. The fixed cells werewashed with PBS and stained with a 4,6-diamidino-2-phenylindile(DAPI, Sigma) solutionfor 10min at roomtemperature. The nucle-ar morphology of the cells was examined by fluorescent micros-copy (Carl Zeiss, Germany).  2.3. DNA fragmentation assay The cells were treated with different concentrations of strepto-chlorin and lysed on ice in a buffer containing 10mM tris–HCl(pH 7.4), 150mM NaCl, 5mM EDTA, and 0.5% triton X-100 for30min. The lysates were vortexed and cleared by centrifugationat 10,000  g   for 20min. The fragmented DNA in the supernatantwas extracted using an equal volume of neutral phenol:chloro-form:isoamylalcohol (25:24:1, v/v/v) and analyzed electrophoret-ically on 1.0% agarose gel containing ethidium bromide (EtBr,Sigma).  2.4. Flow cytometry analysis for measurement of sub-G1 phase The cells were harvested and washed once with PBS, fixed inice-cold 70% ethanol and stored at 4  C. Prior to analysis, the cellswerewashedonceagainwithPBS,suspendedin1mLofacoldpro-pidium iodide (PI, Sigma) solution containing 100 l g/mL RNase A,50 l g/mL PI, 0.1% (w/v) sodium citrate, and 0.1% (v/v) NP-40, andfurther incubated on ice for 30min in the dark. Flow cytometricanalyzes were carried out using a flow cytometer (FACS Caliber,Becton Dikinson, San Jose, CA) and CellQuest software was usedto determine the relative DNA content based on the presence of a red fluorescence.  2.5. RNA extraction and reverse transcription-PCR Thetotal RNAwaspreparedusinganRNeasykit(Qiagen, LaJol-la, CA) and primed with random hexamers to synthesize the com-plementary DNA using AMV reverse transcriptase (AmershamCorp., Arlington Heights, IL) according to the manufacturer’sinstructions. A polymerase chain reaction (PCR) was carried outusing a Mastercycler (Eppendorf, Hamburg, Germany) with theprimers shown in Table 1. The following conditions were used Fig. 2.  Induction of apoptosis by streptochlorin treatment of U937 cells. After being treated withstreptochlorin for five days, the cells were sampled andeither examined byinverted microscopy (A, magnification,   200) or fixed and stained with DAPI so the nuclear morphology could be photographed under fluorescence using a blue filter (B,magnification,  400). (C) Toanalyze the DNAfragmentation, the genomic DNAwas extracted, electrophoresed ina 1.0%agarose gel and thenvisualized by EtBr staining. (D)To quantify the degree of apoptosis induced by streptochlorin, the cells were evaluated for sub-G1 DNA content, which represents the fractions undergoing apoptotic DNAdegradation, using a flow cytometer. Each point represents the mean of representative experiments performed at least three times. The significance was determined by aStudent’s  t  -test (*  p <0.05 vs. vehicle control). C. Park et al./Toxicology in Vitro 22 (2008) 1573–1581  1575  for the PCR reactions: 1  (94  C for 3min); 35  (94  C for 45s;58  C for 45s; and 72  C for 1min) and 1  (72  C for 10min).The amplification products obtained by PCR were separated elec-trophoretically on a 1.0% agarose gel and visualized by EtBrstaining.  2.6. Protein extraction and Western blotting  The cells were harvested and lysed. The protein concentrationswere measured using a Bio Rad protein assay (BioRad Lab., Hercu-les, CA) according to the manufacturer’s instructions. For Westernblot analysis, an equal amount of protein was subjected to elec-trophoresis on SDS–polyacrylamide gel and transferred by elec-troblotting to a nitrocellulose membrane (Schleicher andSchuell, Keene, NH). The blots were probed with the desired anti-bodies for 1h, incubated with the diluted enzyme-linked second-ary antibody and visualized by enhanced chemiluminescence(ECL) according to the recommended procedure (AmershamCorp., Arlington Heights, IL). The primary antibodies were pur-chased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) andCell Signaling Technology, Inc. (Boston, MA). The peroxidase-la-beled donkey anti-rabbit immunoglobulin and peroxidase-labeledsheep anti-mouse immunoglobulin were purchased from Amer-sham Corp.  2.7. Assay of caspase-3, -8 and -9 activity The enzymatic activity of the caspases induced by streptochlo-rin was assayed using a colorimetric assay kit according to themanufacturer’s protocol (R&D Systems, Minneapolis, MN). Briefly,the cells were lysed in a lysis buffer for 30min on an ice bath.The lysed cells were centrifuged at 12,000  g   for 10min, and100 l goftheproteinwasincubatedwith50 l Lofareactionbufferand 5 l L of the colorimetric tetrapeptides, Asp-Glu-Val-Asp(DEVD)-  p -nitroaniline (pNA) for caspase-3, Ile-Glu-Thr-Asp(IETD)-pNA for caspase-8 and Leu-Glu-His-Asp (LEHD)-pNA forcaspase-9, respectively, at 37  C for 2h. The optical density of thereactionmixturewasquantifiedspectrophotometricallyatawave-length of 405nm.  2.8. Mitochondrial membrane potential (MMP,  D W m ) assay The mitochondrial membrane potential (MMP,  D W m ) of intactcells was measured by flow cytometry with the lipophilic cationicprobe 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethyl-imidacarbocyanineiodide (JC-1, Sigma). JC-1 is a ratiometric, dual-emission fluores-cent dye that is internalized and concentrated by respiring mito-chondria and can reflect changes in MMP in living cells. There aretwo excitation wavelengths, 527nm (green) for the monomerform and 590nm (red) for the J-aggregate form. With normalmitochondrial function, MMP is high and the red fluorescence ispredominant. However, when there is mitochondrial injury,MMP is reduced, leading to an increase in green fluorescence(Salvioli et al., 1997). Quantitation of red and green fluorescentsignals reflects whether mitochondria are damaged. For thisstudy, the cells were trypsinized and the cell pellets were resus-pended in 500 l L of PBS, incubated with 10 l M JC-1 for 20min at37  C. The cells were subsequently washed once with cold PBS,suspended in a total volume of 500 l L and analyzed using flowcytometry.  2.9. Statistical analysis The data is expressed as a mean±SD. A statistical comparisonwas performed using one-way ANOVA followed by a Fisher test.The significant differences between the groups were determinedusing an unpaired Student’s  t  -test. A  p  value <0.05 was consideredsignificant. Fig. 3.  EffectsofstreptochlorinonthelevelsofthedeathreceptorandBcl-2familymembersinU937cells.(A)Afterincubationwithstreptochlorinforfivedays,thetotalRNAwasisolatedandreverse-transcribed.TheresultingcDNAwassubjectedtoPCRusingtheindicatedprimers.Thereactionproductsweresubjectedtoelectrophoresisina1.0%agarosegelandthenvisualizedbyEtBrstaining.GAPDHwasusedastheinternalcontrol.(B)ThecellswerelysedandthecellularproteinswerethenseparatedinSDS–PAGE,afterwhichtheyweretransferredontonitrocellulosemembranes. ThemembraneswerethenprobedwiththeindicatedantibodiesandtheproteinswerevisualizedusinganECL detection system. Actin was used as the internal control.1576  C. Park et al./Toxicology in Vitro 22 (2008) 1573–1581  3. Results  3.1. Growth inhibition by streptochlorin in U937 cells In order to determine if streptochlorin decreases cell growth,U937cellswerestimulatedwithvariousconcentrationsofstrepto-chlorin and the cell number and viability were then measured bytryphan blue exclusion method and MTT assay, respectively. Asshown in Fig. 1, treatment with streptochlorin significantly inhib-ited the viability and proliferation of cells, and these effects oc-curred in a time- and concentration-dependent manner. Inaddition, direct observation by inverted microscopy revealed thatnumerous morphological changes occurred in cells treated withstreptochlorin (Fig. 2A).  3.2. Induction of apoptosis by streptochlorin in U937 cells Additional experiments were conducted to determine if theinhibitory effect of streptochlorin on cell growth was the resultof apoptotic cell death. DAPI staining revealed that nuclei withchromatincondensationandapoptoticbodieswereformedincellsthat were cultured with streptochlorin, and that this phenomenonoccurred in a concentration-dependent manner; however, thesefeatures were present in only a few control cells (Fig. 2B). In addi-tion, agarose gel electrophoresis showed that streptochlorin treat-ment induced the progressive accumulation of fragmented DNA,which appeared as a typical ladder pattern of DNA fragmentationdue to internucleosomal cleavage associated with apoptosis, andthat this accumulation occurred in a concentration-dependentmanner (Fig. 2C). The degree of apoptosis was determined by ana-lyzing the amount of sub-G1 DNA in U937 cells that were treatedwith streptochlorin using flow cytometry. As shown in Fig. 2D,the treatment of U937 cells with streptochlorin resulted in the in-creased accumulationof cells in the sub-G1phase. Taken together,these results suggest that cells can undergo apoptosis after beingexposed to streptochlorin. In addition, these results revealed agood correlation between the extent of apoptosis and the inhibi-tion of growth.  3.3. Effects of streptochlorin on the levels of death receptor-related genes and Bcl-2 family members in U937 cells Toinvestigatetheapoptoticpathwaysactivatedbystreptochlo-rin, we used RT-PCR and Western blotting to measure the Fig. 4.  Loss of MMP and activation of caspases by streptochlorin treatment of U937 cells. (A) After being treated with streptochlorin for five days, equal amounts of the celllysates(30 l g)were resolvedbySDS–PAGE, transferredtonitrocellulosemembranesandthenprobedwiththeindicatedantibodies. Theproteins werethenvisualizedusingan ECL detection system. Actin was used as the internal control. (B) Cells grown under the same conditions as (A) were stained with JC-1 and then incubated at 37  C for20min, after whichthemeanJC-1fluorescenceintensitywasdetectedusing aflowcytometer. Datarepresent themean±theSDof representativeexperiments performedatleast three times. The significance was determined by a Student’s  t  -test (*  p  <0.05 vs. untreated control). (C) The cell lysates obtained from cells grown under the sameconditions as (A) were assayed for the  in vitro  caspase-3, -8 and -9 activity using DEVD-pNA, IETD-pNA and LEHD-pNA, respectively, as substrates. The concentrations of thefluorescent products released were then measured. The results are expressed as the mean±the SD of three independent experiments. The significance was determined by aStudent’s  t  -test (*  p <0.05 vs. untreated control). C. Park et al./Toxicology in Vitro 22 (2008) 1573–1581  1577
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