Tumor Inhibition by a Sulfhydryl-blocking Agent Relatedto an Active Principle of Garlic (Allium sativum)*

AUSTINS. WEISBERGERANDJACKPENSKY

(Department of Medicine, University Hospitals and School of Medicine, Western Resene University, Cleveland, Ohio)

This report deals with some anti-tumor effectsobserved with a series of compounds related to theactive principle of garlic (Allium sativum). A studyof these compounds was initiated because of theobservation that an active principle of garlic (al-lylthiosulfinic allyl ester, allicin) can inactivatemany sulfhydryl (-SH) enzymes and can react rapidly with cysteine (10, 43). Such compounds maybe of importance in relation to malignancies andleukemia because the availability of reduced -SHcompounds has often been implicated in the processes of cell growth and division. Observations supporting the importance of -SH compounds in theseprocesses include the following: (a) the demonstration of a high -SH content in proliferating tissues,(6) the increase in soluble thiols within the cellprior to cell division, and (c) the inhibition of celldivision by thiol poisons such as alkylating agentsand heavy metals. Substances which oxidize -SHcompounds (-SH >-S-S-) also may inhibitcell division, whereas reduced -SH compoundsmay stimulate cell growth and division. The inhibition of cell division by -SH poisons can in someinstances be reversed by such -SH compounds ascysteine, glutathione, or thioglycolate. These phenomena have been demonstrated in a wide varietyof plants, tissues, and organisms. The literaturepertaining to these studies has been extensively reviewed by Brächet(8), Contopolous and Anderson(11), and by Ban-on (2, 3).

Voegtlin (32) and Hammett (15) have suggestedthat sulfhydryl compounds are implicated in thegrowth of malignant cells. Tumor tissues havebeen found to contain a higher -SH concentrationthan normal tissues, and an increase in -SH content has been demonstrated during the growth oftumors in plants inoculated with a tumor-produc-

*This investigation was supported bi a research grant(Cy-1678-C6) from the National Cancer Institute, NationalInstitutes of Health, U.S. Public Health Service, and wasaided in part by the Frederick Sands Memorial Fund and theCuyahoga Chapter of the American Cancer Society.

A preliminary report of this study appeared in Science,126:1112, 1957.

Received for publication July 2, 1958.

ing microorganism (6). Low plasma -SH levelshave been found during the growth phase of malignant tumors, and this has been attributed to anincreased demand for -SH by the rapidly proliferating neoplasm (26, 32). The importance of-SH compounds in malignant growth is also suggested by the observation that a decreased availability of these compounds may result in decreasedtumor growth. Thus, diets deficient in the -SHamino acid L-cysteine can suppress malignantgrowth in some animals, whereas the addition ofeither cysteine or glutathione to the diet can resultin an increase in tumor growth (12, 31, 41).

The availability of sulfhydryl compounds hasalso been implicated in both normal and abnormalleukopoiesis. Substances which inactivate -SHgroups such as x-ray, nitrogen mustard, arsenic,gold, and benzol also tend to produce leukopenia.A high -SH content has been reported in severaltypes of leukemic cells by a number of investigators (5, 11, 23, 33); however, this observation hasnot been confirmed by others (17, 18). Low -SHlevels have been found in leukocytes in leukopenicstates (19, 22).

The -SH amino acid L-cysteine appears to be ofparticular importance in relation to leukocytes.Evidence supporting this hypothesis includes theobservation that L-cysteine partially prevents theleukopenia induced by nitrogen mustard and thatthis protective effect is distinctive in that certainstructural characteristics are required (34, 35, 37).Furthermore, marrows cultured on synthetic media deficient in either L-cysteine or L-cystine exhibit rapid degeneration of leukocytes. Addition ofeither of these amino acids to the growth mediumresults in protection of granulocytes (1). The effectof decreased availability of cysteine or cystine onleukocytes is also demonstrated by experiments onthe transplantation of leukemia in animals. It hasbeen shown that a lack of L-cystine in the diet reduces the incidence of transplantable leukemia inanimals, whereas addition of L-cystine increasesthe incidence (42). Similar reduction of other ami-no acids such as lysine or tryptophan has no effecton the incidence of transplantable leukemia. The

1301

Research. on September 13, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

1302 Cancer Research Vol. 18, December, 1958

importance of cysteine is also suggested by the observation that leukocytes from patients with acuteleukemia or chronic myeloid leukemia exhibit amuch more rapid turnover of this amino acid thando normal leukocytes (36, 39). This rapid turnoverof an amino acid by immature leukocytes is probably a manifestation of the increased metabolicdemands generally found in immature cells. However, in view of the other observations on the importance of L-cysteine and L-cystine in relation toleukocytes, the increased avidity of leukemic leukocytes for these amino acids may have added significance.

It is evident from these considerations that substances which inactivate -SH compounds or whichresult in a decreased availability of either L-cysteine or L-cystine might have important effectson leukocytes and that leukemic leukocytes, because of their rapid turnover of cysteine, might bemore susceptible to depletion of these amino acidsthan normal leukocytes. Such an approach wasattempted in previous experiments by replacingthe sulfur of cystine with selenium (diseleno-cystine, selenium cystine) (38, 40). This compounddecreases the influx of radioactive L-cystine intoleukemic leukocytes in vitro and was found to produce a fall in the leukocyte count in some patientswith leukemia (38) . Toxic manifestations and undesirable side reactions prevented an extensivestudy of its potential value. A further search forsubstances capable of inactivating -SH compounds and not as toxic as selenium cystine wastherefore undertaken.

Our interest in substances derived from garlic(allium sativum) was prompted by the observationof Wills (43) that an active principle of garlic inactivates many -SH enzymes without altering thereactivity of several non-SH-dependent enzymes.Extracts of garlic are known to contain a powerfulbactericidal agent, allylthiosulfinic allyl ester (al-licin) (10, 20). This compound is formed by the interaction of an enzyme and substrate present ingarlic bulbs (28, 29). The enzyme, alliinase, is liberated when the garlic bulb is crushed and acts onthe substrate, S-allyl L-cysteine sulfoxide (alliin)as follows (30) :

+H202R-SO-CH2-CH(NH2) COOH -rr - »r

I.

(Alliin) nnase)

R-SO-S-R + 2CH3-CO-COOH + 2NH3(Allicin)

(R=CH2-CH = CH2)

In studying the mechanism of the bactericidalaction of allicin, Wills (43) has shown that it is an-SH inhibitor and that there is a close relationship

between the structure required for bactericidal action and that required for -SH enzyme inactiva-tion. Thus, compounds containing the -SO-S-grouping are effective in both enzyme inhibitionand bactericidal activity, whereas compounds containing the -SO-, -S-S-, or -S- linkage are ineffective. It has been suggested that the -SH inactiva-tion obtained with the alkylthiosulfinic alkyl estermay be the result of a strong combination of thiscompound with cysteine or may be owing to oxidation of -SH to -S-S- by the labile oxygen (10).

Although most studies of allicin have centeredupon its bactericidal action, its reactivity with -SHgroups suggests that it might also have an inhibitory effect on malignant cells. Accordingly, the effect of an alkylthiosulfinic alkyl ester on thegrowth of malignant tumors in animals was investigated. Since the allyl ester of allylthiosulfinicacid which is ordinarily formed in garlic extracts isunstable, the more stable diethyl analog (C2Hs-SO-S-C2Hs) was used in the initial studies. Subsequently, methyl, propyl, and butyl analogs werestudied. This report deals with the effect of thesethiosulfinic acid esters upon the growth of Sarcoma180 tumors in mice.

MATERIALS AND METHODSPREPARATIONOF COMPOUNDS

Enzymatic production of ethylthiosulfinic ethyl ester.—Theenzyme alliinase was prepared from garlic bulbs by the methods described by Wills (43). The enzyme preparation wasstored at 0°C. and was active for at least 48 hours. The enzymeobtained from 25 gm. of garlic was dissolved in 35 ml. of 0.1 Mphosphate buffer, pH 7.O.

S-ethyl L-cysteine sulfoxide (C2HS-SO-CH(NH2)COOH)was prepared by oxidation of S-ethyl L-cysteine1with hydrogenperoxide; 2.57 gm. of S-ethyl L-cysteine was suspended in 20ml. of distilled water; 2.35 ml. of 30 per cent hydrogen peroxidewas added, and the mixture was apitated continuously for 18hours by means of a magnetic stirrer. The sulfoxide was precipitated from the solution by acetone and was recrystallizedfrom aqueous acetone.2 The product was cbromatographicallyhomogeneous and migrated at the same rate as a purifiedsample o S-ethyl L-cysteine sulfoxide3 when tert-butanol-formic acid and water (70:15:15) solvent with ninhydrin wereused as the detecting agent.

Ethyltbiosulfinic ethyl ester (CsH<rSO-S-C2Hs) was prepared enzymatically by the action of alliinase on S-ethyl L-cysteine sulfoxide (II).

+H202C2H6-SO-CHt-CH(NH2) COOH —¿�—— ->-

(S-Ethyl L-oysteine sulfoxide) (Allunase)

2CH,-CO-COOH + 2NH3(Ethylthiosulfinic ethyl ester)

1S-ethyl L-cysteine was obtained from the California Biochemical Foundation.

1Per cent N calculated =8.48; per cent N found = 8.60.1S-ethyl L-cysteine sulfoxide furnished through the courtesy

of Dr. Joseph Seifter, Wyeth Institute for Medical Research,Philadelphia, Pa.

Research. on September 13, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

WEISBERGER ANDPENSKY—Tumor Inhibition by Sulfhydryl-blocking Agent 1303

The reaction mixture contained 2000 Amóles of S-ethyl L-cysteine sulfoxide, 400 /ig. of pyridoxal phosphate, 250 ¿imolesof sodium phosphate buffer, pH 7.0, allanase (87 mg. protein),and water to a total volume of 10 ml. The reaction mixture wasincubated for 1 hour at 37°C. with continuous agitation andwas then stored at —¿�20°C.

Assay of the amount of ethylthiosulfinic ethyl ester formedenzymatically was accomplished by incubation of 0.02 ml. ofthe enzyme-substrate reaction mixture with 0.2 ml. of saturated potassium carbonate in a micro-Conway cell containing0.25 ml. of 2 per cent boric acid in the center well. Incubation at80°C. was continued for 90 minutes to insure complete diffu

sion of ammonia. The amount of ammonia trapped in the boricacid was determined by titration with 0.01 N sulfuric acid withmethyl purple4 as an indicator. The content of etbylthiosulfinicethyl ester was taken as one-half that of the ammonia in thereaction mixture (II).

Synthetic preparation of analogs of Allicin—Ethylthio-

sulfinie ethyl ester was subsequently prepared synthetically bythe oxidation of diethyl disulfide with perbenzoic acid (III) bythe method of Small et al. (27). Dimethyl, dipropyl, and dibutylthiosulfinic esters were also prepared from the respective di-suJfide precursors by this method. These compounds readilypolymerize and are easily further oxidized. The purified preparations were found to be stable when stored at —¿�20°C. Com

pounds diluted in water (2 mg/ml) remained active whenstored at 0°C.

1/2 Oj•¿�C2Hr-SO-S-C2H6 III.

METHODSEMPLOYEDIN STUDYINGTUMORINHIBITIONIN ANIMALS

The tumor-inhibiting effects of these thiosulfiniccompounds were studied both by direct incubationwith tumor cells prior to inoculation and for inhibitory effects when injected intravenously or in-traperitoneally after the tumor was implanted.The effect of preincubation with tumor cells invitro with either enzyme (alliinase) or substrate (S-ethyl L-cysteine sulfoxide) as well as the reactionproduct of these two substances (ethylthiosulfinicethyl ester) on the growth of Sarcoma 180 asciteswas determined as additional controls, because thefinal reaction mixture contained varying amountsof these substances.

The tumors studied included the ascitic andsolid forms of Sarcoma 180 in CFW Swiss mice andthe Murphy-Sturm lymphosarcoma in Wistar rats.In animals given inoculations of the ascitic form ofSarcoma 180, gain in weight and length of survivalwere taken as an index of the amount of ascitesformed and degree of malignancy induced. In thesolid tumors the area of tumor growth was calculated as the index for comparison.

The inhibition of ascites formation by preincubation with these substances was determined asfollows. Swiss mice weighing 20-25 gm. were giveninoculations intraperitoneally of a tumor suspension freshly drawn from donor mice with obvious

* Obtained from the Fleischer Chemical Company, Ben

jamin Franklin Station, Washington 4, D.C

ascites. The donor mice were sacrificed under etheranesthesia, 5-7 ml. of peritoneal fluid was withdrawn and was immediately placed in 2.0 ml. ofsaline and 0.2 ml. heparin (1000 USP units/ml). Atumor cell count was made, and the tumor suspension was then diluted with physiologic saline sothat each inoculum usually contained approximately 5,000,000 cells in 0.2 ml. after dilution witha solution of the substances to be tested. Eachinoculum was incubated either with normal salineor an equivalent volume of the test substance insolution for 10-15 minutes prior to intraperitonealinoculation. In each instance the amount of enzyme, substrate, or reaction product used for preincubation with the inoculum contained equivalent amounts of these substances. The amount ofenzymatically formed ethylthiosulfinic ethyl esterused for preincubation ranged from 0.5 to 2.5/¿moles(.07-.35 mg.) per inoculum of 5,000,000cells. Similar amounts of the synthetically prepared alkylthiosulfinic alkyl esters were used forpreincubation.

Since the thiosulfinic esters are weakly acid(pH 3), the effect of preincubation of tumor cellswith an equivalent volume of .001 N HC1 (pH 3.0)on ascites formation and death was tested in tenmice. In an additional ten mice the tumor cellswere preincubated with propylthiosulfinic propylester, neutralized to pH 7.2 with sodium bicarbonate.

The effect of synthetically prepared alkylthiosulfinic alkyl esters (methyl, ethyl, propyl, orbutyl esters) on the growth of Sarcoma 180 asciteswhen injected after the tumor had been transplanted was determined as follows. CFW Swissmice (20—25gm.) were given inoculations intraperitoneally of 0.2 ml. of a tumor suspension containing 5,000,000 cells as described above. Four-tenths to 0.5 mg. of the alkylthiosulfinic alkyl esterto be tested was injected intravenously 2 hoursafter the tumor was inoculated and daily thereafter for 7 days.

The Sarcoma 180 solid tumors were transplanted by homogenizing the tumor in a WaringBlendor and injecting 0.2 ml. of a 50 per cent tumor suspension subcutaneously into CFW Swissmice. Ten animals received 5 Amólesof the ethylthiosulfinic ethyl ester intraperitoneally 24 hoursafter the tumor was transplanted and daily thereafter for 10 days. Ten animals served as controls.

The Murphy-Sturm lymphosarcoma was transplanted to Wistar rats by homogenizing the tumorin a Waring Blendor and injecting 1.0 ml. of a 5per cent tumor suspension subcutaneously. Seventeen animals were given 3.5 mg. of ethylthiosulfinicethyl ester intraperitoneally daily for 10 days after

Research. on September 13, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

1304 Cancer Research Vol. 18, December, 1958

the tumor was implanted, the first injection beinggiven 24 hours after the transplant. Twenty-twoanimals served as controls.

32

ct¿228H-

Ì26u$iu24

5 22

20

CONTROLSUBSTRATE

o-o

ENZYME + SUBSTRATE

6 8 10 12 14 16TIME (DAYS)

CHAHT1.—Effectof garlic enzyme and substrate on growthof Sarcoma 180 ascites tumor in mice.

Sarcoma 180 ascites tumor cells incubated with saline(control) prior to inoculation grow rapidly. Preincubationcf the tumor cells with either enzyme (alliinase) or substrate(S-ethyl L-cysteine sulfoxide) failed to inhibit tumor growth.However, when the enzyme was allowed to react with thesubstrate prior to incubation with the tumor cells, therewas complete inhibition of tumor growth.

RESULTSIncubation of Sarcoma 180 ascites tumor cells

with enzymaiicatty derived ethylthiosulfinic ethylester prior to inoculation in mice.—Preincubation

of Sarcoma 180 ascites tumor cells with either theenzyme (alliinase) or the substrate (S-ethyl L-cysteine sulfoxide) had no inhibitory effect oneither the rate of tumor growth or mortality(Table 1, Charts 1 and 2). Thus, preincubation of

TABLE 1

THEEFFECTOFGARLICENZYME(ALLIINASE)ANDSUBSTRATE*ONTHEDEVELOPMENTOFSARCOMA

180ASCITESTUMORINMICE

InoculumTumor+salineTumor+substrateTumor-j-enzymeTumor+(substrate+enzyme)Tumor+(substrate+heated

enzyme)

No.oíminiala110252525Percentdevelopingascites1001001000Survivaltime(days)<17<16<16>300

10 100* Substrate = S-ethyl L-cysteine sulfoxide.

tumor cells with saline prior to inoculation in 110control animals resulted in rapid growth of thetumor and death within 17 days in every instance.Preincubation of the tumor cells with alliinase in25 animals or with S-ethyl L-cysteine sulfoxide in

ENZYME+SUBSTRATE

100

90

80

70

§60

¡50

ccw40

30

20

10

CONTROL-»

12 15 18 21 24TIME (DAYS)

27 30>(I20 150 180

CHART2.—Effectof garlic enzyme and substrate on survivalof mice inoculated with Sarcoma 180 ascites tumor.

Preincubation of Sarcoma 180 ascites tumor cells witheither saline (control), enzyme (alliinase), or substrate (S-ethyl

L-cysteine sulfoxide) prior to inoculation results in deathwithin 17 days. Preincubation of tumor cells with the reactionmixture of enzyme and substrate prevented the tumor growthand mortality.

Research. on September 13, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

WEISBERGEB ANDPENSKY—Tumor Inhibition by Sulfhydryl-blocking Agent 1305

25 animals also resulted in rapid growth of thetumor and death within 16 days. However, whenalliinase was allowed to react with S-ethyl L-cysteine sulfoxide to form ethylthiosulfinic ethylester and the resultant mixture was incubatedwith tumor cells, there was complete inhibition oftumor growth. None of the 25 mice inoculatedwith these cells developed ascites, and all are aliveand well after more than 300 days of observation.Inhibition of tumor growth was obtained with 0.5/¿molesof the enzymatically formed thiosulfinicester (0.07 mg.) per inoculum of 5,000,000 cells.Heating the enzyme at 56°C. for 30 minutes be

fore adding it to the substrate prevented formationof the thiosulfinic ester and failed to inhibit tumorgrowth in ten animals.

Preincubation of the tumor cells with iodoace-tate, which is also a sulfhydryl inhibitor, was notas effective as ethylthiosulfinic ethyl ester in preventing tumor growth (Chart 3). Thus, when thetumor cell inoculum was first incubated with 20/¿molesof iodoacetate (compared with 0.5 /¿molesof ethylthiosulfinic ethyl ester), there was a delayin onset of tumor growth, but four of the five animals tested were dead within 24 days (Chart 4).

32

i30

028i—

Ì26ÜJ

uj24

20

CONTROL

SUBSTRATE

6 8 10 12 14 16 18

TIME (DAYS)

CHART3.—Effectof garlic enzyme and substrate comparedwith iodoacetate on growth of Sarcoma 180 ascites tumorin mice.

Preincubation of Sarcoma 180 ascites tumor cells withsaline (control) resulted in rapid growth of the tumor. Preincubation of the tumor cell inoculum with 0.5 /imole ofenzymatically formed ethylthiosulfinic ester (enzyme + substrate) completely prevented tumor growth. Preincubationof the tumor cell inoculum with 20 Amólesof iodoacetatedelayed but did not prevent the growth of the tumor.

18 21

TIME (DAYS)

CHART4.—Effectof garlic enzyme and substrate comparedwith iodoacetate on survival of mice inoculated with Sarcoma180 ascites tumor.

Preincubation of Sarcoma 180 ascites tumor cells withsaline (control) prior to inoculation resulted in the death

of all animals within 16 days. Preincubation of the tumor cellswith 0.5 mole of enzymatically formed ethylthiosulfinic ethylester (enzyme + substrate) completely prevented mortality.Preincubation of the tumor cells with 20 moles of iodoacetatedelayed but failed to prevent mortality.

Research. on September 13, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

1306 Cancer Research Vol. 18, December, 1958

However, N-ethyl maleimide, which is also an -SHinactivator (21), was completely effective in preventing ascites and death when the tumor cellswere preincubated with 1 /mióleof this compoundper inoculum.

Inhibition of Sarcoma 180 ascites tumor growthby synthetically formed alkylthiosulfinic alkyl esters.—¿�Preincubation of Sarcoma 180 ascites tumor

cells with synthetically prepared alkylthiosulfinicalkyl esters was also effective in completely inhibiting ascites formation and death in mice(Table 2). Methyl, ethyl, propyl, and butyl analogs were tested in amounts ranging from 0.5 to2.5 Amólesper inoculum, and all were found to beeffective.

TABLE 2THEEFFECTOFALKYLTHIOSULFINICALKYLESTERS*ON

SARCOMAiso ASCITESTUMORFORMATIONINMICE

No.Animala

11025252525

Per cent Survivaldeveloping time

ascites (days)100 <17

0 >1800 >1800 >1800 >180

No.animals1382200No.surviving2621Per

centwithcompleteinhibition15151016

Inoculum tTumor+salineTumor-j-methyl analogTumor+ethyl analogTumor+propyl analogTumor-j-butyl analog

* R-SO-S-R

t Tumor cells incubated with various compounds in vitroprior to transplantation.

TABLE 3

INTRAVENOUSADMINISTRATIONOFALKYLTHIOSULFINICALKYL*COMPOUNDSAFTERTRANSPLANTATIONOF

SARCOMA180ASCITESTUMORSINTOMICE

CompoundMethyl analogEthyl analogPropyl analogButyl analog

»R-SO-S-R.

Intravenous injection of these synthetic alkylthiosulfinic alkyl analogs after the tumor had beenimplanted intraperitoneally was less effective inpreventing tumor formation. When the tumor inoculum consisted of 5,000,000 cells, no significantreduction in rate of tumor growth or mortalitycould be obtained. The tumor inoculum was thenreduced to 1,000,000 cells, and 0.4 mg. of ethyl-thiosulfinic ethyl ester was injected intravenouslydaily for 7 days, the first injection being given 2hours after the tumor was transplanted. Five of 32animals (15 per cent) treated in this fashion didnot develop ascites and survived (Table 3). In theremainder of the animals there was delay in therate of ascites formation, but all the mice diedwithin 28 days. Mice receiving the methyl, propyl,or butyl analogs exhibited a similar survival rate

(10-16 per cent), as well as delay in the rate of

formation of ascites in the animals which succumbed.

Effect of ethylthiosulfinic ethyl ester on growth ofsolid tumors.—Some inhibition of tumor growth by

ethylthiosulfinic ethyl ester was obtained in thesolid form of Sarcoma 180 in mice and in theMurphy-Sturm lymphosarcoma in rats. The tumors were transplanted subcutaneously, and thethiosulfinic ester was administered intraperitoneally 24 hours after the transplant and daily thereafter for 7 days.

The tumor area at the end of 2 weeks in tenmice transplanted with Sarcoma 180 averaged 6.5sq cm/animal. In a comparable group receiving0.35 mg. of ethylthiosulfinic ethyl ester intraperitoneally for 7 days, the tumor area at the end of 2weeks averaged 2.5 sq cm/animal or 40 per cent ofthe area in the control group. The control animalsgained an average of 2.6 gm. during this period,whereas the animals receiving the thiosulfinic esterlost an average of 0.8 gm.

In 22 rats transplanted with Murphy-Sturmlymphosarcoma, the average tumor area at the endof 2 weeks was 33 sq. cm. In the seventeen ratsreceiving 3.5 mg. of ethylthiosulfinic ethyl esterintraperitoneally for 10 days, the average tumorarea was 21 sq. cm. or 65 per cent of the area in thecontrol group.

Preincubation of either of the tumor suspensionswith the thiosulfinic ester prior to transplantationresulted in complete inhibition of growth in bothinstances.

DISCUSSIONThe absence of demonstrable qualitative bio

chemical differences in malignant cells has been alimiting factor in the rational therapy of cancer.Progress has been made in elucidating vital metabolic pathways in leukemia by the use of anti-metabolites, but additional pathways which maybe vulnerable need to be studied. There are nowconsiderable data suggesting that cell processesdependent upon the availability of reduced sulfhy-dryl groups may be a promising area of investigation. The results obtained in this study with thiosulfinic esters indicate that the ability of thesecompounds to inactivate -SH enzymes and tocombine with cysteine may be correlated with aninhibitory effect on rapidly growing cells and arefurther suggestive of the possible importance ofreduced -SH compounds in tumor growth.

It has been demonstrated that compounds derived from garlic have a bactericidal effect. Otherantibiotics such as various actinomycins, puromy-cin, and sarkomycin have been found to havean anti-tumor effect in animals. Cavallito (9)has shown that many antibiotics react with

Research. on September 13, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

WEISBERGERANDPENSKY—TumorInhibition by Sulfhydryl-blocking Agent 1307

sulfhydryl groups and suggests that the inhibitionof bacterial growth may be due to inactivation of-SH enzymes within the bacteria or to combinationwith -SH groups in polypeptide chains during protein anabolism to form "dead ends." Some anti

biotics such as penicillin react only with certain-SH groups; others such as those derived fromAllium sativum react with almost any -SH groupwith which they come in contact. It is quite possible that the bactericidal effect and tumor-inhibiting effects of some antibiotics are related to thesame mechanism—namely, inactivation of -SHgroups essential for cell growth. It is, therefore,possible that -SH inhibitors with a specific affinity for certain tumor cells could be designed ordiscovered, much as various antibiotics have specific activity against certain classes of bacteria.The current screening program of compounds derived from microbiological filtrates may be helpfulin discovering such chemotherapeutic agents.

Hammett, who was one of the early proponentsof the importance of -SH compounds in normaland malignant growth, also investigated the effectof partially oxidized cystine derivatives on cellularproliferation (15, 16, 24). Cystine disulfoxide (R-SO-SO-R) was found to retard the growth ofspontaneous mammary carcinoma in mice and toinhibit the proliferatile phase of Obelia. Bennett(4), however, found that cystine disulfoxide supported growth in feeding experiments, presumablybecause the compound was unstable and was rapidly converted to either cystine or cysteine. Furthermore, Reimann and Toennies (25) found thatcystine disulfoxide did not inhibit cell division inthe mouse skin. Although inhibition of cellulargrowth by cystine disulfoxide is equivocal, Reimann and Hammett suggested that it might bepossible to discover or design a sulfoxide with highreactive properties and tumor-inhibiting effectswhich at the same time would be sufficiently stableto permit more than transitory existence in thebody (24).

Other partially oxidized sulfur compounds havehad little or no effect on cell growth. Methioninesulfoxide (R-SO-CHs) and cystine sulfinic acid(R-SO2-H) do not inhibit cell division (7). VonEuler (30) obtained an inconstant growth inhibition of the Jensen sarcoma in rats with S-allyl-L-cysteine sulfoxide (R-SO-R'). No tumor inhibition was obtained with S-ethyl L-cysteine sulfoxidein our experiments. However, Boy land obtainedsome slight inhibitory action on the growth of tumors in mice with partially oxidized sulfanilamides(7).

The sulfonic ester, Myleran (1,4-dimethane-sulfonoxybutane, CH3SO2-O-(CH2) 4-O-SO2CH3)inhibits the growth of the Walker rat carcinoma

256 and is effective in the therapy of chronic mye-loid leukemia in man. This compound is believedto produce its effects through chemical alkylationof protein or nucleoprotein in the cell (13, 14). Itis possible that the oxidized sulfur portion of themolecule is responsible for the reactivity with proteins and that at least some of its effectiveness maybe due to -SH inactivation.

The -SO-S- linkage is probably essential for thetumor-inhibitory effects observed with the variousthiosulfinic compounds. This same linkage is essential for the bactericidal effects of these compounds(27) and is also essential for -SH enzyme inhibition(43). In the compounds studied, the length of thealkyl side chains had no appreciable effect on thetumor-inhibiting capacity. Substitution with otheralkyl, aryl, or aromatic side chains may, however,significantly alter the tumor inhibition obtainedwith various thiosulfinic esters. The weakly acidcharacter of these compounds is not a factor inpreventing tumor growth following preincubation,since .001 N HC1 fails to prevent tumor growth.Furthermore, neutralization of the thiosulfiniccompounds with alkali does not alter the tumor-inhibiting effect.

The exposure of tumor cells to the thiosulfinicesters in vitro prior to tumor transplantation is admittedly a very sensitive index for tumor inhibition and may be too sensitive as a screening testfor possible chemotherapeutic agents in man.However, the experiments in this report were not.designed as a screening procedure but were intended to test the hypothesis that a compoundwhich is an -SH inhibitor and reacts with cysteinemight have tumor-inhibiting effects. It is of interest in this connection that N-ethyl maleimide (21)also manifests a tumor-inhibitory effect. This compound is an -SH inactivator and a bactericidalagent which has been shown to inhibit mitosis offibroblasts in tissue culture. It is apparent, however, that not all -SH inactivators are effective inthe test system employed, since iodoacetate delayed but did not prevent tumor growth eventhough used in a relatively high concentration.The ability of these compounds to react with susceptible -SH groups in certain positions in the cellprotein as well as the avidity of these compoundsfor -SH may be a factor in their ability to inhibittumor growth.

SUMMARYExtracts of garlic (Allium sativum) contain a

bactericidal agent which is formed by the action ofan enzyme on a substrate present in garlic bulbs.The active principle formed by this enzymaticprocess is an alkylthiosulfinic alkyl ester (R-SO-S-R) which is an -SH inactivator and reacts rapidly with cysteine. Since cell processes dependent

Research. on September 13, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

1308 Cancer Research Vol. 18, December, 1958

upon the availability of reduced -SH groups havebeen implicated in both normal and malignantgrowth, several related alkylthiosulfinic esterswere investigated for tumor-inhibitory effects.Enzymatically formed ethylthiosulfinic ethyl esteras well as various synthetically prepared alkylthiosulfinic alkyl esters were shown to have tumor-inhibitory effects in the test systems used. Theimplications of tumor inhibition by a compoundwhich is an -SH inactivator are discussed.

REFERENCES1. BALDINI,M., and SACCHETTI,C. L'Effect de la cystine et

de la cysteine sur la moelle osseuse humaine, cultivéeinmilieu carence en amino acides. Rev. Hematol., 8:3, 1953.

2. BARBON,E. S. G. Cellular Metabolism and Growth in theChemistry and Physiology of Growth, pp. 106-134. Princeton, New Jersey: Princeton University Press, 1949.

3. . Thiol Groups of Biological Importance. Adv.Enzymol., 11:201, 1951.

4. BENNETT,M. A. Metabolism of Sulfur. VII. A Quantitative Study of the Replaceability of L-Cystine by VariousSulphur-Containing Amino Acids in the Diet of the AlbinoRat. Biochem. J., 33:885, 1939.

5. BICHEL, J. Blood Glutathione in Disease. Acta. Med.Scandinav. 124:160, 1946.

6. BINET,L., and MAGHOD,J. Glutathion, croissance et cancer des plantes. Compt. rend, de Se., 192:1415, 1931.

7. BOTLAND,E. Experiments on the Chemotherapy of Cancer. 1. The Effect of Certain Antibacterial Substances andRelated Compounds. Biochem. J., 32:1207, 1933.

8. BBACHET,J. Chemical Embryology. New York: Inter-science Publishers, Inc., 1950.

9. CAVALLITO,C. J. Relationship of Thiol Structures to Reaction with Antibiotics. J. Biol. Chem., 164:29, 1946.

10. CAVALIJTO,C. J.; BUCK,J.; and SUTER,C. Allicin, theAntibacterial Principle of Allium Saiivum. II. Determination of the Chemical Structure. J. Am. Chem. Soc., 66:1952, 1944.

11. CONTOPOLOUS,A. N., and ANDERSON,H. H. SulfhydrylContent of Blood Cells in Dyscrasias. J. Lab. & Clin.Med., 36:929, 1950.

12. GOERNER,A., and GOERNER,M. M. The Metabolism ofSulfhydril Compounds in Tumor Tissue. Am. J. Cancer,16:360, 1932.

13. HADDOW,A. Discussion on the Chemotherapy of theReticuloses. Proc. Roy. Soc. Med., 46:692, 1953.

14. HADDOW,A., and TIMMIS,G. M. Bifunctional SulfonicAcid Esters with Radiomimetric Activity. Acta Unióinternat. Contra cancrum, 7:469, 1951.

15. HAMMETT,F. S. An Interpretation of Malignant GrowthBased on the Chemistry of Cell Division. Arch. Path.,8:575, 1929.

16. . Natural Chemical Factors in Growth and Development. Cold Spring Harbor Symp. Quant. Biol., 2:78, 1934.

17. HARDIN,B.; VALENTINE,W. N., and FOLLETTE,J. H.Studies on the Sulfhydryl Content of Human Leukocytes.Am. J. M. Se., 228:73, 1954.

18. KANDEL,E. V., and LsRoY, G. V. The Blood Glutathionein HématologieDisease. J. Lab. & Clin. Med., 24:669,1936.

19. KUZELL,W. C.; KOETS, P.; SCHAFFAHZICK,R. W.; andNAUOLER,W. E. Variation of Blood Glutathione duringNeutropenia. Case Report Stanford M. Bull., 13:284,1955.

20. LEHMANN,F. A. Untersuchungen überAllium Sativum(Knoblauch) Arch. Exper. Path. Pharmak., 147:245,1930.

21. MARHIAN,D. H.; FRIEDMANN,E.; and WARD,J. L. TheAntibacterial Effects of Substances Structurally Resembling Maleimide. Biochem. J., 54:65, 1953.

22. PARKER,F. P., and KRACKE,R. R. Further Studies in Experimental Granulopenia. Am. J. Clin. Path.,6:641, 1936.

23. PLATT, R. The Blood Glutathione in Disease. Brit. J.Exper. Path., 12:139, 1931.

24. REIMANN,S. P., and HAMMETT,F. S. The Reaction ofSpontaneous Mouse Tumors to Cystine Disulfoxide. Am.J. Cancer, 26:554, 1936.

25. REIMANN,S. P., and TOENNIES,G. Reaction of Mouse Skinto Various Reduced and Partially Oxidized Sulfur Compounds. Arch. Path., 29:175, 1940.

26. SCHACTER,B.; ENTENMAN,C.; and SHIMKIN,M. B. BloodPlasma Sulfhydryl Levels during Growth and Regressionof the Murphy Lymphosarcoma of the Rat. J. Nat. CancerInst., 13:647, 1952.

27. SMALL,L. D.; BAILEY,J. H.; and CAVALLITO,C. J. AlkylThiosulfinates. J. Am. Chem. Soc., 69:1710, 1947.

28. STOLL,A., and SEEBECK,E. ÜberAlliin, die genuineMuttersubstanz des Knoblauchöls. Helv. Chim. acta,31:189, 1948.

29. . Die Syntheses des natürlichenAlliins und seinerdrei optisch aktiven Isomeren. Ibid., 34:481, 1951.

30. •¿� . Chemical Investigations on Alliin, the SpecificPrinciple of Garlic. Adv. Enzymol., 11:377, 1951.

31. VOEGTUN,C.; JOHNSON,J. M.; and THOMPSON,J. W.Glutathione and Malignant Growth. Public Health Rep.,61:1689, 1936.

32. VOEGTUN,C. J., and THOMPSON,J. W. Glutathione Content of Tumor Animals. J. Biol. Chem., 70:801, 1926.

33. WEIL,E.; ASCHKENASY,A.; and CAPRON,L. Le Glutathionsanguin dans les polyglobulies, les leukemies et les erthro-blastoses chroniques. Sang, 13:705, 1939.

34. WEISBERGER,A. S., and HEINLE, R. W. The ProtectiveEffect of Cysteine on Leukopenia Induced by NitrogenMustard. J. Lab. & Clin. Med., 36:872, 1950.

35. WEISBERGER,A. S., and LEVINE,B. Some Structural Requirements for the Prevention of Leukopenia by NitrogenMustard. J. Clin. Investigation, 31:217, 1952.

36. . Incorporation of Radioactive L-Cystine by Normal and Leukemic Leukocytes in vivo.Blood, 9:1082,1954.

37. WEISBERGEB,A. S., and STORAASLI,J. P. P. OpticalIsomers in the Prevention of Leukopenia Induced by Nitrogen Mustard. J. Lab. & Clin. Med., 43:246, 1954.

38. WEISBERGER,A. S., and SUHRLAND,L. G. Studies on Analogues of L-Cysteine and L-Cystine. III. The Effect ofSelenium Cystine on Leukemia. Blood, 11:19, 1956.

39. WEISBERGER,A. S.; SUHRLAND,L. G.; and GRIGGS,R. C.Incorporation of Radioactive L-Cystine and L-Methionineby Leukemic Leukocytes in Vitro. Blood, 9:1095, 1954.

40. WEISBERGER,A. S.; SUHRLAND,L. G.; and SEIFTER,J.Studies on Analogues of L-Cysteine and L-Cystine. I. SomeStructural Requirements for Inhibiting the Incorporationof Radioactive L-Cystine by Leukemic Leukocytes. Blood,11:1, 1956.

41. WHITE,J., and EDWARDS,J. E. Effect of Dietary Cystineon the Development of Hepatic Tumors in Rats Fed p-Dimethylaminoazobenzene (Butter Yellow). J. Nat. Cancer Inst., 2:535, 1942.

42. WHITE, J.; MIDEH, G. B.; and HESTON, W. Effect ofAmino Acids on the Induction of Leukemia in Mice. J.Nat. Cancer Inst., 4:909, 1943-1944.

43. WILLS, E. D. Enzyme Inhibition by Allicin, the ActivePrinciple of Garlic. Biochem. J., 63:514, 1956.

Research. on September 13, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

1958;18:1301-1308. Cancer Res   Austin S. Weisberger and Jack Pensky 

)Allium sativumActive Principle of Garlic (Tumor Inhibition by a Sulfhydryl-blocking Agent Related to an

  Updated version

  http://cancerres.aacrjournals.org/content/18/11/1301

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/18/11/1301To request permission to re-use all or part of this article, use this link

Research. on September 13, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from