Sodium selenite alters microtubule assembly and induces apoptosis in vitro and in vivo.

2.3 MB·English

by  Shi, Kejian

Shietal.JournalofHematology&Oncology2013,6:7
http://www.jhoonline.org/content/6/1/7 JOURNAL OF HEMATOLOGY
& ONCOLOGY
RESEARCH Open Access
Sodium selenite alters microtubule assembly and
induces apoptosis in vitro and in vivo
Kejian Shi, Qian Jiang, Zhushi Li, Lei Shan, Feng Li, JiaJia An, Yang Yang and Caimin Xu*
Abstract
Background: Previous studies demonstrated that seleniteinduced cancer-cell apoptosis through multiple
mechanisms; however, effects ofseleniteon microtubules inleukemic cells have not been demonstrated.
Methods: The toxic effect of selenite on leukemic HL60 cells was performed with cell counting kit 8. Selenite
effects oncell cycle distribution and apoptosis inductionwere determined byflow cytometry. The contents of
cyclin B1, Mcl-1, AIF, cytochromeC, insoluble and soluble tubulins were detected withwesternblotting.
Microtubules were visualized with indirect immunofluorescence microscopy. The interaction betweenCDK1 and
Mcl-1 was assessed with immunoprecipitation. Decreasing Mcl-1 and cyclin B1 expression were carried out through
siRNA interference. The alterations of Mcl-1 and cyclin B1in animal model were detected with either
immunohistochemical staining or western blotting. In situ detectionof apoptotic ratio was performed with TUNEL
assay.
Results: Our current results showed thatselenite inhibited thegrowthof HL60 cells and induced
mitochondrial-relatedapoptosis.Furthermore, wefound that microtubule assembly in HL60 cells was altered, those
cellswere arrested at G2/M phase, and Cyclin B1 was up-regulatedand interactedwith CDK1, which led to
down-regulation of the anti-apoptotic protein Mcl-1. Finally, in vivo experiments confirmed the in vitro microtubule
disruption effect and alterations inCyclin B1 and Mcl-1 levels byselenite.
Conclusions: Taken together, the results from our study indicate that microtubules are novel targets of selenite in
leukemic HL60 cells.
Keywords: Sodium selenite, Apoptosis, Microtubule, Cell cycle
Introduction range inhibit tumor formation by acting on antioxidants
Microtubuleshaveimportantrolesin manycellbehaviors and in the inhibition of DNA adduct formation, the pro-
suchascelldivision,organellepositioning,vesiculartrans- motion of cell cycle progression and DNA repair [14-16].
port and cell-shape determination [1-3]. Previous studies However, super-nutritional levels of selenite induce endo-
haveshowed thatmicrotubule dynamics are necessaryfor plasmic reticulum stress, mitochondrial-related apoptosis,
thesefunctionsinvivo[2,4-6].Therefore,chemicalsaffect- DNA strand breaks and cell-cycle arrest [15-19]. There-
ing microtubule dynamics often impact these functions fore, many molecules, such as Akt, GADD153, P53, ERK,
in vivo. On that basis, many anti-tumor agents have been P38, Bad, Bim and Bax [20-24], have been reported to be
developed for their effects on microtubule dynamics and involved in high-dose selenite-induced apoptosis. Add-
cell-cycledistribution[7-12]. itionally, super-nutritional selenite intake has been shown
Selenium(Se)isanessentialtraceelement[13],andap- to be toxic to drug-resistant cancer cells and effective on
propriate selenium intake is necessary for the body to tumor xenografts, which suggests that selenite has poten-
synthesize selenoproteins. Some researchers have shown tialtherapeuticeffects[23-25].
thatseleniteconcentrationsthatarewithinthenutritional In an in-depth study of selenium, selenite was reported
to have strong inhibitory effects on sulfhydryl-containing
*Correspondence:[email protected] proteins [15] such as tubulins, which composed microtu-
StateKeyLaboratoryofMedicalMolecularBiology,Departmentof
bules [26,27], but the effects of selenite on microtubules
BiochemistryandMolecularBiology,InstituteofBasicMedicalSciences,
CAMS&PUMC,Beijing100005,China
©2013Shietal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Shietal.JournalofHematology&Oncology2013,6:7 Page2of9
http://www.jhoonline.org/content/6/1/7
incancercellshadnotbeenproven.Basedonourproteo- participated in selenite-induced apoptosis, we extracted
micsstudy,proteinslinkedtomicrotubuledynamicswere the mitochondrial fraction of HL60 cells after they were
thought to have roles in selenite-triggered apoptosis [28]. exposed to selenite and discovered that cytochrome C
Therefore,ourstudyaimedtoinvestigatetheroleofselen- and AIF were released (Figure 1C), which suggested
iteinmicrotubuleassemblyandinductionofapoptosis. that a mitochondrial-related apoptotic pathway was uti-
lized. Furthermore, we observed nuclear fragmentation
Results after staining with DAPI (Figure 1D).
Sodiumseleniteinhibitsgrowthandinducesapoptosisin
HL60cells Sodiumselenitealtersmicrotubuleassemblyincultured
We first observed that selenite inhibited growth of HL60 leukemiacells
cells.Aftercellswereexposedtodifferingconcentrations We then determined the mechanisms by which selenite
ofsodiumselenite,cellviabilitywasassessedusingCCK-8 inhibited the growth of HL60 cells. Tubulin exists in
kits. Figure 1A showed that 20 μM selenite significantly mainlytwoforms:solubleheterodimersthatarecomposed
reduced cell viability after 24 h. To further investigate if of β- and α-tubulin or insoluble polymers. To detect
growth inhibition was caused by apoptosis, the cells whether selenite induced microtubule depolymerization,
were stained with Annexin V-FITC/PI. Flow cytometry we extracted insoluble and soluble tubulin and discovered
analysis proved that typical apoptosis was elicited by that 20 μM selenite destroyed microtubules. Furthermore,
20 μM selenite (Figure 1B). To identify which pathway we observed a significant decrease of insoluble tubulin,
Figure1Sodiumselenite-induced,cell-proliferationinhibitionandapoptosisinculturedHL60cells.(A)Sodiumseleniteinhibited
proliferationofHL60leukemiacells.Cellsweretreatedwithvaryingconcentrationsofselenitefor24h,andcellviabilitywasdetectedusing
CCK-8kits.Meanvaluesofthreerepetitionsweredisplayed,and*P<0.05,whencomparedwithuntreatedcells.(B)Sodiumseleniteinduced
apoptosisinHL60leukemiacells.Cellswerecollectedafterexposuretovaryingconcentrationsofselenitefor24handlabeledwithAnnexin-V-
FITC/PI,andtheapoptoticratiowasdetectedusingflowcytometry.Theresultswererepresentativeofthreerepetitions,and*P<0.05,when
comparedwithuntreatedcells.(C)SeleniteinducedthereleaseofcytochromeCandAIFfromthemitochondriatothecytoplasm.Aftercells
weretreatedwith20μMofselenitefortheindicatedtimes,fractionsofmitochondriaandcytoplasmwereseparated,andthecytochromeCand
AIFcontentsofeachfractionweredetectedbywesternblotting.Thisexperimentwasrepeatedatleast3times.(D)Sodiumselenite(20μM)
inducednucleusfragmentationofHL60cellsat24h,whichwasdetectedbyimmunofluorescenceafternuclearstainingwithDAPI.Scalebar:
10μm.Thisexperimentwasrepeatedatleast3times
Shietal.JournalofHematology&Oncology2013,6:7 Page3of9
http://www.jhoonline.org/content/6/1/7
whileJurkatcellsreassembledmicrotubulesafter2hofsel- of Cyclin B1 was abrogated (Figure 3C). As mentioned
enite exposure (Figure 2A). Indirect immunofluorescence above,microtubulereorganizationoccurredinJurkatcells;
showed that microtubules presented an integrated, fila- therefore,wedestroyedmicrotubulereassemblythrougha
mentousstructureinuntreatedcellsbutweredispersivein combinationofseleniteandColchicinetreatments(micro-
responsetoseleniteinHL60cells(Figure2B). tubule depolymerization agent) and found that the altera-
tions in Mcl-1 and Cyclin B1 levels were similar to those
Sodiumselenite-treatedHL60cellsarearrestedatG2/M inselenite-treatedHL60cells(Figure3D).
phase
Weaimedtodetermineifcrosstalkbetweenmicrotubule CyclinB1interactedwithCDK1andinduceddown-
disruption and the mitochondrial apoptotic pathway oc- regulationofMcl-1
curred. Because cell cycle arrest could be induced by Alloftheabove-describedobservationsdemonstratedthat
microtubule disruption and some cell cycle-related pro- microtubule depolymerization was induced by selenite
tein kinases could regulate proteins in the mitochondria, andtookpartintheinhibitionofHL60cellcycleprogres-
we analyzed alterations in the cell cycle distribution of sion. Because Cyclin B1 is necessary in G2/M phase for
HL60 cells after selenite treatment. Compared with con- the activity of CDK1, which phosphorylates and destabi-
trol, the number of cells was lower at G0/G1 phase and lizes Mcl-1, we confirmed the cellular interactions
higher at G2/M phase after selenite treated for 24 h betweenCDK1,CyclinB1andMcl-1(Figure4A).Further-
(Figure 3A). In addition, the protein level of Cyclin B1, a more, either inhibition of Cyclin B1 expression by siRNA
G2/M phase marker, was remarkably increased. This interference or inhibition of CDK1/Cyclin B1 activity by
was concomitant with a decrease in Mcl-1, which was Roscovitine rescued the down-regulation of Mcl-1
located in the mitochondria, in a time-dependent man- (Figure 4B,C). To further identify the protective role of
ner (Figure 3B). To illuminate the effect of microtubule Mcl-1, which was consistently altered upon cell cycle ar-
disruption on cell cycle arrest, we employed a combined rest,wedown-regulatedMcl-1bysiRNAinterferenceand
treatment of sodium selenite and Taxol (microtubule observedHL60celldeathbycellcountingusingtheCCK-
stabilizer) in HL60 cells and observed that the alteration 8 kits (Figure 4D, E). Additionally, silencing Mcl-1 after
Figure2Selenitealteredmicrotubuleassembly.(A)Sodiumselenite(20μM)destroyedmicrotubuleintegrity.Afterexposureto20μMof
selenite,HL60andJurkatcellswerecollectedatvaryingtimes,andtheinsolubleandsolublefractionsoftubulinwereharvestedanddetectedby
westernblotting.CellsthatweretreatedwithColchicineandTaxolwereusedaspositivecontrols.Thisexperimentwasrepeatedatleast3times.
(B)Microtubuledepolymerizationwasdetectedbyindirectimmunofluorescence.Thecellswereharvestedafterseleniteexposureforthe
indicatedtimes,andmicrotubuleswereindirectlylabeledwithaβ-tubulinprimaryantibodyandFITC-conjugatedsecondaryantibody.Scalebar:
10μm.Thisexperimentwasrepeatedatleast3times
Shietal.JournalofHematology&Oncology2013,6:7 Page4of9
http://www.jhoonline.org/content/6/1/7
Figure3Thedisruptionofmicrotubules,whichwasinducedbyselenite,causedcellcyclearrestinHL60cells.(A)Analterationinthe
cellcycledistributionwasdetectedbyflowcytometryaftercellswereexposedto20μMofseleniteforvaryingtimes.Theexperimentwas
repeatedthreetimes,themeanvaluewasexhibited,and*P<0.05,whencomparedtountreatedcells.(B)Cellstreatedwithsodiumselenite
(20μM)showedincreasedlevelsofCyclinB1anddecreasedlevelsofMcl-1inatime-dependentmanner.Aftercellswereexposedtoselenite
(20μM)forvaryingtimes,theywerecollected,andalterationsinCyclinB1andMcl-1levelsweredetectedbywesternblotting.Thisexperiment
wasrepeatedatleast3times.(C)TaxolreversedthechangeinCyclinB1andMcl-1levels.CellswerepretreatedwithTaxol(20nM)for1hbefore
beingexposedto20μMsodiumselenite.ThecellswereharvestedandalterationsinCyclinB1andMcl-1levelsweredetectedbywestern
blotting.Thisexperimentwasrepeatedatleast3times.(D)Thedisruptionofmicrotubulereorganizationcauseddown-regulationofMcl-1and
up-regulationofCyclinB1inJurkatcells.Colchicines(20nMor50nM)wereaddedtocellsafterseleniteexposureat2h.Afterthecellswere
treatedwithselenite(20μM)for24h,theywereharvested,andalterationsinCyclinB1andMcl-1levelsweredetectedbywesternblotting.This
experimentwasrepeatedatleast3times
selenite exposure increased the apoptotic ratio of HL60 the TUNEL assay showed that there were more apop-
cells(Figure4F). totic cells after selenite treatment (Figure 5G). These
results suggested that selenite had therapeutic effects on
SeleniteexhibitsinhibitoryeffectsonHL60tumor HL60 xenografts. To determine whether microtubules
xenografts were depolymerized in this model, we separated soluble
Finally, we determined the effects of selenite (1.5 mg/kg/ and insoluble tubulin and found that the amount of in-
day) on HL60 tumor xenografts. When compared with soluble tubulin was decreased (Figure 5E). Furthermore,
the control group, the volume and weight of the tumor results in Figure 5F showed that selenite up-regulated
and spleen were reduced in mice exposed to selenite Cyclin B1 and down-regulated Mcl-1 levels, which was
(Figure 5A, C). Spleen enlargement could be caused by consistent with the in vitro findings. Immunohistochem-
the infiltration of leukemic cells and it reflected the ma- icalstainingalsoindicatedthatCyclinB1andMcl-1levels
lignant degree of tumor cells. CD33 is a known marker werealteredsimilarlytothoseinvitro(Figure5H).
of leukemic cells. Therefore, we detected CD33-positive
cells in the spleen tissue and discovered that CD-33 Discussion
positive cells decreased after selenite treatment, which Selenium isan essentialtrace elementfor animals,and it
also suggested that selenite had antitumor activity has been shown that super-nutritional selenite intake
(Figure 5D). In addition, HE staining result indicated has anti-tumor activity [14,18,19,27,29]. Several reports
that selenite caused nucleus pyknosis (Figure 5B). Fur- have also proved the anti-tumor effects of selenite
thermore, in situ detection of apoptotic tumor cells by in vivo [24]. However, the effects of selenite on
Shietal.JournalofHematology&Oncology2013,6:7 Page5of9
http://www.jhoonline.org/content/6/1/7
Figure4CyclinB1interactedwithCDK1andinducedthedown-regulationoftheanti-apoptoticproteinMcl-1.(A)CDK1interactedwith
CyclinB1andMcl-1.Aftercellsweretreatedwithselenitefor24h,CDK1wasimmunoprecipitatedfromthecelllysatesusingaCDK1antibody,
andtheCDK1,CyclinB1andMcl-1intheprecipitatesweredetectedbywesternblotting.Thisexperimentwasrepeatedatleast3times.(B)
InhibitingtheexpressionofCyclinB1reversedthedecreaseofMcl-1.CellsweretransfectedwithsiRNAstargetingCyclinB1andtreatedwith
20μMselenitefor24h.CyclinB1andMcl-1weredetectedbywesternblotting.Thisexperimentwasrepeatedatleast3times.(C)Inhibitionof
CDK1/CyclinB1reversedthedown-regulationofMcl-1.Roscovitine(10μM)wasaddedtocellsfor1hbeforetheywereexposedtosodium
selenite(20μM)for24h.ThechangesofMcl-1detectedbywesternblotting.Thisexperimentwasrepeatedatleast3times.(D)Theexpression
ofMcl-1wasinhibitedbysiRNAinterference.CellsweretransfectedwithsiRNAstargetingMcl-1andtreatedwithselenite(20μM).Mcl-1levels
weredeterminedbywesternblotting.Thisexperimentwasrepeatedatleast3times.(E)Mcl-1exhibitedaprotectiveroleinHL60cells.AfterMcl-
1wasknockeddown,cellviabilitywasassessedusingtheCCK-8kits.Theexperimentwasrepeatedthreetimes,themeanvaluewasdetermined,
and*P<0.05,whencomparedtountreatedcells.(F)InhibitingtheexpressionofMcl-1increasedtheapoptoticratioofthecells.Cellswere
transfectedwithsiRNAstargetingMcl-1andtreatedwithselenite(20μM)for24h.Theapoptoticratiowasdeterminedbyflowcytometryafter
thecellswerestainedwithAnnexin-VandPI.Thisexperimentwasrepeatedatleast3times.
microtubule dynamics in cancer cells have not been to the cytoplasm, which suggested that selenite-induced
demonstrated. Our study indicated that microtubules apoptosis in HL60 cells might be associated with the
were anovel target ofselenite. mitochondrial apoptotic pathway. Cell cycle-related pro-
Leynadier D et al. first discovered that selenite could teins that were consistently altered with microtubule dy-
directly interact with the sulfhydryl groups of β-tubulin namics could regulate Bcl-2 family members, which
and could inhibit microtubule polymerization in vitro were located in the mitochondria. Therefore, we specu-
[26]. To our knowledge, we are the first to discover that lated that selenite inhibited HL60 cell growth through
selenite also induces microtubule depolymerization in itseffectsonmicrotubules.
HL60 cells and in vivo. However, because microtubules Several reports suggested that microtubule-interfering
reorganized in Jurkat but not in HL60 cells, the apop- drugs affected cell cycle distribution by regulating the
totic mechanisms of the two cell lines differed. We activity of CDKs and, therefore, altering protein phos-
mainly investigated the mechanisms by which selenite phorylation at different cell cycle phases [11]. Mcl-1, a
induced apoptosis. Because tumor cells have a strong Bcl-2 family member, is regulated by the Cyclin B1/
ability to replicate themselves and tubulins, which com- CDK1 complex and is linked to the mitochondrial apop-
pose spindles, are essential for this process, we speculate totic pathway by binding and inhibiting pro-apoptotic
that selenite-induced apoptosis is at least partly proteins [12,30,31]. Our current study proved that selen-
dependent on the effects of selenite on microtubules. ite could induce cell cycle arrest and remarkable altera-
Therefore, the growth inhibitory effect of selenite on tions of Cyclin B1 and Mcl-1 levels in HL60 cells
cultured HL60 cells was assessed, and we discovered through its effect on microtubule depolymerization.
that 20 μM of sodium selenite significantly inhibited cell Interestingly, a combination treatment of Colchicine and
growth. Furthermore, Annexin V-FITC/PI double stain- selenite in Jurkat cells up-regulated Cyclin B1 and
ing assay proved that selenite-induced apoptosis oc- down-regulated Mcl-1. The observations in Jurkat cells
curred and nuclear fragmentation was witnessed in also supported the relationship between microtubule de-
selenite-treated cells. Last, we discovered that cyto- struction and alterations in Cyclin B1 and Mcl-1 after
chrome C and AIF were released from the mitochondria selenite exposure. Cyclin B1 is necessary for the activity
Shietal.JournalofHematology&Oncology2013,6:7 Page6of9
http://www.jhoonline.org/content/6/1/7
Figure5SelenitehadinhibitoryeffectsonaHL60xenografttumormodel.4-week-oldmiceweredividedintotwogroupsrandomlyand
eachgroupwasmarkedandputintoitsownbox.Thetwogroupslivedinthesamecontextandwerefedwiththesamefoodandwater.Then
HL60cellsweregivensubcutaneously.After1weekandtumorsweredetectable,PBSandselenite(1.5mg/kg/day)weregiventothecontroland
selenite-treatedgroupeverytwodaysrespectively.Aftereachgroupwasinjectedfor3weeks,nudemiceweresacrificedforanalysis.(A)Tumor
weightdecreasedafterseleniteexposure.*P<0.05.(B)Selenitecausednuclearpyknosisintumorcells.Thisexperimentwasrepeatedatleast3
times.(C)Spleenweightdecreasedafterseleniteexposure.*P<0.05.(D)ImmunohistochemicalstainingindicatedCD33positiveleukemiacellsin
spleenweredecreasedafterselenitetreated.Scalebarrepresented100μm.Thisexperimentwasrepeatedatleast3times.(E)Seleniteinduced
microtubuledepolymerizationintumorcells.Solubleandinsoluble-tubulinfractionswereseparatedasindicatedaboveanddetectedbywestern
blotting.Thisexperimentwasrepeatedatleast3times.(F)SelenitehadsimilareffectsontheregulationofCyclinB1andMcl-1asitdidinvitro.
Thisexperimentwasrepeatedatleast3times.(G)InsitulabelingofapoptotictumorcellsusingtheTUNELassayindicatedtheappearanceof
apoptoticcellsafterselenitetreatment.Scalebarrepresented100μm.Thisexperimentwasrepeatedatleast3times.(H)Immunohistochemical
stainingindicatedthatalterationsinCyclinB1andMcl-1levelsweresimilartothoseinvitro.Scalebarrepresented25μm.Thisexperimentwas
repeatedatleast3times
of CDK1, which phosphorylates and destabilizes Mcl-1 microtubule assembly and inhibited HL60 cell growth
[32-36]. We observed that Cyclin B1 interacted with throughcellcyclearrestanddecreaseinMcl-1levels.
CDK1. Furthermore, either siRNA interference of Cyclin The above-described experiments indicated that selen-
B1 or inhibition of the CDK1/Cyclin B1 complex with ite altered microtubule assembly and induced cell cycle
Roscovitine rescued the decrease of Mcl-1. Further in- arrest in HL60 cells. To identify the therapeutic activity
vestigation confirmed the protective role of Mcl-1 on of selenite in vivo, we established a HL60-cell-bearing
HL60 cells and suggested that the growth inhibitory nude mice model. Experiments in vivo showed that sel-
effects of selenite might be associated with the down- enite inhibited tumor growth and induced nucleus
regulationofMcl-1. Finally, acombinationofsiRNAtar- pyknosis.Furthermore, we also found that selenite depo-
geting Mcl-1 and selenite treatment caused a higher lymerized microtubules in vivo. Additional experiments
apoptotic ratio than selenite treatment alone. These demonstrated that alterations of Cyclin B1 and Mcl-1
results supported our conclusion that selenite altered levels in the nude mice model were similar to those
Shietal.JournalofHematology&Oncology2013,6:7 Page7of9
http://www.jhoonline.org/content/6/1/7
findings in vitro, which suggested that the mechanisms were resuspended in RIPA lysis buffer (20 nM Tris, pH
demonstrated invitrowerealso active atthetissue level. 7.5; 1 mM EDTA; 1 mM EGTA; 150 mM NaCl; 1% Tri-
ton X-100; 2.5 mM sodium pyrophosphate; 1 mM β-
Conclusions glycerolphosphate; 1 mM Na VO ; 1 mM PMSF; and
3 4
In conclusion, the microtubule destruction that was 1 μg/mL leupeptin) and were submitted to ultrasonica-
induced by selenite stimulated the apoptotic pathway by tion on ice. The lysates were centrifuged at 12,000×g
up-regulating Cyclin B1, which interacted with CDK1 for 20 min at 4°C, and equal amounts of proteins were
and destabilized the anti-apoptotic protein Mcl-1. We separated by SDS-PAGE. The proteins on the PAGE gel
also found that sodium selenite had therapeutic func- were then transferred to a nitrocellulose membrane.
tions in a HL60-cell-bearing nude mice model through After being blocked with 5% non-fat milk, the mem-
its microtubule destruction effects. Importantly, this in- branes were washed withTBSTand incubated overnight
vestigation explored the effects of selenite on apoptosis with primary antibody at 4°C. After being washed three
inadistinct way. times withTBST, the membranes were incubated with a
HRP-conjugated secondary antibody for approximately
Materials and methods 1 h at room temperature. Subsequently, the membranes
Chemicalsandantibodies were washed another three times and probed with
Roscovitin, anti-β-Tubulin (2-28-33) and anti-β-Actin supersignalchemiluminescent substrate.
(AC-15) antibodies were obtained from Sigma-Aldrich.
Anti-Cyclin B1 and anti-Mcl-1 antibodies, which were
Immunoprecipitation
used for western blotting, were obtained from Cell Sig-
Cells (1×107) were harvested and washed twice with
naling Technology. For immunohistochemical staining,
ice-cold PBS. The pellets were resuspended in RIPA buf-
an anti-Cyclin B1 antibody was purchased from Excell,
fer and lysed on ice for 30 min. Subsequently, the lysates
an-anti CD33 antibody was purchased from BIOSS and
were centrifuged at 12,000×g for 20 min at 4°C. A suit-
an anti-Mcl-1 antibody was purchased from Santa Cruz.
able amount of cdc2 antibody was added to the protein
The Cdc2 (1/Cdk1/Cdc2) antibody was purchased from
lysate (200 μg) and rotated overnight at 4°C, while the
BD Biosciences Pharmingen. HRP-conjugated anti-mouse
remaining protein was used as input. Protein A+G was
and anti-rabbit antibodies were purchased from ZSGB-
added,andthemixturewasrotatedforanother3hat4°C;
BIO. A FITC-conjugated anti-mouse antibody was pur-
then, the samples were washed with RIPA buffer three
chasedfromJackson.
times. Finally, the beads were resuspended in 3×SDS
loading buffer and boiled for 10 min. After a short
Cellculture
centrifugation step, the supernatant was collected.
HL60 and Jurkat cells were grown in RPMI 1640
medium containing 10% advanced fetal bovine serum,
100 units/mL penicillin and 100 units/mL streptomycin
siRNAinterference
and incubated in a humidified, 5% CO incubator that 0
2 siRNAs targeting Cyclin B1 (5-CCAAACCTTTGTAGT
wassetat37°C. 0 0 0
GAAT-3), Mcl-1 (5-GGACTGGCTAGTTAAACAA-3)
and negative controls for each sequence were synthe-
Indirectimmunofluorescencemicroscopy sized by GenePharma. Approximately 1×107 cells were
HL60 cells (8×105 total) were harvested. The cells were
collected and washed with Opti-MEM medium (Gibco).
transferred to slides, fixed in 4% paraformaldehyde and
Then, the cells were transfected with 200 nM siRNA
permeabilized using 0.1% Triton X-100. After the slides
and RNAiMAX in Opti-MEM. After transfection for ap-
were blocked with 2% BSA, the cells were incubated
proximately 12 h, the cells were treated with sodium sel-
with β-tubulin antibody overnight at 4°C. After washing
enite for24h.
with PBS three times, the cells were incubated with
FITC-conjugated secondaryantibody for 60minat room
temperature. After a second round of washing, the cells Detectionofcellcycledistribution
were stained with DAPI for approximately 5 min, and Approximately 1×106 cells were collected and fixed in
the slides were washed three times and mounted in anti- 70% ethanol overnight at 4°C. Each sample was centri-
fading medium. Images were visualized using a Zeiss fuged at 1,000×g for 10 min at room temperature and
microscope(Carl Zeiss,Jena,Germany). washed with ice-cold PBS. Subsequently, the cells were
incubatedwith50μg/mlRNaseinPBSfor30minat37°C.
Westernblotting After adding PI to the cells at a final concentration of
Approximately 1×106 cells were collected for each 50 μg/mL, we detected the absorption at 620 nm by flow
treatment. After washing with ice-cold PBS, the cells cytometry.
Shietal.JournalofHematology&Oncology2013,6:7 Page8of9
http://www.jhoonline.org/content/6/1/7
DetectionofapoptosiswithAnnexinV-FITC/PIstaining was dissolved in methanol. Subsequently, the slides were
The cells (1×106) were harvested and washed twice immersed in boiled sodium citrate for antigen retrieval,
with ice-cold PBS. Subsequently, the cells were stained and after being washed three times with 0.01 M PBS for
with AnnexinV-FITC in binding buffer in the dark for 5 min, the slides were incubated with either anti-Mcl-1
15 min. After being centrifuged at 1,000×g for 10 min, or anti-Cyclin B1 antibody overnight at 4°C. The slides
the cells were resuspended in binding buffer containing were incubated with secondary antibody at room
PI. Finally, the apoptotic ratio was determined by an temperature for 3 h, treated with DAB, stained with
Accuri C6 flow cytometry. We calculated the apoptotic Mayer’s hematoxylinfor 2min and washed with running
ratio by calculating the sum of Annexin V+/PI- cells’ water. Slides were then dehydrated with increasing con-
ratio andAnnexinV+/PI+cells’ratio. centrations of ethanol and clarified with xylene. Finally,
theslidesweremountedwith medium.
TheeffectofsodiumseleniteontheviabilityofHL60
cells HEstaining
HL60cellswereseededintoa96-wellplateataconcentra-
After dehydration with decreasing concentrations of
tion of 40,000 cells per well. After treatment with varying
ethanol as described above, the slides were stained with
concentrations of sodium selenite for 24 h, cell viability Harriss hematoxylin for 15 min and washed for 3 min.
was assessed using CCK-8 kits (Dojindo Molecular Tech-
Then, the slides were immersed in 1% hydrochloric acid
nologies,Tokyo,Japan).
dissolved in 75% ethanol for 30 s. Before dehydration,
theslideswerestainedwith eosin for 10min. Finally, the
Theinvivomicrotubulepolymerizationassay slides were clarified with xylene and mounted with
An established method was modified and used to separ-
medium.
ate insoluble tubulin from soluble tubulin [30]. Approxi-
mately 2×106 cells were collected and washed twice.
TUNELassay
The cells were then resuspended in hypotonic buffer at
A FragEL™ DNA Fragmentation Detection Kit was pur-
37°C for 5 min. After centrifugation at 14,000×g for
chased from MERCK. The slides were deparaffinized in
10 min at 25°C, the supernatants, which contained sol-
xyleneand dehydrated with decreasing concentrationsof
uble tubulin, were collected, and the pellets containing
ethanol. After washing with 1×TBSfor 2 min, the slides
insoluble tubulin were resuspended in RIPA buffer and
were incubated with 20 μg/ml proteinase K for 20 min
subjected to ultrasonication on ice. The lysates were
at room temperature. The slides were then washed with
centrifuged at 12,000×g for 10 min at 4°C, and the
1×TBS and incubated with 1×TdT buffer for approxi-
supernatantswere collected.
mately 30 min at room temperature. Subsequently, the
slides were incubated with 57 μl of mix buffer and 3 μl
Xenografttumormodel
of TdT enzyme for 60 min at 37°C. After being washed
At the beginning of the experiment, 4-week-old female
three times with 1×TBS, the slides were mounted with
mice were chosen and divided into control or selenite-
medium.
treated group randomly. Each group was marked and
put into its own box. The two groups lived in the same
context and were fed with the same food and water. Statisticalanalysis
The values were represented as mean ± SEM. Two-tailed
Leukemia HL60 cells were inoculated subcutaneously
students’ t-tests were used for two groups comparison
into female nude mice. After tumors were palpable, an
analysis, and P<0.05 was considered to be significant.
intraperitoneal injection of sodium selenite dissolved in
The bar charts were used to reflect the alterations of ex-
PBS was given to each mouse every 2 days (1.5 mg/kg/
perimentaldata[18,24,37,38].
day) for 3 weeks, and the control group was treated with
PBS for the same period time. At the end of the experi-
Competinginterests
ment, the mice were sacrificed, and the tumors and
Theauthorsdeclarethattheyhavenocompetinginterests.
spleens were rapidly removed and weighed. The Declar-
ation of Helsinki and the guide for Laboratory Animal Authors’contributions
CareandUsewere maintained. KSdesignedandfinishedmostoftheproject,anddraftedthemanuscript.
QJandZLhelpedKSdesigntheproject.QJ,LSandFLhelpedKSestablish
theHL60cellsbearingnudemicemodel.Indirectimmunofluorescence
Immunohistochemicalstaining stainingwasperformedbyJA.StatisticalanalysiswasperformedbyYY.CXis
The slides were deparaffinized in xylene and dehydrated thesupervisorofallauthorsandprovidessomehelpsinmanuscriptediting
andprojectdesign.Allauthorsreadandapprovedthefinalmanuscript.
with decreasing concentrations of ethanol. After the
slides were washed with running water for 2 min, en- Authors’information
dogenous peroxidase was blocked with 3% peroxide that Firstauthor:KejianShi.
Shietal.JournalofHematology&Oncology2013,6:7 Page9of9
http://www.jhoonline.org/content/6/1/7
Acknowledgements 21. ZouY,NiuP,YangJ,YuanJ,WuT,ChenX:TheJNKsignalingpathwayis
Firstofall,wewouldliketospeciallythankDr.YunShen,Dr.QunXuand involvedinsodium-selenite-inducedapoptosismediatedbyreactive
MasterYangYangfortheirselflessaids.Thisworkwassupportedbythe oxygeninHepG2cells.CancerBiolTher2008,7:689–696.
NationalNaturalScienceFoundationofChina(No.31170788andNo. 22. RenY,HuangF,LiuY,YangY,JiangQ,XuC:Autophagyinhibition
30970655),theNationalNaturalScienceFoundationforYoungScholarsof throughPI3K/AktincreasesapoptosisbysodiumseleniteinNB4cells.
China(No.31101018),theNaturalScienceFoundationofBeijing(No. BMBRep2009,42:599–604.
5082015),theStateKeyLaboratorySpecialFund(No.2060204)andthe 23. YangY,HuangF,RenY,XingL,WuY,LiZ,PanH,XuC:Theanticancer
MinistryofEducationofChinaDoctorTrainingUnit(No.20091106110025). effectsofsodiumseleniteandselenomethionineonhumancolorectal
carcinomacelllinesinnudemice.OncolRes2009,18:1–8.
Received:19November2012Accepted:9January2013 24. HuangF,NieC,YangY,YueW,RenY,ShangY,WangX,JinH,XuC,Chen
Published:17January2013 Q:Seleniteinducesredox-dependentBaxactivationandapoptosisin
colorectalcancercells.FreeRadicBiolMed2009,46:1186–1196.
25. HuH,JiangC,SchusterT,LiGX,DanielPT,LuJ:Inorganicselenium
References sensitizesprostatecancercellstoTRAIL-inducedapoptosisthrough
1. DowningKH:Structuralbasisfortheinteractionoftubulinwithproteins superoxide/p53/Bax-mediatedactivationofmitochondrialpathway.Mol
anddrugsthataffectmicrotubuledynamics.AnnuRevCellDevBiol2000, CancerTher2006,5:1873–1882.
16:89–111. 26. LeynadierD,PeyrotV,CodaccioniF,BriandC:Selenium:inhibitionof
2. NogalesE,WangHW:Structuralintermediatesinmicrotubuleassembly microtubuleformationandinteractionwithtubulin.ChemBiolInteract
anddisassembly:howandwhy?CurrOpinCellBiol2006,18:179–184. 1991,79:91–102.
3. McIntoshJR,HeringGE:Spindlefiberactionandchromosome 27. MiL,XiaoZ,HoodBL,DakshanamurthyS,WangX,GovindS,ConradsTP,
movement.AnnuRevCellBiol1991,7:403–426. VeenstraTD,ChungFL:Covalentbindingtotubulinbyisothiocyanates.A
4. WalkerRA,O’BrienET,PryerNK,SoboeiroMF,VoterWA,EricksonHP, mechanismofcellgrowtharrestandapoptosis.JBiolChem2008,
283:22136–22146.
SalmonED:Dynamicinstabilityofindividualmicrotubulesanalyzedby
28. DongH,YingT,LiT,CaoT,WangJ,YuanJ,FengE,HanB,HuaF,YangY,
videolightmicroscopy:rateconstantsandtransitionfrequencies.
JCellBiol1988,107:1437–1448. etal:Comparativeproteomicanalysisofapoptosisinducedbysodium
seleniteinhumanacutepromyelocyticleukemiaNB4cells.JCellBiochem
5. CarlierMF:Roleofnucleotidehydrolysisinthedynamicsofactin
filamentsandmicrotubules.IntRevCytol1989,115:139–170. 2006,98:1495–1506.
6. EricksonHP,O’BrienET:MicrotubuledynamicinstabilityandGTP 29. JiangQ,WangY,LiT,ShiK,LiZ,MaY,LiF,LuoH,YangY,XuC:Heat
hydrolysis.AnnuRevBiophysBiomolStruct1992,21:145–166. shockprotein90-mediatedinactivationofnuclearfactor-kappaB
switchesautophagytoapoptosisthroughbecn1transcriptional
7. MeshkiniA,YazdanparastR:Involvementofoxidativestressintaxol-
inhibitioninselenite-inducedNB4cells.MolBiolCell2011,22:1167–1180.
inducedapoptosisinchronicmyelogenousleukemiaK562cells.Exp
ToxicolPathol2012,64:357–365. 30. YangJS,HourMJ,HuangWW,LinKL,KuoSC,ChungJG:MJ-29inhibits
tubulinpolymerization,inducesmitoticarrest,andtriggersapoptosisvia
8. WilsonL,JordanMA:Microtubuledynamics:takingaimatamoving
target.ChemBiol1995,2:569–573. cyclin-dependentkinase1-mediatedBcl-2phosphorylationinhuman
leukemiaU937cells.JPharmacolExpTher2010,334:477–488.
9. HamelE,LinCM:Reexaminationoftheroleofnonhydrolyzable
31. WangYF,JiangCC,KiejdaKA,GillespieS,ZhangXD,HerseyP:Apoptosis
guanosine50-triphosphateanaloguesintubulinpolymerization:reaction
inductioninhumanmelanomacellsbyinhibitionofMEKiscaspase-
conditionsareacriticalfactorforeffectiveinteractionsatthe
independentandmediatedbytheBcl-2familymembersPUMA,Bim,
exchangeablenucleotidesite.Biochemistry1990,29:2720–2729. andMcl-1.ClinCancerRes2007,13:4934–4942.
10. TanakaE,HoT,KirschnerMW:Theroleofmicrotubuledynamicsin
32. ChenYC,LuPH,PanSL,TengCM,KuoSC,LinTP,HoYF,HuangYC,Guh
growthconemotilityandaxonalgrowth.JCellBiol1995,128:139–155.
JH:Quinoloneanalogueinhibitstubulinpolymerizationandinduces
11. PietenpolJA,StewartZA:Cellcyclecheckpointsignaling:cellcyclearrest
apoptosisviaCdk1-involvedsignalingpathways.BiochemPharmacol
versusapoptosis.Toxicology2002,181–182:475–481. 2007,74:10–19.
12. BhallaKN:Microtubule-targetedanticanceragentsandapoptosis.
33. DomaE,ChakrabandhuK,HueberAO:Anovelroleofmicrotubular
Oncogene2003,22:9075–9086.
cytoskeletoninthedynamicsofcaspase-dependentFas/CD95death
13. BehneD,KyriakopoulosA:Mammalianselenium-containingproteins. receptorcomplexesduringapoptosis.FEBSLett2010,584:1033–1040.
AnnuRevNutr2001,21:453–473.
34. ShinJW,SonJY,KangJK,HanSH,ChoCK,SonCG:Trichosantheskirilowii
14. GuanL,HanB,LiJ,LiZ,HuangF,YangY,XuC:Exposureofhuman tuberextractinducesG2/Mphasearrestviainhibitionoftubulin
leukemiaNB4cellstoincreasingconcentrationsofseleniteswitchesthe polymerizationinHepG2cells.JEthnopharmacol2008,115:209–216.
signalingfrompro-survivaltopro-apoptosis.AnnHematol2009, 35. HarleyME,AllanLA,SandersonHS,ClarkePR:PhosphorylationofMcl-1by
88:733–742. CDK1-cyclinB1initiatesitsCdc20-dependentdestructionduringmitotic
15. BrozmanovaJ,ManikovaD,VlckovaV,ChovanecM:Selenium:adouble- arrest.EMBOJ2010,29:2407–2420.
edgedswordfordefenseandoffenceincancer.ArchToxicol2010, 36. MollinedoF,GajateC:Microtubules,microtubule-interferingagentsand
84:919–938. apoptosis.Apoptosis2003,8:413–450.
16. ZengH:SeleniteandselenomethioninepromoteHL-60cellcycle 37. YedjouC,TchounwouP,JenkinsJ,McMurrayR:Basicmechanismsof
progression.JNutr2002,132:674–679. arsenictrioxide(ATO)-inducedapoptosisinhumanleukemia(HL-60)
17. CaoTM,HuaFY,XuCM,HanBS,DongH,ZuoL,WangX,YangY,PanHZ, cells.JHematolOncol2010,3:28.
ZhangZN:Distincteffectsofdifferentconcentrationsofsodiumselenite 38. ZouL,ZhangH,DuC,LiuX,ZhuS,ZhangW,LiZ,GaoC,ZhaoX,MeiM,et
onapoptosis,cellcycle,andgeneexpressionprofileinacute al:CorrelationofSRSF1andPRMT1expressionwithclinicalstatusof
promyeloyticleukemia-derivedNB4cells.AnnHematol2006,85:434–442. pediatricacutelymphoblasticleukemia.JHematolOncol2012,5:42.
18. LiZ,ShiK,GuanL,CaoT,JiangQ,YangY,XuC:ROSleadstoMnSOD
upregulationthroughERK2translocationandp53activationinselenite-
doi:10.1186/1756-8722-6-7
inducedapoptosisofNB4cells.FEBSLett2010,584:2291–2297. Citethisarticleas:Shietal.:Sodiumselenitealtersmicrotubule
19. GuanL,HanB,LiZ,HuaF,HuangF,WeiW,YangY,XuC:Sodiumselenite assemblyandinducesapoptosisinvitroandinvivo.Journalof
inducesapoptosisbyROS-mediatedendoplasmicreticulumstressand Hematology&Oncology20136:7.
mitochondrialdysfunctioninhumanacutepromyelocyticleukemiaNB4
cells.Apoptosis2009,14:218–225.
20. HanB,WeiW,HuaF,CaoT,DongH,YangT,YangY,PanH,XuC:
RequirementforERKactivityinsodiumselenite-inducedapoptosisof
acutepromyelocyticleukemia-derivedNB4cells.JBiochemMolBiol2007,
40:196–204.