10(2):293. DOI: 10.1128/AAC.10.2.293.

1976,Antimicrob. Agents Chemother. S. S. S. V. Prasad and Y. I. Shethna Ascites Sarcoma Cells

on Yoshidathuringiensis subsp. Bacillus thuringiensis

Proteinaceous Crystal of Antitumor Protein from the Mode of Action of a Purified

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ANTIMICROBIAL AGENTS AND CHZMYrHZRAPY, Aug. 1976, p. 293-298Copyright X 1976 American Society for Microbiology

Vol. 10, No. 2Printed in U.S.A.

Mode of Action of a Purified Antitumor Protein from theProteinaceous Crystal of Bacillus thuringiensis subsp.

thuringiensis on Yoshida Ascites Sarcoma CellsS. S. S. V. PRASADI* AND Y. I. SHETHNA

Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore 560012, India

Received for publication 23 March 1976

A purified antitumor protein from the proteinaceous crystal ofBacillus thu-ringiensis subsp. thuringiensis inhibits the growth of Yoshida ascites sarcomaboth in vivo and in vitro. Exogenous respiration of the tumor cells was unaf-fected by the protein at a concentration as high as 500 Ag/ml. The antitumorprotein inhibits the uptake and incorporation of labeled precursors into macro-molecules. However, the ratio of incorporation over uptake is not affected by theprotein. Further, the protein brings about the leakage of 260-nm-absorbingmaterial, proteins, and 32P-labeled cellular constituents from the Yoshida as-cites sarcoma cells. The results show that the action of the antitumor proteinappears to alter the cellular permeability of the tumor cells.

During sporulation Bacillus thuringiensisproduces a crystalline material which is liber-ated into the medium when the sporangium islysed (6). This unique crystal was composed ofan alkali-soluble protein with a molecularweight of 200,000, which is highly toxic for lepi-dopterous insect larvae (1) and is the least toxicfor mammals (8, 18). Recently, we have re-ported antitumor activity against Yoshida as-cites sarcoma (YAS) with a high therapeuticindex for this crystal preparation (18). We havealso observed that this crystal brings aboutlong-lasting antitumor immunity (21) and en-hances the nonspecific immune response (20).Further, the purification, crystallization, andcharacterization of the biologically active pro-tein subunit of the crystals have been achieved(19).The only biological activity of the crystal

known until recently was its insecticidal activ-ity. The exact insecticidal mode of action of thecrystal is poorly understood because of the un-resolved identity of the toxic protein subunit ofthe crystal. The available evidence suggeststhat the crystal toxin alters the membranepermeability of the insect gut wall, resulting inthe impairment of ion transport and the leak-age of alkaline gut contents into acidic hemo-coel. The resulting pH change brings aboutparalysis and death of the insect larvae (3, 4,16). However, the antitumor mode of action of

I Present address: Department of Developmental Thera-peutics, M.D. Anderson Hospital and Tumor Institute, TheUniversity of Texas System Cancer Center, Houston, Tex.77030.

this toxic protein has not yet been elucidated.The purification of the active protein subunitmade it possible to study its mode of action ontumor cells, which is being reported here.

MATERIALS AND METHODSChemicals. The following radioactive chemicals

were obtained from Bhabha Atomic ResearchCentre, Bombay, India: [3H]thymidine (specific ac-tivity, 10,500 mCi/mmol); ['4C]phenylalanine (spe-cific activity, 312 mCi/mmol); [14C]glucose (specificactivity, 229 mCi/mmol); and [32P]orthophosphoricacid (specific activity, 5 mCi/mmol). ['4C]uridine(specific activity, 55.3 mCi/mmol) was purchasedfrom New England Nuclear Corp., Boston, Mass.;medium 199 with Hanks basal salts solution (HBSS)was purchased from V.P. Chest Institute, NewDelhi, India. All the other chemicals were of ana-lytical grade.

Animals. Inbred Wistar A/Iisc rats weighingaround 100 to 120 g were used in all the studies. Theanimals were given a diet of "pellet" food (Hindu-stan Lever Ltd., Bombay) and water ad libitum.Tumor. YAS, a rapidly developing, chemically

induced tumor (25) was maintained in isogenic Wis-tar rats (A/Iisc) by serial intraperitoneal (i.p.)transfer of 2 x 107 tumor cells, once in 4 days. Thedetails about the morphology and chromosomalcharacteristics of the tumor have been describedearlier (2, 22).

Purification of antitumor protein. Pure antitu-mor protein from crystals was obtained as previ-ously described (19). The lyophilized sample wasdissolved in cold Krebs phosphate buffer at pH 7.4,so that the required concentrations were containedin 0.2 ml of suspension.

Antitumor activity in vivo. Experimental ani-mals were injected i.p. with 3 x 107 actively dividing

293

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294 PRASAD AND SHETHNA

YAS cells. Treatment consisted of a single or multi-ple i.p. dose of protein preparation given 24 h aftertumor transplantation. Regular observations weremade on the weights and the general behavior of theanimals.

Collection of YAS cells. YAS cells were collectedasceptically from the peritoneal cavity of 3-day-old,tumor-bearing rats in 10 volumes of cold saline. Thecells were then washed with cold Krebs phosphatebuffer by repeated centrifugations (1,000 x g) at 00Cuntil the tumor cells were free from contaminatingerythrocytes. The tumor cells were then suspendedin the original volume, and a count was made by thetrypan blue dye exclusion method (17). The concen-tration of YAS cells was adjusted to 3 x 106 viablecells/ml in cold Krebs phosphate buffer.

Cell multiplication. The YAS cell multiplicationwas studied by short-term cell culture as describedearlier (9), using medium 199 with HBSS and 40%heat-inactivated calf serum. Viable YAS cells (105/ml) were inoculated into the medium along withdifferent concentrations of the antitumor protein. Inanother set [3H]thymidine (2 ,uCi/ml) was addedalong with the protein. The culture tubes were thenincubated at 370C, and the viable count and theincorporation of [3H]thymidine into deoxyribonu-cleic acid (DNA) were determined at regular inter-vals. Viable counts were made by the trypan bluedye exclusion method. The incorporation of[3H]thymidine into DNA was estimated by taking a0.2-ml sample and precipitating it with an equalvolume of 10% trichloroacetic acid. The mixture wasthen filtered under suction on Whatman no. 1 fil-ter paper disks, the precipitate was washed with5% trichloroacetic acid, ethanol-ether (1:1), andether successively, and radioactivity was deter-mined in a Beckman LS-100 liquid scintillationcounter.

Respiration studies. Studies on respiration werecarried out using standard manometric techniques(24). YAS cells were washed as described before andsuspended in Krebs phosphate buffer. Warburgflasks contained the following in final volume of 3.2ml: 1 ml of 0.2 M glucose and 3 x 107 YAS cells/ml inthe main compartment. The center well contained0.2 ml of20% KOH. The antitumor protein was usedin appropriate amounts to give the desired concen-trations. The flasks were equilibrated for 10 min at370C, and the oxygen uptake was measured.

Uptake of [14C]glucose by YAS cells. The uptakeof glucose from the medium (medium 199 withoutserum) by YAS cells was determined by adding[14C]glucose (1 ,uCi/ml) to the YAS cell suspension (3x 106 cells/ml) in a final volume of 2 ml. The antitu-mor protein was added at desired concentrations andincubated at 370C in a water bath for 2 h. The periodof incubation was standardized by several prelimi-nary experiments. Samples were taken out at theend of the incubation, and YAS cells were filteredunder suction on Whatman no. 1 filter paper disksand then washed with excess of Krebs phosphatebuffer. The filter paper disks were then dried underan infrared lamp, and the radioactivity was deter-mined.

Uptake and incorporation of labeled precursors

ANTIMICROB. AGENTS CHEMOTHER.

into DNA, RNA, and proteins of YAS cells. TheYAS cells (3 x 106 viable cells/ml) were incubated at370C for 2 h with various concentrations of the anti-tumor protein along with either [3H]thymidine (1,uCi/ml), ['4C]uridine (1 utCi/ml), or [14 Cphenyl-alanine (1 ,uCi/ml) in a total volume of 2 ml. Atthe end of the incubation period the uptake of thelabeled precursors by YAS cells was determinedas described earlier for [14C]glucose. The incor-poration of [3Hlthymidine, [14C]uridine, and [14C]1phenylalanine into DNA, ribonucleic acid (RNA),and protein, respectively, was estimated accord-ing to Mans and Novelli (13). The reaction wasstopped by the addition of an equal volume of 10%cold trichloroacetic acid. The mixture was then fil-tered under suction on Whatman no. 1 filter paperdisks, and the precipitate was washed with 5% tri-chloroacetic acid, ethanol-ether (1:1), and ether suc-cessively, and the radioactivity was determined.

Cell permeability studies. The tumor cells (3 x107 cells/ml) were exposed to the antitumor proteinat various concentrations in a water bath at 370C. Atdifferent time intervals samples were removed, andthe cell exudates were examined for 260-nm-absorb-ing materials by measuring the absorbance. Pro-teins were determined by the method ofLowry et al.(12).

In vivo 32p labeling of tumor cells. Actively grow-ing, 3-day-old tumor cells were labeled in vivo by theadministration of 32p label as ortho-phosphoric acid(0.5 mCi/animal) i.p. After 2 h of 32p administration,rats were sacrificed by cervical dislocation, and thetumor cells were collected in cold saline and proc-essed as described earlier. The labeled tumor cellswere then incubated at 370C for 2 h in the presence ofdifferent concentrations ofthe antitumor protein. Atthe end of the incubation period, the cell exudateswere obtained by centrifugation, and the radioactiv-ity was determined.

RESULTSEffect of purified antitumor protein on

YAS in vivo. The results on the antitumor ac-tivity of the pure protein are presented in Table1. A single dose of 1 mg/kg brought about anincreased survival period of the treated ani-mals, whereas a split dose administration of 0.1mg/kg given for 5 days resulted in the completeregression of the tumor. At a lower dose of 0.05mg/kg, 60% of the treated animals died with aprolonged survival period of28 days, in contrastto 10 days in case of controls.

Effect of antitumor protein on in vitro YAScell multiplication. The results of this experi-ment are depicted in Fig. 1. The viability ofthe cells and the incorporation of the [3H]-thymidine into DNA were adversely affectedin the presence of the protein. Maximum ef-fect was seen at 20 h with a protein concentra-tion of 1 mg/ml. There was 60% reduction in theviable count and 54% decrease in the incorpora-tion of [3H]thymidine into DNA at this concen-

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ACTION OF PROTEIN ON YAS CELLS 295

tration. After 24 h there was a slight increase inboth the viable count and DNA synthesis.

Effect of antitumor protein on respirationof YAS cells. The antitumor protein did notshow a significant effect on the exogenous res-piration ofthe tumor cells. Even at a concentra-tion of 1 mg of protein/ml, there was only a 20%reduction in the total microliters of oxygen con-sumed after 210 min.

TABLE 1. Effect ofpurified antitumor protein fromthe proteinaceous crystal ofB. thuringiensis on

YAS cells in rats

No. ofNo. of Survival period Su.- sur-

Dose" injec- (days) + stand- vorsb vivorstions ard error after

90 days

2.5 1 30 2.0 6/6 01 1 32 1.3 6/6 00.5 5' 90Y' 6/6 60.2 5 90 6/6 60.1 5 90 6/6 60.05 5 28 ± 3.0 6/6 2

Control 1 9 ± 1.2 0/6 05 10 ± 1.5 0.6 0

11 Milligrams of protein per killogram given i.p. 24h after tumor transplantation; controls injected withsaline.

b Survivors at the time of the death of the con-trols.

' Split dose treatment administered at the rate ofone dose per day for 5 days."Surviving until the end of the experiment.

x to54

3

wu

O M

Effect of antitumor protein on the uptakeof ['4C]glucose by YAS cells. The results indi-cate that there was a dose-dependent inhibitionof the ['4C]glucose uptake by YAS cells (Table2). Maximum inhibition of82% was observed ata protein concentration of 1 mg/ml.Effect of antitumor protein on the uptake

and incorporation of labeled presursors intomacromolecules of YAS cells. (i) [3H]thy-midine. The data revealed that there wasan inhibition of both uptake and incorpora-tion of [3H]thymidine into DNA ofYAS cells inthe presence ofthe antitumor protein (Table 3).At a protein concentration of 0.5 mg/ml, thepercentages of uptake and incorporation were31.5 and 38.7%, respectively. However, theprotein did not affect the ratio of incorporationover uptake.

(ii) ['4C]uridine. The protein brought about adecrease in the uptake and incorporation of

TABLE 2. Effect ofantitumorprotein on the uptake of['4C]glucose by YAS cells a

Concn (mg/ cpmb/3 x 106 Uptake (%) Inhibitionml) viable cells Upae()Ihbto0 18,200 100 00.25 15,341 84.3 15.70.5 7,234 39.8 60.21.0 3,262 18 82

a In all the experiments the measurements weremade at the end of 2 h of incubation period at 37°C.

b cpm, Counts per minute.

oe~~~~~~~~~~ o--300

aI

0

w

- 00

IL

_^~~~~ v 2~~00°

~~~z100.

I.

FIG. 1. Effect of antitumor protein on cell multiplication and DNA synthesis of YAS cells in vitro. Solidlines, [3H7thymidine incorporation; dotted lines, cell viability; *, control, 0, 0.5 mg, A, 1.0 mg.

VOL. 10, 1976

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296 PRASAD AND SHETHNA

TABLE 3. Effect ofantitumor protein on [3H1-thymidine uptake and incorporation into DNA of

YAS cells

Uptake (3 x10b Incorporation(3 Ratio of in-Concn (mg/ viable cells) x 106 viablecoprtn

ml) cells) over, up-a ~~~~~~take

cpm" % cpm %

0 3,442 100 435 100 12.70.01 2,513 73.0 393 90.2 15.60.05 2,344 68.1 374 86.0 15.90.1 1,859 54 357 82 19.20.25 1,650 47 311 77.5 18.00.50 1,091 31.5 169 38.7 15.4

a cpm, Counts per minute.

TABLE 4. Effect ofantitumor protein on the uptakeand incorporation of[14C]uridine into RNA of

YAS cells

Uptake (3 x 106 Incorporation (3 Ratio of in-Concn viable cells) x 106 viable cells) corporation(mg/ml) over up-

cpma % cpm % take

0 4,560 100 2,210 100 48.40.01 3,420 75 2,055 93 60.00.05 3,101 68.0 1,858 84.1 55.70.10 2,873 63.0 1,600 72.4 55.60.25 2,374 52.1 1,348 61.0 56.70.50 1,961 43.0 1,090 49.32 55.5

a cpm, Counts per minute.

[14C]uridine into RNA of tumor cells (Table 4).Maximum effect was seen at a concentration of1 mg/ml, wherein the percentages of uptakeand incorporation were 33.5 and 35.7%, respec-tively, of the control. The ratio of incorporationover uptake was not inhibited.

(iii) [14C]phenylalanine. The uptake and in-corporation of [14C]phenylalanine were also in-hibited by the antitumor protein without in-hibiting the ratio of incorporation over uptake(Table 5). At a protein concentration of 1 mg/ml, 54.1 and 53.8% of uptake and incorporationwere observed, respectively.

Effect of antitumor protein on cell permea-bility. The leakage of various cellular constitu-ents like 260-nm-absorbing materials and pro-teins as affected by the antitumor protein areshown in Fig. 2 and 3, respectively. The leak-age of 260-nm-absorbing materials was directlyproportional to the concentration and time ofexposure of the antitumor protein. The viabil-ity of the tumor cells was not affected by theprotein within the incubation period of2 h at allthe doses tested.The leakage ofproteins also showed the same

pattern as the 260-nm-absorbing materials. Theleakage of 32P-labeled cellular constituents is

shown in Table 6. There was a leakage of23.5%of the labeled constituents over the control at aconcentration of 1 mg/ml.

DISCUSSIONThe in vitro and in vivo data presented in

this study clearly reveal the potent tumor in-hibitory activity of the toxic protein from theproteinaceous crystal of B. thuringiensis. Theeffectiveness of the split dose administrationmay be due to the rapid removal of the proteinfrom the system. The detoxification of the pro-tein after 24 h of incubation may also be thereason for the increase in the viable count and

TABLE 5. Effect ofantitumor protein on the uptakeand incorporation of['4C]phenylalanine into protein

of YAS cells

Uptake (3 x 106 Incorporation (3 Ratio of in-Concn (mg/ viable cells) x 106 viable corporation

ml) cells) over up-cpma % cpm % take

0 5,500 100 4,200 100 76.30.01 4,730 86.0 3,868 92.1 81.70.05 4,472 81.3 3,705 88.2 82.80.10 4,070 74 3,465 82.5 85.10.25 3,823 69.5 3,285 78.2 85.90.50 3,480 63.3 3,053 71.6 87.0

a cpm, Counts per minute.

100

I..

-i5

C)

0 30 60 90 12o _TIME (MINJ

FIG. 2. Effect ofantitumor protein on the leakageof 260-nm-absorbing materials from YAS cells.Solid lines, Absorbancy at 260 nm; dotted lines, per-centage of cell viability; 0, control, 0, 0.25 mg, A,0.5 mg, A, 1.0 mg.

ANTimICROB. AGENTS CHEMOTHER.

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ACTION OF PROTEIN ON YAS CELLS 297

TIME (MIN.)

FIG. 3. Effect ofantitumor protein on the leakageofproteins from YAS cells. Solid lines, Protein (mi-crograms per milliliter); dotted lines, percentage ofcell viability; *, control; 0, 0.25; A, 0.5 mg; A,1.0 mg.

TABLE 6. Effect of antitumor protein on the leakageof 32P from in vivo labeled YAS cells

Concn (mg/ Cell viabil- Leakage of np Leakageml) itya (cpm)b %

0 98 40,000 00.25 95 44,100 10.20.5 96.5 46,002 151.0 94.2 49,411 23.5

a Cell viability was determined at the end of theincubation period (2 h) by the trypan blue dye exclu-sion method (17).

b cpm, Counts per minute.

DNA synthesis in vitro. These results suggestthat continuous exposure of the tumor cells tothe toxic protein is necessary for its action.The antitumor protein failed to inhibit exoge-

nous respiration, but there is considerable inhi-bition of the [14C]glucose uptake by the tumorcells. Further, the uptake and incorporation of[3H]thymidine, [14C]uridine, and [14C]phenyl-alanine into DNA, RNA, and protein fractionsof the tumor cells are also inhibited by theantitumor protein. However, the ratio of incor-poration over uptake is not affected in all thecases. Further, there is a selective inhibition ofuptake over the incorporation at all the dosestested. These observations suggest that thetoxic protein may be damaging the integrity ofthe cell membrane. This is further confirmedby the rapid leakage of essential cellular con-

stituents, like proteins and nucleotides fromthe tumor cells exposed to this toxic protein,without affecting the cell viability.A vast amount of literature is available in

the area ofmembrane-active antibacterial (5, 7,15) and antifungal agents (11, 23). However,very few antitumor agents elicit their action byaffecting the membrane function of the cells(10, 14). Membrane-active agents affect themembrane integrity and functions, therebyproducing an initial rapid loss ofhigh- and low-molecular-weight metabolites from the meta-bolic pool ofthe cell. Antifungal antibiotics likepolyenes specifically combine with sterol com-ponents in the membrane of susceptible orga-nisms, resulting in the structural disruption ofmembrane and the loss of essential metabolitesfrom the cell.The data presented here on the purified anti-

tumor protein from the proteinaceous crystal ofB. thuringiensis suggest that its mode of ac-tion is mainly on the cell membrane of thetumor cells, which is similar to the action ofthewhole crystal in the insect system. However,the elucidation of the exact site of action of thispurified protein on the cell membrane of thetumor cells needs further investigation.

ACKNOWLEDGMENTSWe thank University Grants Commission, New Delhi,

for the award of a special grant and the council of Scientificand Industrial Research, New Delhi, for awarding a SeniorResearch Fellowship to one of us (S.S.S.V.P.).

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3. Fast, P. G., and T. A. Angus. 1965. Effect of parasporalinclusions ofBacillus thuringiensis var sotto Ishiwataon the permeability of the gut wall ofBombyx mori(Linnaeus) larvae. J. Invertebr. Pathol. 7:29-32.

4. Fast, P. G., and I. K. Morrison. 1972. The 8-endotoxinof Bacillus thuringiensis. IV. The effect of 8-endo-toxin on ion regulation by mid-gut tissue ofBombyxmori larvae. J. Invertebr. Pathol. 20:208-211.

5. Few, A. V., and I. H. Schulman. 1953. The absorptionofpolymyxin E by bacteria and baterial cell walls andits bactericidal action. J. Gen Microbiol. 9:454-466.

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11. Kinsky, S. C. 1967. Polyene antibiotics, p. 122-141. InD. Gottlieb and P.D. Shaw (ed.), Antibiotics, vol. I.Springer-Verlag, New York.

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13. Mans, R. J., and G. D. Novelli. 1961. Measurement ofthe incorporation of radioactive amino acids into pro-tein by a filter-paper disk method. Arch. Biochem.Biophys. 94:48-53.

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Shethna. 1973. Inhibitory activity of the parasporalcrystal ofBacillus thuringiensis var thuringiensis onYoshida ascites sarcoma. Curr. Sci. 42:568-570.

19. Prasad, S. S. S. V., and Y. I. Shethna. 1974. Purifica-tion, crystallization and partial characterization ofthe antitumor and insecticidal protein subunit fromthe 8-endotoxin of Bacillus thuringiensis var thurin-giensis. Biochim. Biophys. Acta 363:558-566.

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