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Lentinan: Hematopoietic, Immunological, and EfficacyStudies in a Syngeneic Model of Acute MyeloidLeukemiaEmmet McCormack a , Jørn Skavland b , Maja Mujić b , Øystein Bruserud a b & Bjørn Tore

Gjertsen a ba Haukeland University Hospital, Bergen, Norwayb University of Bergen, Bergen, NorwayVersion of record first published: 22 Jun 2010.

To cite this article: Emmet McCormack, Jørn Skavland, Maja Mujić, Øystein Bruserud & Bjørn Tore Gjertsen (2010): Lentinan:Hematopoietic, Immunological, and Efficacy Studies in a Syngeneic Model of Acute Myeloid Leukemia, Nutrition and Cancer,62:5, 574-583

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Nutrition and Cancer, 62(5), 574–583Copyright C© 2010, Taylor & Francis Group, LLCISSN: 0163-5581 print / 1532-7914 onlineDOI: 10.1080/01635580903532416

Lentinan: Hematopoietic, Immunological, and EfficacyStudies in a Syngeneic Model of Acute Myeloid Leukemia

Emmet McCormackHaukeland University Hospital, Bergen, Norway

Jørn Skavland and Maja MujicUniversity of Bergen, Bergen, Norway

Øystein Bruserud and Bjørn Tore GjertsenUniversity of Bergen and Haukeland University Hospital, Bergen, Norway

Lentinan, a β-glucan nutritional supplement isolated from theshitake mushroom (Lentula edodes), is a biological response mod-ifier with immunostimulatory properties. Concomitantly, the roleof β-glucans as chemoimmunotherapeutic in a number of solidcancers has been widely documented. We investigated the effectsof nutritional grade lentinan upon BN rats and in a preclinicalsyngeneic model of acute myeloid leukemia. BN rats supplementeddaily with lentinan exhibited weight gains, increased white bloodcells, monocytes, and circulating cytotoxic T-cells; and had a reduc-tion in anti-inflammatory cytokines IL-4, IL-10, and additionallyIL-6. Lentinan treatment of BN rats with BNML leukemia resultedin improved cage-side health and reduced cachexia in the termi-nal stage of this aggressive disease. Combination of lentinan withstandards of care in acute myeloid leukemia, idarubicin, and cy-tarabine increased average survival compared with monotherapyand reduced cachexia. These results indicate that nutritional sup-plementation of cancer patients with lentinan should be furtherinvestigated.

INTRODUCTIONFungal β-D-glucans are a broad class of bioactive polysac-

charides derived from medicinal mushrooms and recognizedas potent immunostimulators (1). Reports in Chinese medicinehave described their use from immunostimulatory to preventionand treatment of cancer (2). The efficacy of β-glucans was beenwidely demonstrated in multiple preclinical cancer models. Ofnote, oral and intravenous administration of β-glucan with ther-apeutic monoclonal antibodies resulted in higher therapeutic

Submitted 30 March 2009; accepted in final form 31 October 2009.Address correspondence to Bjørn Tore Gjertsen, Institute of

Medicine, Haematology Section, University of Bergen, HaukelandUniversity Hospital N-5021, Bergen, Norway. Phone: +4755975000.Fax: +4755975890. E-mail: bjorn.gjertsen@med.uib.no

efficacy and greater tumor regression (3–5). Similarly, concur-rent use of β-glucan and the fluoropyrimidine anticancer drug,S-1, prolonged survival times in a syngeneic colon cancer modelbut not in athymic nude mice, suggesting a role for β-glucansin stimulating antitumor immunity (6). Furthermore, β-glucanshave also demonstrated a protective effect against genotoxicityand cytotoxicity when administered with the anthracycline an-tibiotic doxorubicin (7). More recently, several human clinicaltrials have demonstrated possible treatment benefits in cancertherapy, particularly in gastric and colorectal cancers, and asadjuvant chemoimmunotherapy in postoperative treatment ofresectable cancers (8–10).

Fungal β-glucans appear to act by stimulating the wholeimmune system, functioning as pathogen associated molecularpatterns (PAMPs) to an array of germ-line encoded moleculescalled pattern recognition receptors (PRRs) (11). In vertebrates,a number of these receptors have been identified such as dectin-1, compliment receptor 3 (CR3), scavenger receptors, lacto-sylceramide, and the toll-like receptor. Of these PRRs, theinteraction between dectin-1, commonly expressed on humanlympho-myeloid lineages, and zymosan [a crude cell wall par-ticulate containing a mixture of β-(1→3)(1→6)-glucans] hasbeen most extensively studied (12). Binding promoted activa-tion of phagocytosis, ROS production, and induction of inflam-matory cytokines (13). However, a similar glucan, lentinan [a(1→3)-β-glucan with (1→6) branches produced from the fruit-ing body of Lentinula edodes], appears to activate CR3 and aug-ments responsiveness of cytotoxic T-lymphocytes (CTLs) andnatural killer cells to IL-2 (14). Nevertheless, although defini-tive conclusion pertaining to their precise immunotherapeuticfunction has yet to be fully elucidated, the clinical applicabil-ity of β-glucans as adjuvant chemoimmunotherapy in a numberof cancers is exciting. Despite these encouraging results, thesupplementation of β-glucans to treatment of acute myeloid

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NUTRITIONAL LENTINAN IN EXPERIMENTAL ANTILEUKEMIC THERAPY 575

leukemia (AML) has never been demonstrated either preclini-cally or clinically.

AML is the most common acute leukemia in adults for whomoverall survival remains poor (15). Conventional chemotherapy,including the use of anthracyclines and cytarabine, is the main-stay of current clinical protocols (16), which is decidedly toxic,eliciting adverse effects upon the host immune system. Accord-ingly, the use of immunomodulatory agents such as targeted an-tibodies (17), cytokine (18), and dendritic cell-based therapies(19) have previously been evaluated as immuno-potentiators orimmuno-effectors in preclinical mouse models of AML (20–22).Thus, we postulated that nutritional supplementation of lentinaneither prior to or in combination with anthracyclins or cytarabinemay elicit a greater antileukemic response in the Brown Norwe-gian (BN) rat syngeneic model of AML, termed BNML (23). Toinvestigate this, we first evaluated the safety, cytokine immuno-logical, and hematological effects of chronic dosing of nutri-tional concentrations of lentinan on normal BN rats over 20 wkor BNML and finally evaluated the chemoimmunotherapeuticpotential of lentinan alone and in combination with “standardsof care” idarubicin and cytarabine in the BNML model.

MATERIALS AND METHODS

RatsMale BN/RijHsd rats were purchased from Harlan, Horst,

The Netherlands, and animals were acclimatized for 1 wk priorto experimentation. All experiments were approved by The Nor-wegian Animal Research Authority and conducted according toThe European Convention for the Protection of Vertebrates Usedfor Scientific Purposes.

Evaluation of Hematopoietic and Cytokine ResponseFollowing Lentinan Administration

To study cytokine production and hematological response ofBN rats to oral administration of lentinan [β-(1→3)-β-(1→6)-D-glucan, Lentinex, generously provided by Glycanova NorgeA/S, Sarpsborg, Norway], BN rats (average weight 270 g; n =10) were dosed with lentinan (p.o.; 12 mg/kg) q.d. for 20 wk.Age matched controls (n = 10) were fed with an equivalentvolume per body weight of lentinan fermentation product fol-lowing removal of lentinan by ethanol precipitation. Weightswere recorded every other day and peripheral blood (500 µl)collected from the lateral tail vein every other week, 1 h fol-lowing lentinan or control vehicle administration. After 20 wk,animals were sacrificed and necropsied.

Blood and Flow Cytometric AnalysisPeripheral blood was collected in microtainer tubes with

EDTA (BD Biosciences, San Jose, CA) and routine hematol-ogy processed (AVIDA 2120 Multispecies Hematology System,Bayer Diagnostics, Tarrytown, NY) in duplicate within 1 h ofcollection. Peripheral blood leukocytes (PBLs) for single andmultilabel flow cytometric analysis were prepared following

incubation of whole blood at 4◦C for 30 min in phosphate-buffered saline (PBS) with the following monoclonal antibod-ies (mAbs) against rat leukocyte markers as FITC, PE or al-lophycocyanin (AP) conjugates: anti-CD8 (clone MRC OX-8);anti-CD4 (clone W3/25), anti-Igκ (clone MRC OX-12), anti-CD11b (clone OX-42), all AbD Serotec, UK and anti-CD3(clone G4.18), anti-CD45RA (clone OX-33), anti-CD172 (SIRPclone OX-41), and anti-CD45 (clone OX-1), all BD Pharmingen(San Diego, CA). Following lysis (NH4Cl; Merck, Whitehous-estation, NJ) for 10 min at room temperature, samples werewashed twice with PBS/BSA (Roche Diagnostics, Mannheim,Germany) and subsequently fixed in 0.5% paraformaldehyde(Sigma, St. Louis, MO). FACS analysis was performed on aFACSCalibur equipped with 488 nm and 635 nm lasers (BectonDickinson, Franklin Lakes, NJ). Data acquisition and analysiswas performed with CellQuestPro software (Becton Dickinson).

Serum Cytokine AnalysisSerum was diluted 1:3 in sample diluent (Rat Serum Diluent

Kit, Bio-Rad, Hercules, CA) and centrifuged at 4◦C for 5 min(9,500 g) to clear the samples of hemolytic debris. Sera wereassayed for cytokines (GM-CSF, IFN-γ , IL-1α, IL-1β, IL-2,IL-4, IL-6, IL-10, TNF-α) using Bio-Plex Rat Cytokine 9-PlexA Panel kit (Bio-Rad Laboratories, Inc.).

BNML LeukemiaOne hundred and twenty male BN/RijHsd rats (average

weight 200–250 g) were injected i.v. via the tail vein with 5 ×106 monocellular BNML cells (500 µl phosphate buffered saline[PBS]; Day 0) derived from the leukemic spleen taken from a ter-minal stage leukemic BN animal. Survival and condition/weightloss of all animals was monitored on a daily basis for the du-ration of experiments. Animals were necropsied when they be-came moribund as defined by the following criteria: weight loss(greater than 15%), dehydration (ruffled fur), hind limb paral-ysis, and lethargy. Lethargic animals were scored accordingly;briefly, once 3 consecutive weight losses were recorded, animalswere placed on lying on their backs on the cage floor and theirability to turn over assessed (where terminal animals cannot turnover). Furthermore, animals were held 10 cm above the floor ofthe holding cage. Healthy animals will normally jump from thisposition, lethargic rats will step onto the cage floor, and terminalanimals will either not move or step with their forelimbs but nothind limbs.

Antileukemic Activity of LentinanIn assessing the preventative, therapeutic, and combinatorial

therapeutic effect, lentinan was administered p.o. q.d. starting atthe time point indicated and for the duration of the experimentat either low (0.5 mg/kg), medium (12 mg/kg), or high dose(20 mg/kg) as specified. Idarubicin (Zavedos; Pfizer, Inc., NewYork, NY) was dissolved in sterile water to a final concentrationof 1 mg/ml and administered p.o. q.d. × 3 from Day 3 at a doseof 1.5 mg/kg as described previously (24). Cytarabine (Pfizer

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576 E. McCORMACK ET AL.

TABLE 1Average weights of Brown Norwegian rats supplemented with lentinan (12 mg/kg) or vehicle controla

Weeks of Age

17 19 21 23 25 27 29 31 33 35 37Group Weight (g)

Cntrl 285 ± 8 303 ± 8 312 ± 8 328 ± 9 338 ± 10 342 ± 10 344 ± 10 347 ± 10 346 ± 9 349 ± 9 348 ± 8LEN 287 ± 7 335 ± 9 349 ± 11 357 ± 11 360 ± 12 368 ± 12 369 ± 14 376 ± 14 376 ± 14 367 ± 14 370 ± 14

aAbbreviations are as follows: Cntrl, vehicle control; LEN, lentinan (12 mg/kg). Weights represented as average ± SEM. ∗ = P < 0.05;∗∗ = P < 0.01; ∗∗∗ = P < 0.001 relative to control.

Inc.) was dissolved in saline to a concentration of 100 mg/mland administered i.p. q.d. × 5 at a dose of 100 mg/kg from Day10 (n = 6), resulting in a total dose similar to that describedpreviously (25) and equivalent to a human dose of 600 mg/kg(26) calculated from formula 1:

Human equivalent dose (mg/kg)

= animal dose (mg/kg) × animal Km/human Km, [1]

Where Km factor is based on the following: human of 60 kg =Km 37 and BSA 1.6 m2 is equivalent to rat of 150 g = Km 6and BSA = 0.025 m2 (27). Control animals received relevantcontrol vehicles via the same route(s) of administration as testanimals (n = 6).

Experimental DesignTo investigate dose effects of lentinan as an oral adjuvant on

BNML, 24 BN rats were inoculated with BNML and dividedinto one of the following 4 groups (n = 6): Group A = control(dosed with vehicle), Group B = low-dose lentinan (0.5 mg/kg),Group C = medium-dose lentinan (12 mg/kg), or Group D =high-dose lentinan (20 mg/kg).

In examination of lentinan’s prophylactic function in BNML,a further 24 rats divided into 4 groups: Group A = control (dosedwith vehicle), Group B = low-dose lentinan (0.5 mg/kg), GroupC = medium-dose lentinan (12 mg/kg), Group D = high-doselentinan (20 mg/kg), of (n = 6) and animals dosed for 2 wk priorto BNML administration.

To explore the potential use of high- or low-dose lentinanas an adjuvant to standard AML therapy, 36 BN rats were in-oculated with BNML and divided into one of 6 groups (n =6): Group 1 = control, Group 2 = lentinan (0.5 mg/kg/day),Group 3 = cytarabine (Ara-C; 100 mg/kg; q.d. × 5), Group 4= Ara-C + lentinan, Group 5 = Ida (1.5 mg/kg; q.d. × 3), orGroup 6 = Ida + lentinan. This study was then repeated with afurther 36 rats with animals divided in the same groups as abovebut supplementing low-dose lentinan for high-dose lentinan(20 mg/kg/day).

Statistical AnalysisResults are expressed as the mean ± SEM. Comparisons be-

tween groups were made using a paired t-test, whereas survival

data was analyzed employing the method of Kaplan and Meierand survival distributions analyzed by the Mantel-Haenszel logrank statistic. Differences where P < 0.05 were consideredstatistically significant. Statistics were made using GraphPadPRISM (version 3.0; GraphPad Software, Inc., La Jolla, CA).

RESULTS

Safety of Continuous Oral DosingWith Lentinan

Chronic administration of lentinan (12 mg/kg) to BN rats(n = 10) over a period of 20 wk resulted in no observabletoxicity during administration or at necropsy following whole-body gross examination. A statistically nonsignificant increasein mean body weight was observed in the lentinan-treated groupwhen compared with the control treated rats for the duration ofthe experiment (Table 1).

Peripheral Blood Cell Counts During Oral Dosing WithLentinan

Comparison of white blood cell (WBC), red blood cell(RBC), platelet (PLT), and hemoglobin (Hb) cell numbers andlevels between the control and lentinan groups suggested thaton the whole, the lentinan group exhibited higher numbers ofcells (Table 2). In particular, WBC counts in the lentinan groupwere statistically higher than controls on 5 of the 9 wk tested.Similarly, RBC counts for the lentinan group demonstrated sta-tistically higher levels than controls on Weeks 23, 29, and 33,whereas Hb and PLT levels were approximately the same be-tween the groups.

Lentinan Effect on Immune Cell PopulationsCD4+ and CD8+ T cells were quantified as percentages of

live CD3+ T cells and Igκ+ B cells as a percentage of totallive lymphocytes and monocytes by flow cytometry (Table 3).In general, there were modest differences in the percentages ofCD4+ T cells between groups; however, lentinan-treated ani-mals were lower on Weeks 27 and 33 (P < 0.01). In contrast,percentages of CD8+ T cells were generally higher in the lenti-nan group in comparison to controls, which was particularlyevident and significant from Week 27 (P < 0.05 to P < 0.001).Interestingly, CD4/CD8 ratios (Fig. 1B) for lentinan-treated

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TAB

LE

2C

ompa

riso

nof

rout

ine

hem

atol

ogic

alpa

ram

eter

sof

BN

rats

supp

lem

ente

dw

ithle

ntin

an(1

2m

g/kg

)or

vehi

cle

cont

rola

Wee

ksof

Age

1921

2325

2729

3133

3537

Para

met

erG

roup

No.

Cel

l

WB

C(1

09 /l)L

EN

8.3

±0.

69.

7∗∗±

0.2

8.3∗ ±

0.9

10.7

∗∗∗ ±

0.7

7.1

±1.

27.

8±

0.7

9.2∗∗

±0.

58.

6±

0.8

8.7∗∗

±0.

710

.3∗ ±

1.1

Cnt

rl8.

6±

0.4

7.2

±0.

56.

3±

0.5

6.1

±0.

46.

8±

0.4

7.6

±0.

37.

9±

0.3

8.0

±0.

45.

6±

0.3

7.7

±0.

5R

BC

(1012

/l)L

EN

8.3

±0.

18.

3±

0.3

8.7∗∗

±0.

18.

6±

0.2

8.7

±0.

18.

6∗ ±0.

18.

7±

0.1

9.0∗∗

±0.

18.

9±

0.1

8.5

±0.

1C

ntrl

8.5

±0.

18.

1±

0.1

7.6

±0.

38.

3±

0.1

8.5

±0.

18.

9±

0.1

8.7

±0.

18.

5±

0.1

8.6

±0.

28.

6±

0.1

Hb

(g/l)

LE

N14

.1±

0.2

14.5

±0.

515

.1∗∗

±0.

215

.0±

0.4

15.0

±0.

214

.4±

0.2

14.8

±0.

115

.2±

0.2

14.9

±0.

314

.3±

0.2

Cnt

rl14

.8∗ ±

0.2

14.3

±0.

113

.7±

0.4

14.7

±0.

214

.9±

0.2

15.4

∗∗±

0.2

15.0

±0.

114

.7±

0.2

15.0

±0.

114

.8±

0.1

PLT

(109 /l)

LE

N74

1±

4878

7±

7887

7∗∗±

2568

8±

8877

4±

6178

0±

4684

3±

1882

2±

2266

5±

7479

6±

35C

ntrl

778

±59

756

±52

787

±16

716

±61

716

±60

672

±70

621

±10

473

6±

3772

3±

2168

6±

78

a Abb

revi

atio

nsar

eas

follo

ws:

LE

N,

lent

inan

(12

mg/

kg);

Cnt

rl,

vehi

cle

cont

rol;

WB

C,

whi

tebl

ood

cells

;R

BC

,re

dbl

ood

cells

;H

b,he

mog

lobi

n;PL

T,pl

atel

ets.

Val

ues

repr

esen

ted

asav

erag

e±

SEM

.∗=

P<

0.05

;∗∗=

P<

0.01

;∗∗∗=

P<

0.00

1re

lativ

eto

cont

rol.

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TAB

LE

3C

ompa

riso

nof

imm

unol

ogic

alpa

ram

eter

sof

BN

rats

supp

lem

ente

dw

ithle

ntin

an(1

2m

g/kg

)or

vehi

cle

cont

rola

Wee

ksof

Age

Para

met

erG

roup

1921

2325

2729

3133

3537

Igκ

+B

cells

bL

EN

9.1∗∗

±0.

911

.4∗∗

±0.

719

.6∗∗

∗ ±1.

415

.4±

0.8

11.4

±1.

119

.4±

1.0

14.8

±0.

510

.0±

1.0

17.1

±1.

5N

/AC

ntrl

6.2

±0.

48.

4±

0.5

4.9

±0.

715

.9±

1.9

9.8

±0.

920

.8±

1.1

12.9

±1.

515

.3∗ ±

1.5

17.9

±0.

9N

/AC

D8+

Tce

llsc

LE

N10

.1±

1.0

9.8∗ ±

0.7

11.2

±0.

812

.7±

1.2

19.3

∗∗±

1.3

10.1

±1.

015

.4∗∗

∗ ±1.

011

.3∗ ±

0.8

9.7

±0.

88.

9±

0.6

Cnt

rl7.

8±

0.3

7.5

±0.

510

.1±

1.2

11.0

±1.

011

.5±

0.7

9.2

±0.

710

.6±

0.5

8.8

±0.

49.

0±

0.4

8.5

±0.

4C

D4+

Tce

llsc

LE

N87

.4±

1.1

88.0

±0.

886

.7±

0.7

85.3

±1.

278

.1±

1.7

88.0

±1.

082

.5±

1.1

86.3

±1.

288

.6±

0.8

89.2

±0.

7C

ntrl

89.6

±0.

390

.2±

0.6

87.4

±0.

386

.6±

1.2

85.8

∗∗±

0.8

88.8

±0.

787

.2∗∗

±0.

589

.3±

0.4

88.9

±0.

489

.2±

0.4

CD

11b+ -

CD

172+b

LE

N19

.0∗∗

∗ ±1.

016

.8∗∗

±0.

918

.0±

0.7

14.7

±1.

019

.8∗ ±

1.2

17.7

±1.

527

.0∗∗

∗ ±1.

820

.8∗ ±

1.4

13.1

±1.

917

.0±

1.7

Cnt

rl10

.7±

0.6

14.0

±1.

6N

/A13

.4±

0.9

16.9

±1.

016

.2±

0.9

11.9

±0.

814

.1±

1.4

15.2

±0.

513

.5±

0.9

a Abb

revi

atio

nsar

eas

follo

ws:

LE

N,l

entin

an(1

2m

g/kg

);C

ntrl

,veh

icle

cont

rol;

N/A

,not

avai

labl

e.Pe

rcen

tage

sre

pres

ente

das

aver

age

±SE

M.∗

=P

<0.

05;∗∗

=P

<0.

01;

∗∗∗=

P<

0.00

1re

lativ

eto

cont

rol.

b As

ape

rcen

tof

lym

phoc

ytes

and

mon

ocyt

es.

c As

ape

rcen

tof

tota

lCD

3+T

cells

.

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NUTRITIONAL LENTINAN IN EXPERIMENTAL ANTILEUKEMIC THERAPY 579

FIG. 1. The effect of daily lentinan nutritional supplementation on monocytes and T cells of BN rats. A: Lentinan treated BN rats demonstrated statisticallysignificant higher levels of circulating monocytes presented as percentage of peripheral blood mononuclear cells (PBMC) than controls. B: Furthermore, theseanimals exhibited decreased ratio of CD4:CD8 cells resultant of increased numbers of CD8+ T cells. Experiments were run for 18 wk. See Material and Methodsfor experimental details. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

animals were lower than controls at all time points and sta-tistically so from Week 27 (P < 0.05 to P < 0.001); however,this did not impact on total cell numbers, as the total percentageof circulating monocytes (Fig. 1A) was higher in the lentinan-treated group in comparison to controls (P < 0.05 to P < 0.001).Finally, lentinan initially appeared to increase the percentagesof B lymphocytes; however, they returned to control levels afterWeek 23.

Influence of Oral Dosing of Lentinan on Serum CytokinesIn general, there was a significant suppressive effect upon

the serum cytokine levels of IL-4, IL-6, IL-10, and GM-CSFwith continuous dosing of lentinan over the 20-wk period (Fig.2). Levels of TNF-α and IL-1α were comparable between thegroups, whereas levels of IFN-γ were on average higher inthe lentinan treated group, although not statistically so. Inter-estingly, IL-1β levels, which for the control group decreasedwith age, were stabilized in the lentinan-treated group and ac-tually increased substantially from Week 29 in comparison tothe natural decline in IL-1β levels experienced by controls (P <

0.05–0.01). Finally, whereas IL-2 serum levels were practicallynonexistent in control BN rats, which is consistent with previ-ous reports (28) in this strain of rat, lentinan-treated animalsdemonstrated sporadic increases of IL-2.

Impact of Lentinan on BNML Survival as OralImmunotherapeutic Agent

The oral effect of lentinan as an antileukemic chemothera-peutic agent was assessed in the syngeneic BNML rat AMLmodel. BN rats were either dosed p.o. q.d. from 2 wk prior toinoculation with BNML cells (5 × 106 cells; i.v.) or from in-oculation (p.o. q.d.) with low- or high-dose lentinan (0.5 or 20mg/kg) until moribund. A further group were treated with a me-dian dose (12 mg/kg q.d. p.o.) consistent with the chronic dosingregime (Table 4). There was no statistically significant increasein survival in any of groups administered lentinan, although

groups administered lentinan from 14 days prior to receivingBNML did, on average, survive longer.

Lentinan as a Combinatorial Adjuvant to Standard AMLChemotherapy

The use of idarubicin and cytarabine are the standard of carefor patients being treated for AML; thus to investigate the poten-tial of lentinan as an adjuvant to these treatments in an experi-mental model of AML, BN rats inoculated with BNML receivedeither cytarabine (100 mg/kg) ± lentinan (0.5 or 20 mg/kg) oridarubicin (1.5 mg/kg) ± lentinan (0.5 or 20 mg/kg). Both cy-tarabine and idarubicin mono-treated groups responded to treat-ment, resulting in a statistically significant increase in averagesurvival in comparison to controls (3.5–4.4 days, respectively;P < 0.01). The addition of low-dose lentinan to these treatmentsdid not have any impact on survival. However, administrationof high-dose lentinan in combination with cytarabine and idaru-bicin did, in both cases, increase average survival in comparisonto mono-treated groups, although not statistically. Whereas theeffect was strongest in combination with idarubicin (furtheraverage increase in survival of 1.4 days vs. idarubicin alone),the combination of cytarabine and high-dose lentinan increasedthe statistical significance of cytarabine-induced survival fromP < 0.01 for cytarabine monotherapy vs. controls to P < 0.001for combination therapy vs. controls. Finally, animals treatedwith the combination of lentinan and cytarabine (P < 0.001) oridarubicin (P < 0.05), demonstrated significantly reduced treat-ment induced weight loss at sacrifice compared with controls(Fig. 3).

DISCUSSIONNutritional supplementation, including β-glucans, have pre-

viously been demonstrated to cause antagonism in cancer andwhen combined with chemotherapy (29). Thus, evaluation ofsupplementation is critical prior to clinical application (30).

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580 E. McCORMACK ET AL.

TABLE 4Survival time (days) of treated vs. control BN rats inoculated with BNMLa

Treatment Group Schedule Survival

Cntrl q.d. 19.3 ± 0.3Ara-C (100 mg/kg) Day 10→14 22.5∗∗ ± 0.4IDA (1.5 mg/kg) Day 3→5 22.3∗∗ ± 0.3LEN (12 mg/kg) q.d. 19.0 ± 0.5

(Day −14) q.d. 19.8 ± 0.3LEN (0.5 mg/kg) q.d. 19.0 ± 0.5

+ (Day −14) q.d. 20.3 ± 0.2+ Ara-C (100 mg/kg) Day 10→14 22.0∗∗ ± 0.4+ IDA (1.5 mg/kg) Day 3→5 22.2∗∗ ± 0.5

LEN (20 mg/kg) q.d. 19.8 ± 0.2+ (Day −14) q.d. 20.0 ± 0.3+ Ara-C (100 mg/kg) Day 10→14 22.8∗∗∗ ± 0.3+ IDA (1.5 mg/kg) Day 3→5 23.7∗∗ ± 0.8

aAbbreviations are as follows: IDA, idarubicin; Ara-C, cytarabine; Cntrl, control; LEN, lentinan; q.d., every day. Survivaltime (days) is represented as average ± SEM. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 relative to control.

FIG. 2. Result of chronic lentinan nutritional supplementation on cytokine profile of BN rats vs. controls. See Materials and Methods for experimental details.TNF-α, tumor necrosis factor alpha; IL, interleukin; INF-γ , interferon gamma; GM-CSF, granulocyte-macrophage colony stimulating factor. ∗, P < 0.05; ∗∗, P <

0.01; ∗∗∗, P < 0.001.

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NUTRITIONAL LENTINAN IN EXPERIMENTAL ANTILEUKEMIC THERAPY 581

FIG. 3. The effect of adjuvant lentinan (LEN) upon BNML and standard AML chemotherapy induced weight loss. A: Control BN rats (�) administered withBNML cells demonstrates leukemia-induced cachexia of approximately 10% at morbidity. Daily treatment with lentinan (�, 20 mg/kg) improved cage-side healthand D: significantly reduced observed cachexia at necropsy (P < 0.01). B: Similarly, lentinan cotreatment with standards of care Ara-C (�100 mg/kg); and C:Idarubicin [(IDA) �1.5 mg/kg) reduced chemotherapy-induced cachexia when compared with Ara-C (P < 0.001;�) and IDA-only (P < 0.05; �) treated BNMLrats (D).

We therefore investigated the safety of nutritional supplementof lentinan in healthy BN rats and in a syngeneic model ofAML both as a single agent and in combination with standardchemotherapy.

Conventional chemotherapy against AML (15), including theuse of anthracyclines and cytarabine, is the mainstay of currentclinical protocols (16) that elicit adverse effects on the hostimmune system in patients who already exhibit disease relatedimmune defects (31). Thus, the potential use of agents, whichpotentiate the immune system, particularly T-cell activity in im-mune recognition of leukemia cells, or aid restoration of normalhematopoiesis, would be useful in AML therapy.

Disease progression in the BNML model, as in human AML,results in cancer cachexia at terminal stage disease. BN ratschronically administered lentinan over a 20-wk period demon-strated a net mean weight gain over controls for the durationof the study (Table 1). Furthermore, BN rats inoculated withBNML and treated either with a monotherapy of high-dose lenti-

nan or in combination with idarubicin or cytarabine exhibited re-duced weight loss as compared with controls and minimized thecachexic effects of chemotherapy (Fig. 3). Previous reports es-tablished that lentinan protected against 5-fluorouracil inducedtoxicity and associated weight loss in mice 32) and partiallynormalized TNF-α induced cachexia in rats but only at a highdose (33). Although there are no differences between plasmaTNF-α levels in healthy donors and AML patients, elevatedTNF-α levels in AML patient’s bone marrow inhibits normalhematopoiesis (34). In our study, lentinan suppressed the pro-duction of the cachexia mediator IL-6 (35) (Fig. 2) in healthyBN rats. Mice transgenic for the expression of high levels ofIL-6 demonstrate marked decreases in growth rate (50–70%)to wild-type littermates (36). Cumulatively, one may postulatethat weight gains, and moderated cachexia observed in BNMLanimals, resultant of lentinan administration, may be a result ofIL-6 ablation, warranting further investigation of bone marrowcytokine levels.

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582 E. McCORMACK ET AL.

Similarly, the increased number of peripheral blood cells(Table 2), particularly in WBC and monocytic populations (Fig.1A), point to hematopoietic potentiation by lentinan. Similarobservations have previously been determined in experimentalmodels of myelotoxic injury whereby prophylactic administra-tion of the β-glucan, poly-(1,6)-β-D-glucopyranosyl-(1,3)-β-D-glucopyranose (PCG) was shown to accelerate recovery ofPBLs in mice and monkeys following myeloblative and myelo-suppresive doses of cyclophosphamide (37) or following sub-lethal irradiation (38). The effect of lentinan on T-cell popu-lations is particularly interesting. Although there was minimalimpact on CD4+ populations (Table 3), there was a significantincrease in the percentage of circulating CD8+ cells as previ-ously observed in broilers fed β-glucan with concurrent weightgain (39).

The requisite of CD8+ T-cells for lentinans antitumor func-tion has been demonstrated by Suzuki and coworkers (40) in apreclinical lymphoma model. Clinically, the lentinan effect, andincreased survival in responding patients, appeared to dependupon modulation of the CD8+ T-cell subset together with a re-duction in IL-6, GM-CSF, and PGE2 levels following lentinantreatment (41,42). The CD4/CD8 ratio, which is naturally highin BN rats, was significantly reduced by lentinan to more nor-mal levels as observed in other rat strains (43). This is ratherinteresting, as BN rats are known to have approximately 3 timesless CD8 T cells than other rat strains, resulting in the highCD4:CD8 ratio.

Generally β-glucans, including lentinan, appear to induceproduction of Type 1 cytokines, IL-2, and INF-γ (44). In ourstudy, chronic dosing of BN rats with lentinan did not signif-icantly alter the production of IL-2 and INF-γ ; however, theGM-CSF, IL-4, IL-6, and IL-10 production was significantlyimpaired (Fig. 2). Murata et al. (44) previously demonstratedthat lentinan reduced IL-4 production in CD4 T cells derivedfrom lentinan-treated mice and elicited decreased productionof IL-6, IL-10, and PGE2 in peritoneal macrophages. Similarmechanisms may explain our observations.

It is interesting to note that reductions of cytokine levelsand CD4:CD8 ratios were transient, trending to control levelsfrom Week 33 (Fig. 2 and Fig. 1B). Oral dosing of lentinanpreviously demonstrated modulated systemic immune functionpreclinically (45). However, continued and prolonged adminis-tration resulted in diminished immune effects, suggesting tol-erance possibly through downregulation of pattern recognitionreceptors (46).

Analysis of survival curves revealed that dosing BNML an-imals with lentinan at 0.5, 12, or 20 mg/kg had no statisticalimpact on survival (Table 4). Furthermore, pretreating rats toreceive BNML with lentinan at the doses employed did notincrease survival. Combination of lentinan with “standards ofcare” in AML therapy, idarubicin and cytarabine, again did notyield a statistical significance in survival over idarubicin or cy-tarabine alone. However, it must be noted that the combinationof lentinan with idarubicin did increase the survival of this group

by 1.5 days over idarubicin-treated animals, whereas BNML rats(which are normally lethargic and cachexic) were less so whentreated with lentinan (Fig. 3). In particular, anthracyclin-treatedcells are exceptionally effective in eliciting an anticancer T-cellinvolved immune response via rapid, preapoptotic transloca-tions of calreticulin to the cell surface (47), possibly explainingthe increased survival and limited cachexia in this group.

In conclusion, lentinan supplementation to the diets ofhealthy and leukemic BN rats results in distinct immunologicalresponses, pointing toward enhanced T-cell function. Nutritionaldoses of lentinan, in cotreatment with conventional chemother-apy, intimate beneficial results in this preclinical model of AML,with no negative effects observed. Thus, these results suggestthat further evaluation of lentinan as a nutritional supplement topatients undergoing intensive chemotherapy should be explored.

ACKNOWLEDGMENTSThis work was supported by Medimush AS, Hørshold, Den-

mark, Helse Vest (grant numbers 911388 and 911182 to E.McCormack). B. T. Gjertsen and Ø. Bruserud were supportedby the Norwegian Cancer Society. The authors wish to thankthe following for their invaluable contribution to the productionof this article: Maren Boge, Kjetil Jacobsen, Lars Erik Pindard,Henriette Jensen, and Marianne Enger.

REFERENCES1. Mantovani MS, Bellini MF, Angeli JP, Oliveira RJ, Silva AF, et al.: Beta-

glucans in promoting health: prevention against mutation and cancer. MutatRes 658, 154–161, 2008.

2. Sullivan R, Smith JE, and Rowan NJ: Medicinal mushrooms and cancertherapy: translating a traditional practice into Western medicine. PerspectBiol Med 49, 159–170, 2006.

3. Cheung NK, Modak S, Vickers A, and Knuckles B: Orally administeredbeta-glucans enhance anti-tumor effects of monoclonal antibodies. CancerImmunol Immunother 51, 557–564, 2002.

4. Hong F, Hansen RD, Yan J, Allendorf DJ, Baran JT, et al.: Beta-glucanfunctions as an adjuvant for monoclonal antibody immunotherapy by re-cruiting tumoricidal granulocytes as killer cells. Cancer Res 63, 9023–9031,2003.

5. Hong F, Yan J, Baran JT, Allendorf DJ, Hansen RD, et al.: Mechanism bywhich orally administered beta-1,3-glucans enhance the tumoricidal activityof antitumor monoclonal antibodies in murine tumor models. J Immunol173, 797–806, 2004.

6. Mushiake H, Tsunoda T, Nukatsuka M, Shimao K, Fukushima M, et al.:Dendritic cells might be one of key factors for eliciting antitumor effect bychemoimmunotherapy in vivo. Cancer Immunol Immunother 54, 120–128,2005.

7. Tohamy AA, El-Ghor AA, El-Nahas SM, Noshy MM: Beta-glucan inhibitsthe genotoxicity of cyclophosphamide, adriamycin, and cisplatin. MutatRes 541, 45–53, 2003.

8. Nakano H, Namatame K, Nemoto H, Motohashi H, Nishiyama K, et al.: Amulti-institutional prospective study of lentinan in advanced gastric cancerpatients with unresectable and recurrent diseases: effect on prolongation ofsurvival and improvement of quality of life. Kanagawa Lentinan ResearchGroup. Hepatogastroenterology 46, 2662–2668, 1999.

9. Munemoto Y, Iida Y, Abe J, Saito H, Fujisawa K, et al.: Significanceof postoperative adjuvant immunochemotherapy after curative resection of

Dow

nloa

ded

by [

Uni

vers

itets

bibl

iote

ket i

Ber

gen]

at 1

0:10

28

Nov

embe

r 20

12

NUTRITIONAL LENTINAN IN EXPERIMENTAL ANTILEUKEMIC THERAPY 583

colorectal cancers: association between host or tumor factors and survival.Int J Oncol 20, 403–411, 2002.

10. Ito K, Nakazato H, Koike A, Takagi H, Saji S, et al.: Long-term effect of5-fluorouracil enhanced by intermittent administration of polysaccharide Kafter curative resection of colon cancer. A randomized controlled trial for7-year follow-up. Int J Colorectal Dis 19, 157–164, 2004.

11. Brown GD and Gordon S: Immune recognition of fungal beta-glucans. CellMicrobiol 7, 471–479, 2005.

12. Brown GD: Dectin-1: a signalling non-TLR pattern-recognition receptor.Nat Rev Immunol 6, 33–43, 2006.

13. Willment JA, Marshall AS, Reid DM, Williams DL, Wong SY, et al.: Thehuman beta-glucan receptor is widely expressed and functionally equivalentto murine Dectin-1 on primary cells. Eur J Immunol 35, 1539–1547, 2005.

14. Vetvicka V, Thornton BP and Ross GD: Soluble beta-glucan polysaccharidebinding to the lectin site of neutrophil or natural killer cell complementreceptor type 3 (CD11b/CD18) generates a primed state of the receptorcapable of mediating cytotoxicity of iC3b-opsonized target cells. J ClinInvest 98, 50–61, 1996.

15. Lowenberg B, Downing JR and Burnett A: Acute myeloid leukemia. N EnglJ Med 341, 1051–1062, 1999.

16. Tallman MS, Gilliland DG and Rowe JM: Drug therapy for acute myeloidleukemia. Blood 106, 1154–1163, 2005.

17. Abutalib SA and Tallman MS: Monoclonal antibodies for the treatment ofacute myeloid leukemia. Curr Pharm Biotechnol 7, 343–369, 2006.

18. Hatfield KJ, Olsnes AM, Gjertsen BT, and Bruserud O: Antiangiogenictherapy in acute myelogenous leukemia: targeting of vascular endothelialgrowth factor and interleukin 8 as possible antileukemic strategies. CurrCancer Drug Targets 5, 229–248, 2005.

19. Bruserud O and Wendelboe O: Biological treatment in acute myelogenousleukaemia: how should T-cell targeting immunotherapy be combined withintensive chemotherapy? Expert Opin Biol Ther 1, 1005–1016, 2001.

20. Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, and Dick JE: Targeting ofCD44 eradicates human acute myeloid leukemic stem cells. Nat Med 12,1167–1174, 2006.

21. Tavor S, Petit I, Porozov S, Avigdor A, Dar A, et al.: CXCR4 regulatesmigration and development of human acute myelogenous leukemia stemcells in transplanted NOD/SCID mice. Cancer Res 64, 2817–2824, 2004.

22. Weigel BJ, Panoskaltsis-Mortari A, Diers M, Garcia M, Lees C, et al.: Den-dritic cells pulsed or fused with AML cellular antigen provide comparablein vivo antitumor protective responses. Exp Hematol 34, 1403–1412, 2006.

23. McCormack E, Bruserud O, and Gjertsen BT: Animal models of acutemyelogenous leukaemia—development, application, and future perspec-tives. Leukemia 19, 687–706, 2005.

24. Gausdal G, Gjertsen BT, McCormack E, Van Damme P, Hovland R, et al.:Abolition of stress-induced protein synthesis sensitizes leukemia cells toanthracycline-induced death. Blood 111, 2866–2877, 2008.

25. Hubeek I, Comijn EM, Van Der Wilt CL, Merriman RL, Padron JM, et al.:CI-994 (N-acetyl-dinaline) in combination with conventional anti-canceragents is effective against acute myeloid leukemia in vitro and in vivo.Oncol Rep 19, 1517–1523, 2008.

26. Carella AM, Carlier P, Pungolino E, Resegotti L, Liso V, et al.: Idarubicin incombination with intermediate-dose cytarabine and VP-16 in the treatmentof refractory or rapidly relapsed patients with acute myeloid leukemia. TheGIMEMA Cooperative Group. Leukemia 7, 196–199, 1993.

27. Reagan-Shaw S, Nihal M, and Ahmad N: Dose translation from animal tohuman studies revisited. FASEB J 22, 659–661, 2008.

28. Saoudi A, Bernard I, Hoedemaekers A, Cautain B, Martinez K, et al.:Experimental autoimmune myasthenia gravis may occur in the context ofa polarized Th1- or Th2-type immune response in rats. J Immunol 162,7189–7197, 1999.

29. Deng G, Lin H, Seidman A, Fornier M, D’Andrea G, et al.: A phase I/II trialof a polysaccharide extract from Grifola frondosa (Maitake mushroom) in

breast cancer patients: immunological effects. J Cancer Res Clin Oncol135, 1215–1221, 2009.

30. Spierings EL, Fujii H, Sun B, and Walshe T: A Phase I study of the safetyof the nutritional supplement, active hexose correlated compound, AHCC,in healthy volunteers. J Nutr Sci Vitaminol (Tokyo) 53, 536–539, 2007.

31. Chan L, Hardwick NR, Guinn BA, Darling D, Gaken J, et al.: An immuneedited tumour versus a tumour edited immune system: prospects for immunetherapy of acute myeloid leukaemia. Cancer Immunol Immunother 55,1017–1024, 2006.

32. Akimoto M, Ueki H, Nakajima Y, Matano S, Ewasaki H, et al.: Preventionof 5-FU induced toxicity in C3 H/HE mice with interferon or with inter-feron inducers (poly 1: C, OK-432, Lentinan). Gan To Kagaku Ryoho 11,1462–1467, 1984.

33. Tamura R, Tanebe K, Kawanishi C, Torii K, and Ono T: Effects of lentinanon abnormal ingestive behaviors induced by tumor necrosis factor. PhysiolBehav 61, 399–410, 1997.

34. Iversen PO and Wiig H: Tumor necrosis factor alpha and adiponectin inbone marrow interstitial fluid from patients with acute myeloid leukemiainhibit normal hematopoiesis. Clin Cancer Res 11, 6793–6799, 2005.

35. Kurzrock R: The role of cytokines in cancer-related fatigue. Cancer 92,1684–1688, 2001.

36. De Benedetti F, Meazza C, and Martini A: Role of interleukin-6 in growthfailure: an animal model. Horm Res 58(1 Suppl), 24–27, 2002.

37. Patchen ML, Vaudrain T, Correira H, Martin T, and Reese D: In vitro andin vivo hematopoietic activities of Betafectin PGG-glucan. Exp Hematol26, 1247–1254, 1998.

38. Cramer DE, Allendorf DJ, Baran JT, Hansen R, Marroquin J, et al.: Beta-glucan enhances complement-mediated hematopoietic recovery after bonemarrow injury. Blood 107, 835–840, 2006.

39. Chae BJ, Lohakare JD, Moon WK, Lee SL, Park YH, et al.: Effects ofsupplementation of beta-glucan on the growth performance and immunityin broilers. Res Vet Sci 80, 291–298, 2006.

40. Suzuki M, Kikuchi T, Takatsuki F, and Hamuro J: Curative effects ofcombination therapy with lentinan and interleukin-2 against establishedmurine tumors, and the role of CD8-positive T cells. Cancer ImmunolImmunother 38, 1–8, 1994.

41. Matsuoka H, Seo Y, Wakasugi H, Saito T, and Tomoda H: Lentinan poten-tiates immunity and prolongs the survival time of some patients. AnticancerRes 17, 2751–2755, 1997.

42. Matsuoka H, Yano K, Seo Y, Saito T, Tomoda H, et al.: Usefulness oflymphocyte subset change as an indicator for predicting survival time andeffectiveness of treatment with the immunopotentiator lentinan. AnticancerRes 15, 2291–2296, 1995.

43. Damoiseaux JG, Cautain B, Bernard I, Mas M, van Breda Vriesman PJ,et al.: A dominant role for the thymus and MHC genes in determining theperipheral CD4/CD8 T cell ratio in the rat. J Immunol 163, 2983–2989,1999.

44. Murata Y, Shimamura T, Tagami T, Takatsuki F, and Hamuro J: The skew-ing to Th1 induced by lentinan is directed through the distinctive cytokineproduction by macrophages with elevated intracellular glutathione content.Int Immunopharmacol 2, 673–689, 2002.

45. Hanaue H, Tokuda Y, Machimura T, Kamijoh A, Kondo Y, et al.: Effectsof oral lentinan on T-cell subsets in peripheral venous blood. Clin Ther 11,614–622, 1989.

46. Hong M, Ryan KR, Arkwright PD, Gennery AR, Costigan C, et al.: Patternrecognition receptor expression is not impaired in patients with chronicmucocutanous candidiasis with or without autoimmune polyendocrinopa-thy candidiasis ectodermal dystrophy. Clin Exp Immunol 156, 40–51,2009.

47. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, et al.: Calreti-culin exposure dictates the immunogenicity of cancer cell death. Nat Med13, 54–61, 2007.

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