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Halocynthiaxanthin and Peridinin Sensitize ColonCancer Cell Lines to Tumor Necrosis Factor–RelatedApoptosis-Inducing Ligand

Tatsushi Yoshida,1 Takashi Maoka,3 Swadesh K. Das,4 Kazuki Kanazawa,4 Mano Horinaka,1

Miki Wakada,1 Yoshiko Satomi,2 Hoyoku Nishino,2 and Toshiyuki Sakai1

Departments of 1Molecular-Targeting Cancer Prevention and 2Biochemistry and Molecular Biology,Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji,Kamigyo-ku; 3Research Institute for Production Development, Kyoto, Japan; and 4Department ofLife Science, Graduate School of Science and Technology, Kobe University, Hyogo, Japan

AbstractCarotenoids are compounds contained in foods and

possess anticarcinogenic activity. Tumor necrosis

factor–related apoptosis-inducing ligand (TRAIL) is a

promising candidate for cancer therapeutics due to its

ability to induce apoptosis selectively in cancer

cells. However, some tumors remain tolerant to

TRAIL-induced apoptosis. Therefore, it is important to

develop agents that overcome this resistance. We show,

for the first time, that certain carotenoids sensitize

cancer cells to TRAIL-induced apoptosis. Combined

treatment with halocynthiaxanthin, a dietary carotenoid

contained in oysters and sea squirts, and TRAIL

drastically induced apoptosis in colon cancer DLD-1

cells, whereas each agent alone only slightly induced

apoptosis. The combination induced nuclear

condensation and poly(ADP-ribose) polymerase

cleavage, which are major features of apoptosis.

Various caspase inhibitors could attenuate the

apoptosis induced by this combination. Furthermore,

the dominant-negative form of a TRAIL receptor could

block the apoptosis, suggesting that halocynthiaxanthin

specifically facilitated the TRAIL signaling pathway.

To examine the molecular mechanism of the synergistic

effect of the combined treatment, we did an RNase

protection assay. Halocynthiaxanthin markedly

up-regulated a TRAIL receptor, death receptor 5 (DR5),

among the death receptor–related genes, suggesting a

possible mechanism for the combined effects. Moreover,

we examined whether other carotenoids also possess

the same effects. Peridinin, but not alloxanthin,

diadinochrome, and pyrrhoxanthin, induced DR5

expression and sensitized DLD-1 cells to TRAIL-induced

apoptosis. These results indicate that the combination

of certain carotenoids and TRAIL is a new strategy

to overcome TRAIL resistance in cancer cells.

(Mol Cancer Res 2007;5(6):615–25)

IntroductionCarotenoids are biologically active compounds contained in

plants and microorganisms, but they cannot be synthesized in

animals. We obtain carotenoids from our diet, and the

carotenoids in our blood and peripheral tissue reflect our

dietary intake (1, 2). Various natural carotenoids have been

proven to have anticarcinogenic activity by epidemiologic

studies and animal studies of colon, liver, skin, and lung cancer

models (2, 3). Colon cancer is considered to be related to

dietary habits, and numerous studies suggest that a high intake

of fruits and vegetables that are rich in carotenoids has been

associated with decreased risk of colon cancer (4, 5).

Halocynthiaxanthin is a kind of carotenoid isolated from sea

squirt Halocynthia roretzi and inhibits the growth of human

malignant tumor cells (6). Moreover, halocynthiaxanthin

possesses an inhibitory effect on EBV activation activity of a

tumor promoter in Raji cells, which is widely used as a primary

screening test for antitumor-promoting activity (7) and shows

the highest suppressive effect on the generation of free radicals

among 19 natural carotenoids (8).

A death ligand, tumor necrosis factor–related apoptosis-

inducing ligand (TRAIL), is one of the most promising new

candidates for cancer therapeutics, due to its ability to induce

apoptosis selectively in cancer cells in vitro and in vivo , with

little or no toxicity toward normal cells (9-11). Death receptor 5

(DR5; also called TRAIL-R2) is a receptor for TRAIL (12-15).

Interaction between TRAIL and DR5 forms a protein complex

with an adapter protein Fas-associated death domain (FADD)

and activates initiator caspases containing caspase-8 and

caspase-10 (16). Initiator caspases cleave and activate effector

caspases, like caspase-3, and the activated effector caspases

disrupt a variety of substrates and lead to apoptosis (16). In some

cell types, as a secondary effect, initiator caspases cleave Bid

and the truncated Bid stimulates mitochondria followed by

caspase-9 activation, effector caspase activation, and apoptosis

(17). Soluble recombinant TRAIL is in a phase I clinical trial for

Received 2/14/06; revised 2/27/07; accepted 3/22/07.Grant support: The Japanese Ministry of Education, Culture, Sports, Scienceand Technology.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Toshiyuki Sakai, Department of Molecular-TargetingCancer Prevention, Graduate School of Medical Science, Kyoto PrefecturalUniversity of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566,Japan. Phone: 81-75-251-5339; Fax: 81-75-241-0792. E-mail: [email protected] D 2007 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-06-0045

Mol Cancer Res 2007;5(6). June 2007 615on June 16, 2020. © 2007 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

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the treatment of solid tumors (18), but some tumor types exhibit

resistance to TRAIL (19). Thus, it is important to overcome this

resistance in tumor cells. Conventional antitumor agents, such as

doxorubicin, cisplatin, and 5-fluorouracil, sensitize malignant

tumor cells to TRAIL-induced apoptosis (20-22), although these

agents lead to DNA damage in cells. Therefore, to develop more

safe TRAIL sensitizers, we have been searching for candidates

among food factors.

FIGURE 1. Halocynthiaxanthin (Halo-cyn) sensitizes DLD-1 cells to TRAIL-induced apoptosis. A. DLD-1 cells weretreated with 40 Amol/L halocynthiaxanthinfor 24 h and subsequently incubated with10 ng/mL TRAIL for 12 h. The DNAcontents of cells were analyzed by flowcytometry. M1 shows the populations ofapoptotic cells with sub-G1 DNA con-tents. Percentages of sub-G1. Columns,mean (n = 3); bars, SD. B. Analysis ofthe synergistic effect of the combinationwith halocynthiaxanthin and TRAIL.

Yoshida et al.

Mol Cancer Res 2007;5(6). June 2007

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In this report, we show for the first time that dietary

carotenoids act as sensitizers for TRAIL-induced apoptosis.

ResultsDietary Carotenoid Halocynthiaxanthin Sensitizes Colo-rectal Cancer DLD-1 Cells to TRAIL-Induced Apoptosis

We investigated the effect of combined treatment with

halocynthiaxanthin and TRAIL on apoptosis by measuring

the sub-G1 population (Fig. 1A). Halocynthiaxanthin or

TRAIL as single agents slightly induced apoptosis in

colorectal cancer DLD-1 cells. However, interestingly, the

combination of halocynthiaxanthin and TRAIL drastically

induced apoptosis. These results suggest that halocynthiax-

anthin functions as a sensitizer for TRAIL-induced apopto-

sis. In this study, we used recombinant human TRAIL

without any tag protein because histidine-tagged TRAIL is

toxic to normal hepatocytes (11). To examine whether the

apoptosis induced by the combined treatment of halocyn-

thiaxanthin and TRAIL is synergistic or additive effect,

combination index (CI) values were analyzed by the method

of Chou and Talalay (Fig. 1B; Tables 1 and 2; ref. 23). CI value

was 0.45 or 0.102, when fraction affected by the dose (Fa) was

0.2 or 0.8, respectively. An additive effect is reflected by

CI = 1, a synergistic effect is reflected by CI < 1, and an

antagonistic effect is reflected by CI > 1 (24). Thus, the CI

values suggest that the combination of halocynthiaxanthin and

TRAIL possesses synergistic effect.

Combination of Halocynthiaxanthin and TRAIL InducesNuclear Condensation and Fragmentation

To confirm that sub-G1, as shown in Fig. 1, represents

apoptosis, we examined the morphologic change in nuclei using

4¶,6-diamidino-2-phenylindole staining. As shown in Fig. 2A,

nuclear condensation was detected in cells treated with

halocynthiaxanthin and TRAIL. Moreover, the fragmentation

of nuclei was detected by treatment with halocynthiaxanthin

and TRAIL, as indicated in Fig. 2A (arrows). These features

represent typical apoptotic cells. Poly(ADP-ribose) polymerase

(PARP) is a substrate of caspase and is cleaved by caspase in

apoptotic cells (25). Therefore, we detected PARP cleavage

in cell lysate treated with halocynthiaxanthin and TRAIL

(Fig. 2B). Of interest, PARP was clearly cleaved only in the

combined treatment of halocynthiaxanthin and TRAIL. This

result indicates that the combined treatment drastically induces

apoptosis, compared with treatment with a single agent, and

is consistent with the result of Fig. 1. In addition, DR5/Fc

chimeric protein, a dominant-negative form of a TRAIL

receptor, completely blocked the cleavage of PARP, suggesting

that the activation of apoptosis occurs through receptors of

TRAIL.

Combination with Halocynthiaxanthin and TRAIL InducesCleaved Caspases

Next, we examined the statuses of various caspases by

Western blotting. The cleaved forms of caspase-8, caspase-10,

caspase-9, and caspase-3 were strikingly detected following

combined treatment with halocynthiaxanthin and TRAIL

(Fig. 3A). Moreover, all procaspases were decreased by the

combined treatment. Bid is cleaved by caspase-8, and

subsequently, the cleaved Bid mediates the apoptotic signal

from the TRAIL receptor to mitochondria (17). Indeed,

combined treatment with halocynthiaxanthin and TRAIL

cleaved Bid (Fig. 3B). A dominant-negative form of the

TRAIL receptor, DR5/Fc chimeric protein, attenuated the

cleaved caspases and Bid (Fig. 3A and B).

Caspase Inhibitors and DR5/Fc Chimeric Protein BlockApoptosis Induced by the Combination of Halocynthiax-anthin and TRAIL

We investigated whether caspase inhibitors could block the

apoptosis induced by the combined treatment of halocyn-

thiaxanthin and TRAIL. Inhibitors of caspase-3, caspase-10,

Table 1. Apoptosis (Sub-G1 DNA Contents) Was Detected by Flow Cytometry in DLD-1 Cells Treated with VariousConcentrations of Halocynthiaxanthin and/or TRAIL

Halocynthiaxanthin (Mmol/L) 5 10 15 20 30 40Apoptosis (%) 0.97 1.21 1.83 2.64 4.66 9.46

TRAIL (ng/mL) 1.25 2.5 3.75 5 7.5 10Apoptosis (%) 2.59 3.64 3.84 5.64 8.02 11.02

Halocynthiaxanthin (Mmol/L) + TRAIL (ng/mL) 5/1.25 10/2.5 15/3.75 20/5 30/7.5 40/10Apoptosis (%) 1.45 2.2 4.75 8.35 20.06 46.30

NOTE: The values shown are the average of triplicate data.

Table 2. CI Values Were Analyzed from the Results ofTable 1

Fa CI EstimatedSD

Halocynthiaxanthin(Amol/L)

TRAIL(ng/mL)

0.02 2.432 0.5487 7.82427 1.956070.05 1.237 0.1745 12.7606 3.190150.1 0.749 0.1368 18.7679 4.691980.15 0.558 0.132 23.8313 5.957830.2 0.45 0.1277 28.5264 7.13160.25 0.379 0.1233 33.0943 8.273570.3 0.327 0.1191 37.6794 9.419850.35 0.287 0.1151 42.3922 10.5980.4 0.255 0.1112 47.3341 11.83350.45 0.228 0.1075 52.6132 13.15330.5 0.205 0.1039 58.3566 14.58920.55 0.184 0.1004 64.727 16.18180.6 0.166 0.0968 71.9459 17.98650.65 0.149 0.0932 80.3331 20.08330.7 0.133 0.0894 90.3808 22.59520.75 0.117 0.0854 102.903 25.72570.8 0.102 0.081 119.38 29.84510.85 0.087 0.076 142.9 35.7250.9 0.071 0.0696 181.453 45.3633

NOTE: An additive effect is reflected by CI = 1, a synergistic effect is reflected byCI < 1, and an antagonistic effect is reflected by CI > 1.Abbreviation: Fa, fraction affecting by dose.

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caspase-8, and pan-caspase blocked the apoptosis (Fig. 4).

These results indicate that the apoptosis depends on caspases.

In addition, DR5/Fc chimeric protein markedly blocked the

apoptosis, indicating that the apoptosis is induced through a

specific interaction of TRAIL and its receptor.

Halocynthiaxanthin Specifically Induces DR5 Expressionamong Death Receptor–Related Genes

Next, we examined the mechanism for the sensitization of

TRAIL by halocynthiaxanthin. Previous reports have shown

that the exogenous expression of TRAIL receptor using

expression vector can overcome TRAIL resistance (26, 27).

Therefore, we hypothesized that halocynthiaxanthin may

modulate the expression of TRAIL receptor. We carried out

an RNase protection assay to examine the expressions of death

receptor–related genes. Consequently, halocynthiaxanthin spe-

cifically up-regulated DR5, a receptor of TRAIL, among death

receptor–related genes (Fig. 5A). We confirmed the increase of

DR5 mRNA by halocynthiaxanthin using Northern blotting.

Halocynthiaxanthin up-regulated DR5 mRNA in a dose-

dependent manner (Fig. 5B). Furthermore, halocynthiaxanthin

increased DR5 protein in a dose- and time-dependent manner

(Fig. 5C and D). We prepared subcellular fractions and carried

out Western blotting of DR5. Halocynthiaxanthin markedly

induced DR5 protein in membrane fraction (Fig. 5E).

Interaction between TRAIL and DR5 forms a protein complex

with FADD (16). We did a pull-down assay in DLD-1 cells

treated with halocynthiaxanthin and recombinant TRAIL and

examined TRAIL-induced complex. Halocynthiaxanthin treat-

ment enhanced TRAIL-induced formation of the complex

containing DR5 and FADD (Fig. 5F).

DR5 Small Interfering RNA Blocks the Sensitizing Effectof Halocythiaxanthin to TRAIL-Induced Apoptosis

To examine the relation between DR5 up-regulation and

the sensitization to TRAIL by halocynthiaxanthin, we used

DR5 small interfering RNA (siRNA). DR5 siRNA blocked the

up-regulation of DR5 by halocynthiaxanthin at a total protein

level (Fig. 6A). In addition, DR5 siRNA decreased cell

surface DR5 protein (Fig. 6B). DR5 siRNA effectively

prevented the sensitizing effect to TRAIL (Fig. 6C),

suggesting that the sensitizing effect of halocynthiaxanthin

on TRAIL-induced apoptosis at least partly depends on the

FIGURE 2. Halocynthiaxanthin and TRAIL cooperate to activateapoptotic signaling. A. Morphologic features of cells stained with 4¶,6-diamidino-2-phenylindole. Arrows, nuclear fragmentation. B. Westernblotting of PARP. DLD-1 cells were treated with 40 Amol/L halocynthiax-anthin for 24 h and then treated with 10 ng/mL TRAIL for 12 h. DR5/Fcprotein (1 Ag/mL) was added at the same time of halocynthiaxanthinaddition.

FIGURE 3. Combination with halocynthiaxanthin and TRAIL inducescaspase cleavage. A. Cleaved caspases in DLD-1 cells treated withhalocynthiaxanthin (40 Amol/L) and/or TRAIL (10 ng/mL). DR5/Fc protein(1 Ag/mL) was added when halocynthiaxanthin was added. The celllysates were analyzed by Western blotting with anticaspase antibodies.B. Western blotting of Bid. h-Actin is a loading control. The bandintensities were measured with NIH Image software (NIH) and normalizedby h-actin. The protein levels relative to control were described at thebottom of the blot.

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up-regulation of DR5 by halocynthiaxanthin. Next, we

investigated the effect of halocynthiaxanthin in other colon

cancer HT29 cells. Halocynthiaxanthin induced DR5 expres-

sion and enhanced TRAIL-induced apoptosis in HT29 cells as

well as DLD-1 cells (Fig. 7).

Carotenoids Induce DR5 Expression and Sensitize DLD-1Cells to TRAIL-Induced Apoptosis with Specificity

We next investigated whether carotenoids universally

induce DR5 expression. We used four additional carotenoids

contained in corbicula clams (Fig. 8A; ref. 28). Interestingly,

peridinin also up-regulated DR5 expression, but alloxanthin,

diadinochrome, and pyrrhoxanthin did not induce DR5

expression (Fig. 8B). It has been reported that peridinin

also exhibits antitumor and anticarcinogenic activity (29, 30).

These results indicate that carotenoids do not universally,

but rather, specifically, up-regulate DR5 expression. Fur-

thermore, we examined the sensitizing effect of carote-

noids for TRAIL-induced apoptosis. Halocynthiaxanthin and

peridinin enhanced TRAIL-induced apoptosis (Fig. 8C).

However, alloxanthin, diadinochrome, and pyrrhoxanthin

only showed an additive effect or less than that when

together with TRAIL. Of interest, these carotenoids, which

sensitized DLD-1 cells to TRAIL-induced apoptosis,

accorded with those that up-regulated DR5 expression

(Fig. 8B and C).

FIGURE 4. Caspase inhibitors andDR5/Fc chimeric protein block TRAIL-induced apoptosis sensitized by hal-ocynthiaxanthin. DLD-1 cells weretreated with halocynthiaxanthin andTRAIL as shown in Fig. 1. Caspaseinhibitors (20 Amol/L) or DR5/Fc pro-tein (1 Ag/mL) was added at the sametime of treatment with halocynthiax-anthin. The DNA contents of cellswere analyzed by flow cytometry. M1shows the populations of apoptoticcells with sub-G1 DNA contents. Per-centages of sub-G1. Columns, mean(n = 3); bars, SD. C3, caspase-3inhibitor; C10, caspase-10 inhibitor;C8, caspase-8 inhibitor; VAD, pan-caspase inhibitor; Fc, DR5/Fc.



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DiscussionIn this study, we showed for the first time that dietary

carotenoids sensitize cancer cells to TRAIL-induced apoptosis.

Moreover, we first showed that dietary carotenoids up-regulate

the expression of DR5, a TRAIL receptor. Of interest, only

some carotenoids possess these activities. The carotenoids that

induced DR5 expression are the same as those that sensitized

DLD-1 cells to TRAIL, suggesting that the DR5 up-regulation

is a possible mechanism for the sensitization to TRAIL by

carotenoids. Moreover, DR5 siRNA partly prevented the

enhancement of TRAIL-induced apoptosis by halocynthiax-

anthin accompanied with the inhibition of DR5 up-regulation

by halocynthiaxanthin. Indeed, DR5 overexpression enhances

the sensitivity of cancer cells to TRAIL (26, 27). In addition,

our results suggest the possibility that more effective

carotenoids than halocynthiaxanthin or peridinin could be

detected among several carotenoids and that more effective

agents may be developed through insight into the common

chemical structure.

To date, it has been reported that combined treatment

with TRAIL and conventional antitumor agents, such as

doxorubicin, cisplatin, and 5-fluorouracil, is a promising

strategy against malignant tumors (20-22). However, the

action of these conventional antitumor agents is accompa-

nied by DNA damage. In this regard, carotenoids are

considered to be safer than these conventional antitumor

agents. Thus, carotenoids may substitute for conventional

antitumor agents in combined treatment with TRAIL against

malignant tumors.

We show here that DR5 expression is specifically induced

by carotenoids among death receptor–related genes. To our

knowledge, reports of carotenoid-regulated genes in human

cells are rare. Especially, this is the first report about genes

regulated by halocynthiaxanthin and peridinin. Therefore, our

FIGURE 5. Halocynthiax-anthin up-regulates DR5expression.A. Halocynthiaxan-thin induces DR5 expressionamong death receptor– relatedgenes. DLD-1 cells were treat-ed with DMSO or 40 Amol/Lhalocynthiaxanthin for 24 h andan RNase protection assay wascarried out.B.Northernblotting.DLD-1 cells were treated withvarious concentrations of halo-cynthiaxanthin for 24 h. Ethi-dium bromide–stained 28S and18S rRNA are loading controls.C and D. Western blotting.DLD-1 cells were treated withvarious concentrations of halo-cynthiaxanthin for 24 h (C) andtreated with 40 Amol/L halocyn-thiaxanthin for the indicatedtime (D). h-Actin is a loadingcontrol. CT, treated with DMSO.E. Halocynthiaxanthin inducesDR5 protein in membrane frac-tion. DLD-1 cells were treatedwith 40 Amol/L halocynthiaxan-thin or DMSO for 24 h. Cytosol,membrane, and nuclear proteinfractions were separated andanalyzed by Western blottingof DR5. Coomassie brilliant bluestaining of the gel (CBB ) isshown to ensure equal loadingof each fraction. F. Halocyn-thiaxanthin treatment enhancesTRAIL-induced formation of thecomplex containing DR5 andFADD.DLD-1 cells were treatedwith 40 Amol/L halocynthiaxan-thin or DMSO for 24 h andincubated with 1 Ag/mL histidinetagged-TRAIL for 1 h. Celllysate was prepared as shownin Materials and Methods.TRAIL-induced complex waspurified by Ni-NTA agaroseand analyzed with Western blot-ting of DR5 and FADD. �,medium change only (B andC) or treated with DMSO (Dand E).

Yoshida et al.


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results may be useful for the elucidation of the molecular

mechanisms for the function of carotenoids in humans. Some

carotenoids, such as h-carotene, possess provitamin A activity

(2, 3). Previous reports showed that retinoic acids also up-

regulate DR5 expression (22). Because the carotenoids we used

in this study do not possess provitamin A activity, the

mechanism of DR5 induction by carotenoids may be different

from that by retinoic acids. Moreover, as an important point, a

previous report showed that DR5 down-regulation by siRNA

promotes tumor growth in an in vivo xenograft model and

confers resistance to the antitumor agent 5-fluorouracil (31).

From this point of view, it may be important for cancer

prevention that carotenoids up-regulate DR5 expression, as

shown in our present study.

DR5 is a downstream gene of the tumor suppressor p53

gene (32-34). However, halocynthiaxanthin up-regulates DR5

FIGURE 6. Down-regulation of DR5 bysiRNA partially prevents the sensitizingeffect of halocynthiaxanthin on TRAIL-induced apoptosis. A. Western blotting.DR5 siRNA or control LacZ siRNA weretransfected into DLD-1 cells. Twenty-fourhours after the transfection, 40 Amol/Lhalocynthiaxanthin or DMSO was treatedfor 24 h. OF, treated with Oligofectaminewithout any siRNA. h-Actin is a loadingcontrol. Arrowheads, DR5 protein bands.*, a nonspecific signal because the bandwas not affected by DR5 siRNA. B. Cellsurface expression. Forty-eight hours aftertransfection of DR5 siRNA or control LacZsiRNA with FITC-oligonucleotides, cellsurface levels of DR5 and DR4 weredetected with phycoerythrin-conjugatedanti-DR5 (DR5 Ab ), anti-DR4 (DR4 Ab ),or anti-mouse IgG1n isotype control (CTAb ) antibody by flow cytometry. Cellsurface level was analyzed in FITC-positive cells. Clear histograms, controlLacZ siRNA– transfected cells; gray histo-grams, DR5 siRNA– transfected cells. C.DLD-1 cells were treated with 40 Amol/Lhalocynthiaxanthin and/or 10 ng/mLTRAIL after transfection of siRNA andFITC-oligonucleotides. The DNA contentsof cells were analyzed by flow cytometry.M1 shows the populations of apoptotic cellswith sub-G1 DNA contents in FITC-positivecells. Percentages of sub-G1. Columns,mean (n = 3); bars, SD. *, P < 0.05.



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independent of p53 because we used DLD-1 cells, which

contain a mutation of the p53 gene. More than half of

malignant tumors possesses an inactivating mutation in the

p53 gene (35, 36). Halocynthiaxanthin and peridinin slightly

induced apoptosis in DLD-1 cells, indicating that the

apoptosis by these carotenoids is independent of p53.

Furthermore, these carotenoids can sensitize cancer cells to

TRAIL-induced apoptosis in a p53-independent manner. Thus,

our results suggest that the combination of carotenoids and

TRAIL is useful for cancer treatment, regardless of the p53

status.

Recently, we reported that flavonoid luteolin sensitizes

cancer cells to TRAIL-induced apoptosis (37). Flavonoids and

carotenoids are abundantly contained in foods and have

been shown to have anticarcinogenic effects (3, 38). It is

remarkable that both of these famous food factors are capable

of enhancing TRAIL activity. TRAIL is a cytokine existing at

concentrations in the order of nanograms per milliliter in the

blood or urine of the human body due to the stimuli of IFN or

Bacillus Calmette-Guerin (39, 40). These and our data

together raise the possibility that TRAIL-DR5 signaling may

be responsible for the anticarcinogenic effects of carotenoids

and flavonoids.

Materials and MethodsReagents

Halocynthiaxanthin was derived from fucoxanthin according

to the method described previously (41), and peridinin,

alloxanthin, diadinochrome, and pyrrhoxanthin were prepared

as described previously (28). These carotenoids were dissolved

FIGURE 7. Halocynthiaxanthin induces DR5 expression and enhancesTRAIL-induced apoptosis in HT29 cells. A. Western blotting. HT29 cellswere treated with 40 Amol/L halocynthiaxanthin for 24 h. h-Actin is aloading control. Arrowheads, DR5 protein bands. *, a nonspecific signal.�, treated with DMSO. B. HT29 cells were treated with 40 Amol/Lhalocynthiaxanthin and/or 10 ng/mL TRAIL as shown in Fig. 1. The DNAcontents of cells were analyzed by flow cytometry. M1 shows thepopulations of apoptotic cells with sub-G1 DNA contents. Percentages ofsub-G1. Columns, mean (n = 3); bars, SD.

FIGURE 8. Halocynthiaxanthin and peridinin induce DR5 expressionand enhance TRAIL-induced apoptosis. A. Structure of carotenoids. B.Western blotting. DLD-1 cells were treated with 40 Amol/L of a variety ofcarotenoids for 24 h. h-Actin is a loading control. �, medium change only.

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in DMSO. Soluble recombinant human TRAIL/Apo2L was

purchased from PeproTech. Human recombinant DR5 (TRAIL-

R2)/Fc chimera protein and caspase inhibitors, zVAD-fmk,

zDEVD-fmk, zIETD-fmk, and zAEVD-fmk, were purchased

from R&D Systems.

Cell CultureHuman colorectal cancer DLD-1 cells were maintained in

RPMI 1640 supplementedwith 10% fetal bovine serum, 2mmol/L

glutamine, 100 units/mL penicillin, and 100 Ag/mL strepto-

mycin. Human colon cancer HT29 cells were maintained in

DMEM supplemented with 10% fetal bovine serum, 4 mmol/L

glutamine, 100 units/mL penicillin, and 100 Ag/mL streptomy-

cin. Cells were incubated at 37jC in a humidified atmosphere of

5% CO2.

Detection of ApoptosisCells stained with 4¶,6-diamidino-2-phenylindole were

analyzed using a fluorescent microscope as described previously

(42). For the detection of sub-G1, cells were harvested from

culture dishes and washed with PBS. The cells were suspended

with PBS containing 0.1% Triton X-100 and RNase A (Sigma

Chemical). The nuclei were stained with propidium iodide

before the DNA content was measured using FACSCalibur

FIGURE 8 Continued . C.DLD-1 cells were treated with40 Amol/L of the indicated car-otenoids for 24 h, then treatedwith 10 ng/mL TRAIL, andincubated for the following 12 h.The DNA contents of cells wereanalyzed by flow cytometry. M1shows populations of apoptoticcells with sub-G1 DNA contents.Percentagesofsub-G1.Columns,mean (n = 3); bars, SD.



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(Becton Dickinson). For each experiment, 10,000 events were

collected. The CellQuest software (Becton Dickinson) was used

to analyze the data.

Northern Blot Analysis and RNase Protection AssayTotal RNA from the cells was extracted using the Sepasol-

RNA I (Nacalai Tesque). Total RNA (10 Ag) was separated withelectrophoresis on a 1% agarose gel and transferred to a nylon

membrane (Biodyne B, Pall). A full-length DR5 cDNA was

used as a probe for the Northern blot analysis. Hybridization

was carried out with a 32P-labeled probe in PerfectHyb PLUS

Hybridization buffer (Toyobo) at 68jC for 16 h and the

membrane was washed at 68jC in 2� SSC containing 0.1%

SDS. The blot was exposed to X-ray Films (Kodak). The

RNase protection assay was done with an RPA III kit (Ambion).

hAPO3d (death receptor and death ligand) template sets (BD

Biosciences PharMingen) were labeled with [a-32P]UTP and

MAXI script (Ambion). The labeled RNA probes were

hybridized with 10 Ag total RNA from DLD-1 and digested

with RNase. The remaining probes were analyzed on 5% urea-

polyacrylamide gel.

Western Blot AnalysisThe cell lysate was prepared as described previously (43).

Cell lysate containing 50 Ag protein was separated on 10% or

12.5% SDS-polyacrylamide gel for electrophoresis and blotted

onto polyvinylidene difluoride membranes (Millipore). Rabbit

polyclonal anti-DR5 antibody (Cayman Chemical); mouse

monoclonal anti-Bid, anti-caspase-8, anti-caspase-9, and anti-

caspase-10 antibodies (MBL); mouse monoclonal anti-procas-

pase-3 antibody (Immunotech); rabbit monoclonal cleaved

caspase-3 antibody (Cell Signaling); mouse monoclonal anti-

PARP antibody (Santa Cruz Biotechnology); and mouse

monoclonal anti-h-actin antibody (Sigma Chemical) were used

as the primary antibodies. The signal was detected with an

enhanced chemiluminescence Western blot analysis system

(Amersham Biosciences). For subcellular fractionation, subcel-

lular proteome extraction kit (Calbiochem) was used.

Analysis of TRAIL-Induced Complex FormationDLD-1 cells were treated with 40 Amol/L halocynthiax-

anthin or DMSO for 24 h. Cells were harvested and 7 � 106

cells were treated with 1 Ag/mL recombinant human histidin-

tagged TRAIL (R&D Systems) at 37jC for 1 h with gentle

shake. Cells were washed with PBS and lysed for 15 min on

ice with lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4),

150 mmol/L NaCl, 0.2% NP40, and 10% glycerol. The lysate

was cleared by centrifugation at 13,000 rpm for 10 min at 4jC.The soluble fraction was incubated with Ni-NTA agarose

(Qiagen) and 10 mmol/L imidazol (Qiagen) at 4jC for 30 min.

After three washes with lysis buffer containing 10 mmol/L

imidazole, the bound proteins were eluted by boiling for 3 min

in loading buffer and resolved by SDS-PAGE. DR5 and FADD

proteins were detected by Western blotting.

siRNAThe DR5 and control LacZ siRNA (100 nmol/L) were

transfected into DLD-1 cells with Oligofectamine (Invitrogen) as

described previously (42). The volume of Oligofectamine was

two thirds the recommended volume in the manufacturer’s

protocol. For flow cytometry analysis, 100 nmol/L FITC-oligo-

nucleotides (FITC-5¶-gacccgcgccgaggtgaagtt) was cotransfectedwith siRNA. FITC-positive cells were selected and analyzed.

Detection of DR5 and DR4 Expressions on Cell SurfaceDLD-1 cells were transfected with 100 nmol/L FITC-

oligonucleotides and 100 nmol/L DR5 siRNA or control LacZ

siRNA. Forty-eight hours after transfection, cells were harvested,

washed with PBS, and suspended in PBS containing 1% bovine

serum albumin. Then, 3 AL of phycoerythrin-conjugated anti-

DR5, anti-DR4, or mouse IgG1n isotype control antibody

(eBiosciences) were added to cells and incubated on ice for

30 min. After wash with PBS, DR5 and DR4 expressions on cell

surface were analyzed in FITC-positive cell population by flow

cytometry.

Statistical AnalysisData represent means F SD of triplicate data. Data were

analyzed using a Student’s t test and differences were

considered significant from controls when P < 0.05. The CI

was calculated by the method of Chou and Talalay (23) using

Calcusyn software (Biosoft). Synergism is defined as more than

the expected additive effect with CI < 1 (24). An additive effect

is reflected by CI = 1 and an antagonistic effect is reflected by

CI > 1.

AcknowledgmentsWe thank Dr. Taka-aki Matsui for helpful advice.

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2007;5:615-625. Mol Cancer Res Tatsushi Yoshida, Takashi Maoka, Swadesh K. Das, et al. Apoptosis-Inducing Ligand

Related−Cell Lines to Tumor Necrosis Factor Halocynthiaxanthin and Peridinin Sensitize Colon Cancer

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