2005-Characterization of Apoptosis induced by fucoxanthin=FX

Characterization of Apoptosis Induced by Fucoxanthin

in Human Promyelocytic Leukemia Cells

Eiichi K OTAKE -N ARA ,Masaru T ERASAKI ,and Akihiko N AGAO y

National Food Research Institute,2-1-12Kannondai,Tsukuba,Ibaraki 305-8642,Japan

Received August 10,2004;Accepted October 21,2004

Apoptosis induced by fucoxanthin in HL-60cells was

associated with a loss of mitochondrial membrane

potential at an early stage,but not with an increase in

reactive oxygen species.Fucoxanthin treatment caused

cleavages of procaspase-3and poly (ADP-ribose)poly-

merase without any e?ect on the protein level of Bcl-2,

Bcl-X L ,or Bax.Apoptosis induction by fucoxanthin may

be mediated via mitochondrial membrane permeabili-

zation and caspase-3activation.

Key words:apoptosis;caspase;fucoxanthin;HL-60;

mitochondria

Epidemiological studies suggest that fruit and vege-

table consumption reduces the risk of certain cancers

and other degenerative diseases.Phytochemicals such as

carotenoids,?avonoids,and isothiocyanates present in

such foods have been thought to have bene?cial e?ects

on human health.Carotenoids,which are pigments with

conjugated polyene structure,are considered to work as

antioxidants with radical scavenging and singlet oxygen

quenching activities in biological tissues.Moreover,the

cancer-preventive potential of carotenoids has been

indicated in studies with cultured cells as well as

experimental animals.Carotenoids have been shown to

suppress the propagation of cancer cells in vitro by

inducing di?erentiation and apoptosis,enhancing gap-

junctional communication,and arresting the cell cycle.

Since Muto et al.1)discovered that -carotene induces

apoptosis in cervical dysplastic cells via down-regula-

tion of an epidermal growth factor receptor,several

carotenoids such as lycopene, -cryptoxanthin,lutein,

and canthaxanthin have been reported to induce apop-

tosis in certain cancer cells.Hosokawa et al.2)found that

HL-60human promyelocytic leukemia cells underwent

apoptosis by fucoxanthin,which has characteristic

structures of 5,6-monoepoxide and an allenic bond.In

our previous studies,3,4)?fteen dietary carotenoids were

evaluated for their e?ects on propagation of PC-3

human prostate cancer cells.In addition to fucoxanthin,

neoxanthin,which has a structure similar to fucoxanthin,

remarkably reduced cell viability by inducing apoptosis.These two carotenoids have also been shown to suppress chemically induced carcinogenesis in experimental animals.Fucoxanthin and neoxanthin are the major carotenoids present in chloroplasts of brown algae and higher plants respectively.Our recent studies indicate that these carotenoids orally administered to mice were absorbed and underwent metabolic conversion.5)Thus fucoxanthin and neoxanthin are noteworthy dietary carotenoids with respect to cancer prevention,but the detailed mechanisms underlying apoptosis induction by these carotenoids remain unknown.In the present study,we characterized the apoptosis induced by fucoxanthin in HL-60cells.HL-60,HP50-2,and HP100-1human promyelocytic leukemia cells were obtained from the Riken Gene Bank (Tsukuba,Japan).HP50-2and HP100-1cells are hydro-gen peroxide-resistant cells derived from HL-60cells and have 3.9and 17.6-fold higher catalase activity than HL-60cells respectively.6)These cell lines were cultured in an RPMI1640medium supplemented with 10%heat-inactivated fetal bovine serum,4m M L -glutamine,and antibiotics (40U/ml penicillin and 40m g/ml streptomycin)at 37 C in a humidi?ed atmo-sphere of 5%CO 2in air.Treatment with fucoxanthin was conducted by incubating the cells at a density of 2?105cells/ml medium containing 10m M fucoxanthin for the indicated period.Fucoxanthin,prepared as previously reported,3)was dissolved in distilled tetrahy-drofuran (THF)and added to the culture medium.The ?nal concentration of THF was 0.25%(v/v),and the control culture received only THF (vehicle alone).The percentage of apoptotic cells among those treated with 10m M fucoxanthin for 24h was estimated by ?uores-cence-activated cell sorting (FACS)and DNA ladder formation by agarose gel electrophoresis,as described previously.7)The intracellular levels of reactive oxygen species (ROS)were evaluated with diacetoxymethyl 6-carboxy-20,70-dichlorodihydro?uorescein diacetate (DCF)as a ?uorescence probe.Brie?y,HL-60cells were preincu-y To whom correspondence should be addressed.Tel:+81-29-838-8039;Fax:+81-29-838-7996;E-mail:nagao@nfri.a?rc.go.jp

Abbreviations :DCF,diacetoxymethyl 6-carboxy-20,70-dichlorodihydro?uorescein diacetate;FACS,?uorescence-activated cell sorting;MMP,mitochondrial membrane permeabilization;PARP,poly (ADP-ribose)polymerase;PBS,phosphate-bu?ered saline;ROS,reactive oxygen species;THF,tetrahydrofuran Biosci.Biotechnol.Biochem.,69(1),224–227,2005

Note

bated with the medium containing10m M DCF at37 C for30min.Thereafter,the culture was diluted two-fold with the medium containing20m M fucoxanthin,and the cells were further incubated at37 C for1h.The treated cells were washed with phosphate-bu?ered saline(PBS) and resuspended in0.25ml lysis solution(0.5%SDS in PBS).The?uorescence intensity(Ex.510nm,Em. 534nm)of the oxidized DCF in the lysate was then measured.

The e?ect of fucoxanthin on mitochondrial membrane potential was evaluated with rhodamine123as a probe. Cells treated with10m M fucoxanthin for1h were incubated with10m M rhodamine123in PBS at37 C for15min.Thereafter,the cells were suspended in 0.25ml of0.1%bovine serum albumin solution.They were subjected to FACS.Data were analyzed with Cellquest software(BD Immunocytometry Systems,San Jose,CA,U.S.A.).

The e?ect of fucoxanthin on caspase activities was evaluated with substrates speci?c to the inpidual caspases.HL-60cells treated with10m M fucoxanthin for24h were lysed in50m l of lysis bu?er(50m M HEPES–KOH,pH7.4,100m M NaCl,0.1%CHAPS, 10m M DTT,100m M EDTA,10%glycerol,1m M PMSF, and10mg/ml each of antipain,chymostatin,leupeptin, and pepstatin A(Peptide Institute,Osaka,Japan). Aliquots(50m g protein)of the cell lysates were incubated in the dark at37 C for1h with10m M caspase substrates as follows:Ac-DEVD-MCA(MCA: 4-methyl-1-coumaryl-7-amide)for caspase-3,Ac-IETD-MCA for caspase-8,and Ac-LEHD-MCA for caspase-9. Afterwards,?uorescence intensity(Ex.360nm,Em. 465nm)was measured.

The e?ect of fucoxanthin on the levels of apoptosis-related proteins was evaluated by western blot analysis. The proteins of the cells treated with fucoxanthin for 24h as described above were extracted with a lysis bu?er(0.5M Tris–HCl,pH6.8,10%SDS,10%glycer-ol,and0.6% -mercaptoethanol).Total proteins(50m g) of the lysate were subjected to western blot analysis as described previously.4)The primary antibodies were as follows:mouse anti-Bcl-2monoclonal antibody (1:1000;B3170,Sigma Aldrich,St.Louis,MO, U.S.A.),rabbit anti-Bcl-X S/L polyclonal antibody (1:200;Santa Cruz Biotechnology,Santa Cruz,CA, U.S.A.),rabbit anti-Bax polyclonal antibody(1:200;N-20,Santa Cruz Biotechnology),rabbit anti-caspase-3 polyclonal antibody(1:1000,H-227,Santa Cruz Bio-technology),rabbit anti-cleaved caspase-3polyclonal antibody(1:1000;#9661,Cell Signaling Technology, Beverly,MA,U.S.A.),rabbit anti-poly(ADP-ribose) polymerase(PARP)polyclonal antibody(1:1000; #9542,Cell Signaling Technology),and mouse anti- -actin monoclonal antibody(1:50000;AC-15,Sigma Aldrich).The proteins were probed with alkaline phosphatase-conjugated secondary antibodies.

The prooxidant action of -carotene and enhancement of intracellular ROS associated with an activation of NF- B has been reported to mediate apoptosis in several cancer cells,including HL-60cells.8)ROS in the bio-logical tissues play pivotal roles in mediating some cellular signals.In particular,enhancement of ROS has been associated with apoptosis induced by certain stimuli.With regard to HL-60cells,the food components curcumin and quercetin as well as the cytotoxic drugs duocarmycin A and cisplatin have been reported to induce apoptosis via enhanced generation of ROS at the early stage.In order to elucidate the possible involve-ment of ROS in apoptosis induction by fucoxanthin, HP50-2and HP100-1cells,which are hydrogen per-oxide-resistant cell lines derived from HL-60cells,were exposed to10m M fucoxanthin for24h.Fucoxanthin treatment induced apoptosis in the resistant cells as well as in HL-60cells,although the percentage of apoptotic cells evaluated as SubG1phase was lower in the resistant cell lines than in the parental cells(Fig.1A).DNA ladder formation was also con?rmed in the three cell lines treated with fucoxanthin(Fig.1B).However,the apop-tosis induced by50m M hydrogen peroxide was abolished in HP50-2and HP100-1cells(Fig.1A).We examined whether exposure of HL-60cells to fucoxanthin could enhance the intracellular level of ROS.The DCF-treated HL-60cells were exposed to fucoxanthin for1h.The relative?uorescence intensity of the oxidized DCF in the cells treated with vehicle alone,50m M hydrogen per-oxide,and10m M fucoxanthin was100?7:1,386:4?26:4,and94:7?5:1%(n?3)respectively.Thus fucox-anthin treatment did not a?ect the ROS level at all.These results suggest that ROS is not involved in a major pathway of apoptosis induced by fucoxanthin.

The mitochondria play an important role in regulating the apoptotic process.Mitochondrial membrane perme-abilization(MMP)followed by the loss of membrane potential occurs at the early stage of apoptosis via the mitochondria9)and leads to the release of apoptogenic proteins such as cytochrome c,AIF,and EndoG to cytosol.Oxidative stress is known to cause MMP-associated apoptosis via an enhancement of ROS.In fact,treatment with50m M hydrogen peroxide reduced the mitochondrial membrane potential in HL-60cells after1h incubation,but had no e?ect in the H2O2-resistant cell lines in the present study(data not shown). On the other hand,treatment with10m M fucoxanthin for 2h reduced the mitochondrial membrane potential in both HL-60cells and the resistant HP100-1cells(Fig.2). Therefore,these results suggest that fucoxanthin causes the loss of mitochondrial membrane potential at an early stage in a manner di?erent from oxidative stress,which thereafter leads to apoptotic pathways via the mitochon-dria.

Bcl-2family proteins regulate MMP and the release of cytochrome c from mitochondria to cytosol.The released cytochrome c leads to apoptotic cell death via activation of caspase-9and-3.In the present study, treatment of HL-60cells with10m M fucoxanthin for 24h did not a?ect the levels of the anti-apoptotic

Apoptosis Induced by Fucoxanthin225

proteins,Bcl-2and Bcl-X L ,or the pro-apoptotic protein

Bax (Fig.3).Thus the Bcl-2family proteins tested were

not involved in MMP and apoptosis induced by

fucoxanthin.The fucoxanthin treatment induced clea-

vages of procaspase-3and PARP.The activities of

caspase-3,-8,and -9were respectively higher by

8:52?0:36,2:74?0:26,and 5:11?0:65times (n ?

3)in HL-60cells treated with 10m M fucoxanthin for 24h

than in the cells treated with the vehicle alone.The

remarkable enhancement of caspase-3and the cleavage

of procaspase-3to an active fragment (Fig.3)indicate

that the apoptosis induced by fucoxanthin was caspase-3

dependent.Furthermore,the marked enhancement of

caspase-9activity suggests the release of cytochrome c

from the mitochondria to cytosol and the subsequent

activation of the caspase cascade.Taken together,the results in the present study suggest that apoptosis induced by fucoxanthin in HL-60cells is mediated via the reduction of mitochondrial membrane potential,followed by caspase-9and -3activation.The reduction of mitochondrial membrane potential was not associated with the Bcl-2family protein levels and ROS gener-ation.Fucoxanthin,which was taken up readily by the cells (data not shown),may directly cause dysfunction of mitochondira by highly accumulating in mitcondrial membranes,leading to the reduction of its membrane potential.The action of fucoxanthin on mitochondria and the detailed mechanism of subsequent apoptosis induction deserve future

study.Fig.2.E?ect of Fucoxanthin on Mitochondrial Membrane Potential.

HL-60(A)and HP100-1(B)cells were incubated with 10m M

rhodamine 123for 15min at 37 C after 2h exposure to THF alone

(gray line)and 10m M fucoxanthin (solid line).The ?uorescence

intensity of rhodamine 123in the cells was analyzed by

FACS.

Fig.3.Western Blot Analysis of Apoptosis-Related Proteins Ex-pressed in HL-60Cells Treated with Fucoxanthin.The HL-60cells were incubated with 10m M fucoxanthin for the indicated times.Levels of -actin are shown as equal loading

controls.Fig.1.Apoptosis Induction by Fucoxanthin in HL-60,HP50-2,and HP100-1Cells.

(A)HL-60,HP50-2,and HP100-1cells were incubated in the presence of THF alone (open column),50m M H 2O 2(oblique line column),and 10m M fucoxanthin (solid column)for 24h.The cells in SubG1phases were counted as apoptotic cells by FACS and expressed as a percentage of the total cell number.The values represent means ?SD (n ?3).Values not sharing a common letter in each cell line are signi?cantly di?erent (P <0:05)by Tukey’s test.(B)Agarose gel electrophoresis of DNA extracted from HL-60cells (lanes 1and 5),HP50-2cells (lanes 2and 6),and HP100-1cells (lanes 3and 7)treated with THF alone (lanes 1–3),and 10m M fucoxanthin (lanes 5–7)for 27ceeb08168884868662d64cne 4and 8,DNA marker ( -Hind III).

226 E.K OTAKE -N ARA et al.

Acknowledgments

This study was supported in part by the Food Research Project of the Ministry of Agriculture,Forest-ries,and Fisheries of Japan.

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