Studies on the in Vitro Antitumor Activity of Fatty Acids

I . 1O-.Hydroxy-2--decenoic Acid from Royal Jelly*

GORDON F. TOWNSEND, JOSEPH F. MORGAN, SUSAN TOLNAI, BARBARA HAZLETF,

HELEN J. MORTON, AND R. W. SHUEL

(Department of Apiculture, Ontario Agricultural College, Guelph; Laboralory of Hygiene, Department of National Health andWelfare, Ottawa; and Department of Therapeutics, Univer@rityof Toronio, Toronto, Canada)

SUMMARY

Whole royal jelly, when premixed with tumor cells before inoculation, has beenshown to inhibit completely the development of transplantable AKR leukemia and ofthree lines of mouse ascitic tumors. Fractionation studies have established that all, ornearly all, of the in vitro antitumor activity of whole royal jelly can be accounted for inthe major component of the ether-soluble fatty acid fraction, 10-hydroxy-2-decenoicacid. Concentration studies have shown that 1.5 mg. of 10-hydroxy-2-decenoic acid or40.0 mg. whole royal jelly per ml. of cell suspension completely prevented tumor formation with all four types of tumors. The in vitro antitumor activity has been shown to bedemonstrable only at low pH values. No activity with either compound could be shownabove pH 5.6. Time studies have shown that the reaction between the tumor cells and10-hydroxy-2-decenoic acid was completed within 6 minutes. Some variations in susceptibility to royal jelly or 10-hydroxy-2-decenoic acid have been demonstrated within thefour test tumors.

Royal jelly is a thick milky material secretedby the pharyngeal glands of young worker beeswhich, when fed as the sole nutrient to larvae,causes the development of sexually mature queenbees. The unusual nature of this material hasprompted many investigations into its pharmacological properties (7), but no direct evidencefor specific activity has yet been obtained. Aspart of a long-term study on the chemistry andbiological activity of royaljelly (15), an investigation of its possible antitumor properties has beencarried out (16). It is the purpose of the presentcommunication to report that admixture of royaljelly with tumor cells before inoculation completelysuppressed the development of a transplantablemouse leukemia and the formation of ascitic tumors in mice. Fractionation studies establishedthat this in vitro antitumor activity was associatedwith the main fatty acid component of royal jelly,10-hydroxy-2-decenoic acid. The pH of the reaction mixture was shown to be a determiningfactor in promoting the antitumor activity invitro.

I Presented in part at the annual meeting of the American

Association for Cancer Research, Inc., Atlantic City, New Jersey, April 10—12,1959.

Received for publication November 4, 1959.

MATERIALS AND METHODSMouse leukemia studies.—Antileukemia studies

were carried out by an early method of Burchenaland associates (3), using female mice, 5—6weeksof age, of the AKR strain.' Cells were suspendedin Gey's balanced salt solution (5) by mincingthe spleen with a ground glass homogenizer. Thesuspension was then filtered by gentle suctionthrough cotton and diluted to the approximatecell concentration required. The first transfer wasmade from a mouse dying from spontaneous leukemia, and successive transfers were made as themice showed signs of dying from leukemia. Tothese cell suspensions were added whole royal jellyor various fractions of royal jelly. In pH experiments, the cell suspensions in Gey's solution wereadjusted to acid ranges with 0.1 M citric or phosphone acid. The pH of each mixture was measuredwith a Beckman pH meter. The mixture was thenshaken and injected subcutaneously into the rightside at a dose of 0.2 ml/mouse. Each mouse received 2—5million tumor cells, as measured by

1 The AKR and CSH mice used in these experiments were

purchased from the Roseoe B. Jackson Memorial Laboratory,Bar Harbor, Maine. The Connaught mice were purchased fromthe Connaught Medical Research Laboratories, University ofToronto.

503

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504 Cancer Research Vol. 20, May 1960

direct count in a hemocytometer. The numberof cells administered per mouse was constant within any one experiment. Injections were normallycompleted within 2 minutes of preparing the testmixtures. Control mice received either tumor cellsalone or royal jelly alone at the experimentallevels.

The criterion used in these experiments wassurvival : the mice either developed leukemia orwere protected. Each dead mouse was examinedgrossly ; sections were then prepared from theliver and spleen to determine the extent of leukemia infiltration. First transfers from mice withspontaneous leukemia caused death in approximately 50 days, but successive transfers causeddeath more rapidly, in some cases within 14 days.The experiments were considered terminated atthe end of 90 days in order to distinguish betweentransplanted leukemia and spontaneous leukemia.In most cases the mice actually lived more than150 days.

Ascitic tunwr studies.—In vitro antitumor studies were conducted in three lines of ascitic tumors:the 6CSHED lymphosarcoma, the TAS mammarycarcinoma, and the Ehrlich carcinoma. Initially,these tumors were carried in the tumor-specificmouse strains, but in later experiments all threetumors were carried in the nonspecific Connaughtmouse line, following the freezing procedure ofMorgan, Guerin, and Morton (9). An exceptionto this procedure was found desirable with the6CSHED lymphosarcoma. Routine serial passagewas carried out in CSH mice, but the experimentswere performed in Connaught mice. Careful comparisons showed that results with the 6C3HEDcells were similar in CSH and Connaught mice.The stock tumors were carried by routine serialpassage at weekly intervals. Ascitic cells wereharvested by intraperitoneal puncture 7 days afterinoculation. The cells and accompanying fluidwere mixed with two volumes of deionized waterto lyse any red cells present, and the mixture wascentrifuged at 2000 r.p.m. for 3 minutes. Thesupernatant fluid was decanted and discarded,and the packed tumor cells were resuspended inHanks' balanced salt solution (6) to the desiredcell count, as measured by a hemocytometer. Thissuspension was mixed with appropriate amountsof whole royaljelly or fraction of royaljelly, and thepH was determined with a Beckman pH meter.The reaction mixture was then administered intraperitoneally to groups of at least ten mice in0.5-mi. doses. Each mouse normally received 5—8million tumor cells. Inoculation of the test animalswas completed within 30 minutes of preparing

the reaction mixtures. Male mice, 20—22gm. inweight, were used throughout these studies.

As in the antileukemia experiments, the critenon was survival : control mice died from ascitictumors in less than 14 days, while mice receivingappropriate mixtures of cells and royal jelly failedto develop tumors. One hundred per cent deathof the control mice in approximately 14 days wasobtained consistently with all three ascitic tumors.Mice protected by royal jelly were kept underobservation for 90 days from the start of theexperiment. This period was found necessary todistinguish between compounds which protectedthe mice completely and those which affordedonly partial protection by slowing the rate oftumor development. At the end of the 90-dayperiod, the experiments were terminated, and random mice were autopsied to confirm the absenceof tumors. In one case, protected mice were keptunder observation for 15 months and showed notumors at the end of this period.

Royal jelly.—The royal jelly used in these studies was collected at the Department of Apicultureof the Ontario Agricultural College. To supplythe amounts required, special methods for largescale production were devised (14). The antitumoractivity in vitro was found to be approximatelyequivalent in lots produced over a 3-year period.The royal jelly was stored in a deep freeze imniediately after production, and small portions werethawed shortly before use. Fractionation of freshroyal jelly was carried out by the procedures ofAbbott,2 whereas lyophilized royal jelly was fractionated by the methods of Townsend and Lucas(15).

RESULTS

In vitro an.titumor activity of whole royal jelly.—Initial experiments indicated that admixture ofwhole royal jelly with tumor cells prior to inoculation completely prevented the development oftransplanted mouse leukemia or ascitic tumors.To determine the minimal effective concentration,graded amounts of royal jelly were mixed withtumor cells and aliquots of the mixtures inoculatedinto groups of mice. Typical data from theseexperiments are presented in Tables 1 and 2.

It is evident (Table 1) that concentrations ofwhole royal jelly equal to or greater than 30.0mg/nil of cell suspension have completely prevented development of the transplantable leukemia. In the case of the 6C3HED ascitic tumor(Table 2), concentrations of whole royal jellygreater than 40.0 mg/mI of cell suspension have

I Ø@D. Abbott. Royal Jelly—unpublished data, College ofAgriculture, University of Florida, Gainesville, Florida.

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ROYAL JELLYAVERAGE

SURVIVAL

(DAYS)No.

SURVIVORSAT90DAYS(mg/mi

cellsusp@ension)Test mixtureCellcontrol20

406080

10019t

co:t

a@m13

131313124/9

10/108/810/1047/47

ROYAL JELLYAVERAGE

SURVIVAL

(DAYS). No.SURVIVORS AT

90DAYS(mg/micell

suspension)Test mixtureCellcontrol755

1550305045.0605080.023

21cut21

21214848250/7

0/77/75/55/5

19/19

TOWNSEND et al.—AntiturnorActivity of 1O-Hydroxy-2-decenoic Acid 505

compound were isolated from royal jelly,3 andthe activity was tested against ascitic tumors andtransplantable leukemia. The results of these experiments are summarized in Table 3.

It is apparent (Table 3) that 1.5 mg. of 10-hydroxy-2-decenoic acid per ml. of cell suspension

TABLE 2

EFFECT OF GRADED LEvsLs OF WHOLE RoYAL

JELLY WHEN MIXED WITH 6CSHEDLYMPHOSARCOMA CsiLs PRIOR TO INocu

LATION INTO MIcE1

completely prevented development of the tumor.Half this concentration has afforded partial protection, while smaller quantities were found tohave no demonstrable effect. Similar studies withthe Ehrlich and TA3 tumors showed that thissame concentration (40.0 mg/nil of cell suspension)was completely protective in both cases.. Isolation ofactive component of royal jelly.—W ith

the in vilro antituinor activity of whole royaljelly established, attention was directed towardisolating the active component. Royal jelly wasfractionated as described previously (15), andthe activity of each fraction was tested against6CSHED cells at concentrations equal to, above,and below the level normally present in whole

S TABLE 1

EFFEcT OF GRADED L1v@s oi@WHOLE RoYAi JEuxWREN MIXED WITH LEUKEMIC CELLs PRIOR TO

INocuLATIoN INTO MIcE1

I Each mouse received 5—8million tumor cellseither alone (cell clrntrol) or mixed with royaljelly (test mixture).

t Calculatedfromtimesof death offive out ofnine mice which failed to survive.

@ Represents survival beyond the 90-day testperiod.

4/ERPGE SURViVALINI@YS0 30 60 90

CELLCONTROL

CHART 1.—In vUro antitumor activity of whole royal jellyand its component fractions when mixed with 6C3HED lyrephosarcoma cells prior to inoculation into mice. Results expressed as average survival time in days of groups of ten to 24mice in each test mixture. All fractions were tested at concentrations equivalent to those occurring in fresh whole royaljelly.

I The 10-hydroxy-2-decenoic acid used in these experimentswas isolated and purified by Dr. W. H. Brown, of the Department of Chemistry, Ontario Agricultural College, whose cooperation is gratefully acknowledged.

I Each mouse received 2—5 million leukemiccells either alone (cell control) or mixed withroyal jelly (test mixture).

t Represents survival beyond the 00-daytestperiod.

royal jelly. The results of these experiments aresummarized in Chart 1. It is evident that thein vitro antitumor activity of the whole royaljelly was contained almost entirely in the ethersoluble acid fraction.

Additional experiments were carried out inwhich royal jelly was lyophilized and the drypowder sterilized by treatment with ethylene oxide. This material, after reconstitution in deionizedwater, proved fully as active as the untreatedmaterial.

In vitro antitumor activity of 1O-hydroxy4?-deceiwic acid.—The major component of the ethersoluble fatty acid fraction of royal jelly was originally isolated by Townsend and Lucas (15) andwas later identified by Butenandt and Rembold(4) as 10-hydroxy-2-decenoic acid, while confirmation of the structure has been reported recently(1). Accordingly, considerable quantities of this

F@DVALJELLY

@rER-SOLU8LENEUTRAL

@@TER-SOWBLEACID

PROTEIN

ETHER-W6@TERSOLUBLE

ErHER-SOLUOLENEUTRAL

@HER-SCLUBLEACID

Research. on December 14, 2020. © 1960 American Association for Cancercancerres.aacrjournals.org Downloaded from

rHAVERAGE

SURVIVAL

(DAYS)

Test ControlNo.

SURVIVORS

AT 90DAYS4.2

4.75.66.16.57.3cut

21312324

242422242410/10

10/1010/100/60/100/10

1O-IFrnRoxTDECENOIC ACID

(mg/mi cellsuspension)AVF.RAGR

SURVIVAL

(DAYS)

Test Cellmixture controlNo.

SURVIVORS AT

90DAYS0.5

1.01.52.0

0.61.02.0Le

4569cDt

SC

231:

mukewic

cc

54545454

SHED cell

121213us:

0/50/55/5

20/20

s:

1/1010/1015/15

@HAVERAGE

SURVIVAL

(DAYS)

Test ControlNo.

SURVIVORS

AT 90DAYS3.6

4.04.55.25.96.9@cnt

24111716

1612

8121310/10

9/910/100/150/100/10

506 Cancer Research

has completely prevented the development oftransplantable leukemia in AKR mice. With the6C3HED ascitic tumor, a level of 0.6 mg/mlof cell suspension showed slight activity, while1.0 mg/mi conferred complete protection. Similarresults were obtained with the Ehrlich carcinomaand the TA3 mammary carcinoma. Since wholeroyal jelly contains approximately 3 per centof 10-hydroxy-2-decenoic acid, on a wet weightbasis (15), the activity of this component accountsalmost completely for the activity of the wholeroyal jelly.

TABLE 3

EFFECT OF GRADED LEVELS OF lO-HYDROXYDECENOIC AcID WnEN MIXED WITH LEuKEMIC Cm@s OR 6C3HED LYMPH0SA1t-COMACELI5SPRIOR TO INOCULATION INTOMICE

cell suspensions, and aliquots of the mixtureswere adjusted to different pH values with either0.1 N HCI or 0.1 N NaOH. Cell controls, without10-hydroxy-2-decenoic acid, were also brought tothe same pH values with either 0.1 N HCI or0.1 N NaOH. Groups of mice were then giveninoculations of portions of the mixtures in theusual manner, and the survival times of the mice

were recorded. The results of these experimentsare summarized in Tables 4 and 5.

It is evident (Tables 4 and 5) that the protectiveeffect of 10-hydroxy-2-decenoic acid was exerted

TABLE 4

EFFECT OF PH ON THE ACTIVITY OF lO-HYDROXYDECENOIC ACID AGAINST TRANS.

PLANTABLE LEUKEMIC CEus1

I All reaction mixtures contained 2.0 mg. 10.hydroxydecenoic acid per ml. cell suspension.Each mouse received subcutaneously 0.2 ml. ofmixture containing 2—Smillion leukemic cells.

t Represents survival beyond the 90-day testperiod.

TABLE 5

EFFECT OF PH ON THE AcTWITY OF 10.Hy.DROXYDECENOIC ACID AGAINST 6C3B.ED

LYMPHOSARCOMA CELLS1

I Each mouse received 2—5 million leukemic

cells or 5-8 million lymphosarcoma cells eitheralone (cell control) or mixed with lO-hydroxydecenoic acid (test mixture).

t Represents survival beyond the 90-daytestperiod.

@ Calculated from the times of death of nineout of ten mice which failed to survive.

pH Dependency of antitumor activity in vitro.—The tumor cell suspensions used in the presentstudies were prepared in either Gey's or Hanks'balanced salt solutions (5, 6), which are bufferedwith phosphate and bicarbonate to pH 7.2. Theaddition of royal jelly or 10-hydroxy-2-decenoicacid caused a sharp drop in the pH of the reactionmixtures, as shown by the yellow color of thephenol red indicator. Accordingly, it became ofinterest to determine the relationship betweenpH and biological activity. A level of lO-hydroxy2-decenoic acid previously shown to be effectiveagainst transplantable leukemia and 6C3IIEDlymphosarcoma cells was mixed with the tumor

I All reaction mixtures contained 1.0 mg. 10-hydroxydecenoic acid per ml. of cell suspension.Each mouse received intraperitoneally 0.5 ml. ofmixture containing 5—8million tumor cells.

t Represents survival beyond the 90-day testperiod.

@ At this pH, the concentration of 10.hydroxydecenoic acid was doubled to detect possible small effects.

Vol.20,May@

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TreatmentAverage.

survival

(days).

No. survivorsat 90daysCell

control(untreated 6CSHEDcells)120/10Reaction

mixture (6CSHED cells plus1.0 mg. lO-hydroxydecenoic acid perml. cellsuspension)I9/9Aliquot

removed 3 minutes after mixing, centrifuged 3 minutes. Packedcells washed twice in Hacks' solution, pH 7.2, resuspended in Hanks'saline, and injected intomice.10/10Supernatant

from previous step, fil.tered, untreated 6CSHED cellsadded, and injected into mice.260/10

TOWNSEND et al.—Antitumor Acticity of 1O-Hydro@y-2-decenoie Acid 507

only at low pH values. With transplantable leukemia cells, no protection was afforded at pHvalues above 5.6. With the 6C3HED lymphosarcoma cells, the protective effect was exertedat pH values of 4.5 and lower. At pH 5.2, someretardation of the rate of tumor formation wasobserved, but none of the test mice survived.At pH values higher than 5.2, no protective effectwhatever was obtained. Even double the normallyeffective concentration of 10-hydroxy-2-decenoicacid was completely ineffective at neutrality. Similar experiments with whole royal jelly showedthat this material, too, was active only at pHvalues below 5.0. The pH relationships with theEhrlich and TAB ascitic tumors were found tobe identical with those of the 6C3HED lymphosarcoma.

In all cases, control studies were performedin which cell suspensions without 10-hydroxy-2-decenoic acid were adjusted to pH values corresponding to those of the test mixtures. Thesecell suspensions, at pH values ranging from 6.9to 3.6, invariably caused death of inoculated micewithin the normal 14-day period. These controlexperiments established that a low pH, by itself,did not interfere with the tumor-inducing capacityof the cell suspensions. The protective effect ofthe test mixtures, accordingly, could not be attributed to cell damage under the acid conditionsof the test but must be related to an activityof 10-hydroxy-2-decenoic acid itself.

Time relationships of antitumor activity in vitro.—The early experiments were carried out withsmall numbers of mice and were completed within10 minutes of mixing the tumor cells and royaljelly. With more complex experiments involvinggreater numbers of mice, the time intervals between preparing the test mixtures and completingthe inoculations became progressively longer. Itbecame necessary, accordingly, to determine thetime relationships of the antitumor activity invitro. To this end, a reaction mixture was prepared,containing 1.0 mg. of 10-hydroxy-2-decenoic acidper ml. of 6C3IIED cell suspension. Three minutesafter preparation of the mixture, an aliquot wasremoved and immediately centrifuged for 3 mmutes at 2000 r.p.m. The packed cells were washedtwice in Hanks' solution at pH 7.2, resuspendedin fresh Hanks' solution to the original cell count,and injected into a group of ten mice. To thesupernatant fluid removed from the packed cellswere added fresh untreated 6CSHED cells tothe original cell count, and the mixture was injected into mice. The results of this experiment,with the appropriate controls, are shown in Table 6.

It can be seen (Table 6) that the 6CSHEDcells exposed to 10-hydroxy-2-decenoic for 8—6

minutes were unable to form tumors on subsequent

inoculation into mice. The supernatant fluid fromthese cells showed only slight activity when mixedwith fresh untreated 6C8HED cells. It appeared,therefore, that the reaction between the tumorcells and the 10-hydroxy-2-decenoic acid was es

sentially complete within 3-6 minutes and that

the greater part of the 10-hydroxy-2-decenoic acidhad disappeared from solution within that time.Three repetitions of this experiment yielded identical results.

In additional experiments, the reaction mixturewas held at room temperature for 60 minutes,

TABLE 6

ACTIvITY OF VARIous COMPONENTS OF TEST SYSTEMWm@N SEPARATED 6 MINUTES AFTER ADMIXTURE OF

6CSHED CELLSAND10-HYDROXYDECENOICACID

S Represents survival beyond the 90-day test period.

aliquots were then removed and separated as

before into packed cells and supernatant fluidfor inoculation into mice. The results of these

experiments were in complete agreement with theprevious ones and established that the reactionbetween the tumor cells and 10-hydroxy-2-decenoic was as complete within 6 minutes as in 60minutes. Further experiments were carried outin which the pH of the reaction mixture was raisedfrom 4.2 to 7.8 with 0.1 N NaOH within 1 minuteof mixing the tumor cells and the fatty acid. Thismixture was found to be completely ineffectivein preventing tumor formation on subsequent inoculation of mice. From these results, it appearedthat a time interval of from 1 to 6 minutesis required to complete the reaction between the6C3HED tumor cells and 10-hydroxy-2-decenoicacid.

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TUMOR1O-HyDaoxyDECENOICACID (Mo/ML)Avxa&oa

SURVIVAL(DAYS)

Test ControlNo.

SURVIVORSAT 90DAYSTransplantable

leukemia(AKR)

@CSHED

Ehrlich

TA31

.0I .5

0.61.0

1.02.0

1.02.069

co@

23m

25

32m54

54

1212

1111

15150/5

5/5

1/1010/10

2/1010/10

2/1010/10

508 Cancer Research Vol.20,May 1960

Variation in tumor susce'ptibihty.—The markedeffectiveness of royal jelly and 10-hydroxy-2-decenoic acid against the 6C3HED lymphosarcomamade it of interest to determine whether differences in susceptibility existed between the severaltumors available for study. Accordingly, gradedlevels of 10-hydroxy-2-decenoic acid were testedagainst the various tumors, with the results suminarized in Table 7.

It is evident (Table 7) that slightly lower concentrations of 10-hydroxy-2-decenoic acid arerequired to inhibit the transplantable leukemiaand the lymphosarcoma than in the case of theother two tumors. Similar experiments with whole

TABLE 7

DIFFERENCES IN SUSCEPTIBILITY OF EXPERIMENTALTUMORS TO 10-HYDROXYDECENOIC ACID1

different laboratories over a 3-year period, inexperiments involving several thousand mice andfour different experimental tumors.

The demonstration that nearly all, if not all,of the in vitro antitumor activity of whole royaljelly can be attributed to 10-hydroxy-%-decenoicacid is of interest in view of the recent reportthat this compound exhibits antibiotic activityagainst a range of bacteria and fungi (2). Thepresent studies have shown that this fatty acidis active against tumor cells only under acidconditions, and this suggests that only the undissociated fatty acid molecule is effective. Thereis no evidence at the present time that the invitro antitumor activity of 10-hydroxy-%-decenoicacid bears any relationship to the morphogeniceffect of royal jelly on bee larvae.

The mechanism by which 10-hydroxy-2-decenoic acid inhibits tumor formation has not yetbeen elucidated. Time studies have shown thatthe reaction between the tumor cells and thefatty acid is complete within 3—6minutes, but isnot complete after I minute. These studies haveshown that the fatty acid disappears from solutionrelatively quickly in the presence of tumor cellsbut do not distinguish between simple adsorptionon the cell surface and actual penetration withinthe tumor cells. It was found, however, thatwashing inhibited cells with buffer at neutralpH did not restore the tumor-incuding capacityof these cells, whereas raising the pH of the reactionmixture to neutrality prevented the activity of10-hydroxy-2-decenoic acid in solution. From theseobservations, it appears most probable that the10-hydroxy-2-decenoic acid either is firmly boundto the cell surface or has actually penetratedwithin the tumor cells. Studies with chromatography and dye exclusion methods are now in progressto investigate these possibilities.

The highly acid conditions required to demonstrate the activity of 10-hydroxy-2-decenoic acidhave made it difficult to study the action of thiscompound on normal and malignant cells in tissueculture. The observation that the reaction betweenthe fatty acid and the tumor cells is completedwithin a few minutes, however, makes possiblea study of the effects of short-term exposureof tissue cultures to the active compound. Thesestudies are now in progress and will form the basisof a subsequent communication. Tests with wholeroyal jelly have shown that this material is relatively nontoxic on intraperitoneal inoculation intomice. From 125 to 250 mg. per mouse was requiredto cause a toxic reaction, an amount 6—12 times

that normally administered in the antitumor experiments. While it is generally recognized that

S Tumor cells mixed with specified concentrations of 10-liydroxydecenoic acid at pH 4.5, and aliquots containing 5—8million tumor cells inoculated into mice.

t Represents survival beyond the 90-day test period.

royal jelly showed that a concentration of 40.0

mg/mi cell suspension was completely protectivewith all four tumors, but that the effectivenessof lower concentrations differed, depending uponthe tumor used. In general, the transplantableleukemia and the 6C3HED cells were most susceptible, followed by the Ehrlich carcinoma, whereasthe TA8 mammary carcinoma cells were somewhatmore resistant.

DISCUSSIONThe role of royal jelly in the development of the

queen bee has stimulated many investigationsinto its possible therapeutic use in a variety ofhuman diseases, including leukemia (17). In mostinstances, such reports have been incomplete and@difficultto assess. The present results appear tooffer the first unequivocal demonstration of anin vitro antitumor activity in royal jelly. Theseresults have been confirmed independently in two

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TOWNSEND et al.—Anti@umor Activiiy of 1O-Hydroxy-@-decenoic Acid 50@

many substances, such as alkylating agents, aretoxic to suspended neoplastic cells, these materialsalso exert an appreciable toxicity on the host animal. The relative nontoxicity of both royal jellyand 10-hydroxy-2-decenoic acid to the test miceis an important feature of the present experiments.

The four tumors used in these studies showedsome differences in susceptibility to both wholeroyal jelly and purified 10-hydroxy-2-decenoicacid. Thus, the AKR transplantable leukemia andthe 68CHED lymphosarcoma were relatively moresusceptible than were the Ehrlich carcinoma orthe TAS mammary carcinoma. With sufficientquantities of the antitumor agents, all four tumorscould be completely inhibited. It must be emphasized, however, that inhibition was obtained onlyof the transplantable AKR leukemia. Mice protected against this transplantable leukemia stilldeveloped spontaneous leukemia at the normalperiod of 10-12 months. Studies on transplantableleukemia are complicated by the possible involvement of viral agents. For this reason, each criticalexperiment was carried out on the three lines ofascitic tumors, as well as on the transplantableleukemia. In addition, all mice in the ascitic tumorexperiments that were protected by royal jellyor 10-hydroxy-2-decenoic acid have been held under observation for a 10—12-month period beyondthe 90-day experimental period. At the end ofthis time, random mice have been autopsied andan attempt made to demonstrate polyoma virusthrough inoculation of young mice with splenicextracts. Up to the present time, only negativeresults have been obtained.

While the present experiments have establishedthe in vitro antitumor activity of royal jelly and10-hydroxy-2-decenoic acid, they have also established two drawbacks against the therapeutic ap-.plication of these findings. The first drawback isthe necessity of premixing with the tumor cellsbefore inoculation into the test mice, since bothagents have so far proved ineffective in preventingtumor development if administered after the tumors have become established. The second drawback is the necessity of an acid pH to demonstratethe antitumor activity. Attempts to overcomeboth these objections are now in progress throughtests on other fatty acids. These studies, basedon 62 fatty acids of varying chain length and degree of unsaturation, have indicated that the abiity to inhibit ascites tumor cells in vitro is possessedby a wide range of compounds. The distributionof this activity and the relationship of chemicalstructure to antitumor activity in vitro will formthe basis for subsequent communications in thisseries.

The results presented in this communicationemphasize the possible importance of fatty acidsas antitumor agents. This idea is not entirelynew. In a series of papers published between 192@and 1925, Nakahara (10—iS) reported the valueof olive oil and of fatty acids in increasing theresistance of mice to several forms of transplantable tumors. Similar observations were reported(8) on the beneficial effect of fatty acids in retarding tumor development following application ofcoal tar derivatives to the skin of mice. The basicconcept in these early publications is essentiallysimilar to the present findings with royal jellyand 10-hydroxy-2-decenoic acid. It would appear,therefore, that the role of fatty acids as antitumoragents warrants intensive investigation.

ACKNOWLEDGMENTS

We wish to acknowledge our indebtedness to Mr. D. E.Ainley and Mr. R. Scantland, whose technical assistancehelped materially in the completion of these experiments.

We wish also to expressour appreciation to the PathologyDepartment of the Ontario Veterinary College, Guelph, forpreparation of theliver and spleen sections from the AKR mice.

REFERENCES

1. BARKER,S. A.; FosTan, A. B.; Lasre, D. C.; and HODOSON@N. Identification of 10-Hydroxy-@'-decenoic Acid inRoyal Jelly. Nature, 183:996—97, 1959.

2. BLUM, M. S.; Nov@tx, A. F.; and TABER, S. III. lO-Hydroxy-z@-Decenoic Acid, an Antibiotic Found in RoyalJelly.Science,130:452—53,1959.

S. BURCEENAL,J. H.; LxsTnn, R. A.; Ruzx, J. B.; andRao@us, C. P. Studies on the Chemotherapy of Leukemia.I. Effect of Certain Nitrogen Mustards and Carbamateson Transmitted Mouse Leukemia. Cancer, 1:399-412,1948.

4. BUTENANDT,A., and Rmmow, H. lYber den Weiselzellenpuffersalt der Honigbiene. I. Isolierung, Konstitutionsermittlung und Vorkommen der 10-HydroxydeeensUure.Hoppe-Seyler's Ztschr. f. Physiol. Chem., 308:284—89,.1957.

5. GEY, G. 0., and Gxr, M. K. The Maintenance of HumanNormal Cells and Tumor Cells in Continuous Culture. I.Preliminary Report: Cultivation of Mesoblastic Tumorsand Normal Tissue and Notes on Methods of Cultivation.Am. J. Cancer, 27:45—76,1936.

6. HANICS,J. H., and W@.u@cz, R. E. Relation of Oxygenand Temperature in the Preservation of Tissues by Refrigeration. Proc. Soc. Exper. Biol. & Med., 71:196—200,.1949.

7. JORANSSON, S. K. Royal Jelly. Bee World, 36:3—iS, 21-32,.1955.

8. LECLOUX, J. Recherches sur l'infiuence des graisses Sw lecancer au goudron de la souris. Compt. rend. Soc. de Biol.Med., 93:832—33,1925.

9. MORGAN,J. F.; Gumux, L. F.; and MouroN, H. J. TheEffect of Low Temperature and Storage on the Viabilityand Mouse Strain Specificityof AsciticTumor Cells.Can-.cerResearch,16:907—11,1956.

10. N@x@4n@tiu,W. Studies on Lymphoid Activity. VI. Immunity to Transplanted Cancer Induced by Injection ofOlive Oil. J. Exper. Med., 35:493-507, 1922.

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510 Cancer Research

11. NAKAHARA,W. Effect of Fatty Acids on the Resistance ofMice to Transplanted Cancer. Ibid., 40:363—73,1924.

12. ——. Resistance to Spontaneous Mouse Cancer Inducedby Injections of Oleic Acid. Ibid., 41:347—57,1925.

is.@-@. Production of Tumor Resistanceby Fatty Acids.Gann, 19:1—4,1925.

14. SMITH, M. V. Some Facts about Royal Jelly Production.Gleanings in Bee Culture, 82:713—15, 1954.

15. TOWNSEND, G. F., and Luc@ts, C. C. The Chemical Natureof Royal Jelly. Biochem. J., 34: 1155—62, 1940.

16. Towiismw, G. F.; MORGAN, J. F.; and HAZLE'Z'r,B. Activity of lO-Hydroxydecenoic Acid from Royal Jellyagainst Experimental Leukaemia and Ascitic Tumors. Nature, 183:1279-71, 1959.

17. WIu.aoN, R. B. Royal Jelly. A Review. Am. Bee J., 97:356—59,396—99,1957.

Vol. @0,May 1960

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1960;20:503-510. Cancer Res   Gordon F. Townsend, Joseph F. Morgan, Susan Tolnai, et al.   10-Hydroxy-2-decenoic Acid from Royal Jelly

Antitumor Activity of Fatty Acids: I.in vitroStudies on the

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