Effect of Core Lipopolysaccharides from Salmonella ...cancerres.aacrjournals.org/content/canres/35/3/628.full.pdf · aThe abbreviationsusedare:Hep, l-glycero-n-mannoheptose;KDO,...

[CANCER RESEARCH 35, 628-633. March 1975]

SUMMARY

Injection of a homologous series of bacterial core lipopolysaccharides obtained from Salmonella minnesota Rmutants to Ehrlich solid tumor-bearing mice results in anincrease of survival times of treated animals. Lower chainlength favors greater antitumor activity. Oligosaccharidesand polysaccharides derived from lipopolysaccharides werefound to be ineffective in increasing survival time oftumor-bearing mice. Smaller-sugar-chain-length core lipopolysaccharides were found to be better adjuvant than werethose with longer sugar chains. Implication of adjuvantaction of lipopolysaccharides in the elicitation of antitumoractivity is suggested.

INTRODUCTION

Endotoxins and lipopolysaccharides from certaingram-negative bacteria have been shown to have a tumornecrotizing property (I I—IS,18—21,26, 27, 34, 35, 38—41).Since such preparations are ill-defined crude materials andsince most contain significant amounts of protein, it is notknown which component (lipid, polysaccharide, or protein)elicits tumor-necrotizing action. Hartwell et a!. (18) andIkawa et a!. (2 1) associate necrotizing activity with intactlipopolysaccharides, and Mihich et a!. (27) associate it withthe lipid component. Rathgeb and Sylvén(34, 35) suggestthat it is due to a lipid-polysaccharide-protein complex,whereas Creech et a!. (1 1) discount the necessity of lipid forthe retention of antitumor activity.

With the availability of core lipopolysaccharides fromrough mutants of S. minnesota, which are of knowncomposition, are practically free of proteins, and contain nonucleic acid, I have investigated their tumor-damagingpotential as determined by an increase in the survival timesof mice bearing Ehrlich solid tumor. A comparison with thelipopolysaccharide isolated from Serratia marcescens andfrom the S form of S. minnesota has also been made. Sincecore lipopolysaccharides from various rough mutants of S.minnesota form a structural homologous series, one canalso assessthe potential of various groups in the elicitationof antitumor activity.

I Supported by grants from the National Cancer Institute of Canada.

2 Research Associate of the National Cancer Institute of Canada.

Present address: Dêpartement de Biologic Cellulaire, Facultê de Mêdecine,Universitê de Sherbrooke, Sherbrooke, Quebec, Canada.

Received March 8, 1973: accepted November 27, 1974.

In a recentstudy, Chiller et a!. (9) haveobservedthat corelipopolysaccharides form Escherichia coli mutants as wellas lipid A are more active than are lipopolysaccharides fromthe smooth strain ofE. coliOl I I in eliciting an adjuvanticityeffect for antibody formation against bovine serum albumin. This is consistent with our observations, which showthat shorter chain core lipopolysaccharides are more effective in enhancing the life of tumor-bearing animals than arelipopolysaccharides containing 0-antigen.

In other studies, it has been observed that certain plantpolysaccharides (36, 42) as well as synthetic glyceryl ethersof small-chain fatty acids (2) have the ability of damagingtransplantable tumors and increasing the survival times ofanimals bearing these tumors. In my experiments I havefailed to observe antitumor activity in the oligosaccharide orpolysaccharide part of the lipopolysaccharides whenamounts equivalent to the lipopolysaccharide were injectedin to tumor-bearing animals. Thus, my results (as well asthose of others in this field) tend to favor a major role forthe lipid part of lipopolysaccharides in the elicitaion ofantitumor activity.

MATERIALS AND METhODS

Lipopolysaccharides. The lipopolysaccharides from thesmooth form and from the rough mutants of S. minnesotawere kindly supplied by Dr. 0. Luderitz, Max-PlanckInstitute fur Immunbiologie, Freiburg, West Germany.Lipopolysaccharide from S. marcescens was obtained fromDifco Laboratories, Detroit, Mich. Lipopolysaccharidesfrom the smooth forms were isolated by the phenol method(43). They contain approximately 4% protein. Core lipopolysaccharides from the rough mutants were isolatedaccording to the method of Galanos et a!. (17). They containless than 0. 1% protein. Chemical data on the compositionand purity of some of these compounds have been described(17, 25). Based on their composition and known structuralfeatures, Table I gives a general view of structures ofvarious core lipopolysaccharides and the lipopolysaccharidefrom the smooth form of S. minnesota.

It must be emphasized, however, that the criteria ofpurity of bacterial lipopolysaccharides are far from established. Although it has once been suggested that the absenceof nucleic acid was the best criterion for their purity (37), ithas become obvious that lipopolysaccharides can be heterogenous in size (37) and that the lipid portion can be very

628 CANCER RESEARCH VOL. 35

Effect of Core Lipopolysaccharides from Salmonella minnesota RMutants on the Survival Times of Mice Bearing Ehrlich Tumor'

Vijai N. Nigam2

Laborazoire de Recherche, Institut du Cancer de Montréal, Hbpital Notre Dame. Montréal, Quebec, Canada

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ChemotypeTrivial nameProbablestructure―RemR595(KDO)3-Lipid

ARd3mR3Hep-(KDO),-Lipid

ARdIP

—mR7Hep-Hep-(KDO)3-LipidARd1P+mRzHep-Hep-(KDO)3-Lipid

A

P,ETNRcPmRSGlc-Hep-Hep-(KDO)3-Lipid

ARbmR345Glc-Gal-Glc-Hep-Hep-(KDO)3-Lipid

AI@

GalP,ETNRamR6OG1cNac-Glc-Gal-Glc-Hep-Hep-(KDO)3-Lipid

AI

GalP,ETNII

(S form)mRSO-antigen-GIcNac-Glc-Gal-Glc-Hep-Hep-(KDO),-Lipid AI@Gal P,ETN

Antitumor Action of Bacterial Core Lipopolysaccharides

Table I

Structure of lipopolysaccharides

aThe abbreviationsusedare: Hep, l-glycero-n-mannoheptose;KDO, 2-keto-3-deoxyoctonicacid: Glc,glucose: GlcNac, N-Acetylglucosamine: Gal, galactose: P. phosphate; ETN, ethanolamine: 0 antigen.(mannose-rhamnose-Gal)@-mannose-rhamnose-ga1actose (n = 30); lipid A, glucosaminyl-fl- I ,6-glucosaminewith fatty acid substituted to hydroxyl and amino groups. Fatty acids found in lipid A are $-hydroxymyristicacid, myristic acid, palmitic acid, and lauric acid.

complex (3 1). Since lipopolysaccharides form aggregates,the molecular weight of the lipopolysaccharide from thesmooth form of S. minnesota has been described as I to 20million (24). Alkali treatment results in considerable disaggregation with the loss of some of the fatty acids and areduction of the molecular weight to approximately200,000 (24). Similarly, core lipopolysaccharides also formaggregates to give molecular weights in the range ofhundreds of thousands. In order to see the effect ofmolecular size on antitumor activity, I have used bothuntreated and alkali-treated core lipopolysaccharidemR595. Recently, Chen et a!. (8) have separated corelipopolysaccharide mR595 into 4 polymeric forms andreport an average molecular weight of 4000, as determinedby vapor pressure depression method.

Alkali-treated core lipopolysaccharide mR595 was prepared by treating 4 mg of the material with 0. 1N NaOH for18 hr at room temperature (25°).The solution was thenneutralized with 0.1 ml of I N HC1 and brought to 10 mlwith the addition of 0.85% NaCI.

Lipid-free oligosaccharide and polysaccharide from corelipopolysaccharides and lipopolysaccharide from smoothform of S. minnesota were obtained by treating 4 mg of thematerial with 1 ml of 0. 1 N acetic acid at l000 for 40 mm.The resulting lipid A precipitate was removed by centrifugation and washed with isoosmotic 0.9% NaCI solution. Thesupernatants were combined, adjusted to pH 7.0 with I NNaOH, and brought to 10 ml with the addition of 0.85%NaCI solution. The supernatant contains all the 2-keto-3-deoxy-octonate present in the lipopolysaccharides.

Animals. White Swiss mice weighing 20 to 25 g, normally

used for carrying Ehrlich ascites tumor, were obtained fromCharles River Breeding ç0@,St. Constant, Quebec, Canada.

Implantation of Tumors. Twenty mice were given injections of 0.5 ml of ascitic cell suspension from Ehrlich ascitestumor-bearing mice. After 7 days all animals showedwell-developed ascites tumor in their peritoneal cavities.The ascites cell suspension obtained from these mice wasused to inoculate s.c. (0.5 ml/mouse) on the back of 100to 250 mice for different set ofexperiments. The mice werekept in groups of 10/cage and were allowed food and waterad libitum. After 6 days mice having no tumors or havingvery small tumors were removed and, out of the remaining mice, the required number was used for the experiments, as described in the text.

Injection of Lipopolysaccharides. Four mg of each lipopolysaccharide were suspended in 10 ml of 0.85% NaCIsolution. The suspension was thoroughly mixed and thecontents were heated for 3 to 10 mm in a boiling water bath.The lipopolysaccharides from S. marcescens and corelipopolysaccharides mR3 and mRS were soluble in cold0.85% NaCI, whereas other lipopolysaccharides dissolvedwhen heated. Solutions of varying degrees of opalescencewere obtained with different lipopolysaccharides.

Two experiments were performed with intact lipopolysaccharides. In the 1st experiment, groups of 8 tumor-bearingmice were given i.p. injections of 0. 1 and 0.2 ml of thesolutions of each lipopolysaccharide. Controls received 0.1and 0.2 ml of isoosmotic 0.9% NaC1 solution. In the 2ndexperiment, groups of 20 mice received i.p. 0.2 ml of 0.85%NaCI (control) per mouse or 0.2 ml of solution of each ofthe lipopolysaccharides per mouse. The number of surviving

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LipopolysaccharideNo.

ofanimals Iiving/totalno. ofanimals in the experimentat dayMean life-span(days) of mice

living < 30daysI46101317202530Control(0.9%NaClsolution)20/2020/2012/205/200/200/2000/200/209.2±

2.9―mR59519/2018/2018/2015/2013/2013/204/204/200/2018.5±8.2―mR319/2015/2012/2011/2010/2010/204/204/202/20'17.3±

11.0'mR720/2020/2020/2010/204/204/200/200/200/2014.1±4.3―mRz18/2018/2018/2015/2015/2014/207/203/203/20'@19.3±7.7―mRS20/2017/2017/2012/2010/202/200/200/200/2013.5±4.7―mR34518/2018/2017/2010/2010/204/202/200/200/2014.9±5.7―mR6O20/2018/2015/2013/207/203/202/200/200/2013.1±6.2emRS18/2015/2014/209/209/204/200/200/200/2012.5±6.7'S.marcescens18/2014/2014/208/207/203/200/200/200/2011.9±

4.9'

V. N. Nigam

animals was recorded every 2 to 3 days.In the experiment with the oligosaccharide or polysac

charide mRS9S, groups of 8 mice received 0.2 ml solution ofuntreated core lipopolysaccharide mRS9S or 0.2 ml solutionofalkali-treated core lipopolysaccharide mRS95 per mouse.

In the experiment with the oligosaccharide or polysaccharide portion of lipopolysaccharides, groups of 8 micereceived i.p. 0.2 ml solution of 0.85% NaCI containingsodium acetate per mouse or a 0.2 ml solution each of thelipid-free oligosaccharide from core lipopolysaccharidesmR59S, mRS. mR6O, and polysaccharide from lipopolysaccharide mRS. These were prepared as described earlier.

The number of survivors was recorded every 2 days.AdjuvancyEffectof CoreLipopolysaccharidesmR595and

mR6O and Lipopolysaccharide mRS. This experiment wascarried out in collaboration with Dr. R. Lallier and Dr. C.Brailovsky. Wistar rats (Flow Laboratories. Rockville,Md.) of 70 g body weight received i.p. 108sheep red cells onDays I, 4, and 7. On Day 7, groups of 3 rats received eithera 0.5 ml solution of phosphate-buffered saline or 0.5 mlsolution of 20 .cg/ml of core lipopolysaccharide mR595 ormR6O or lipopolysaccharide mRs in phosphate-bufferedsaline. The composition of phosphate-buffered saline wasas follows (g/liter of distilled water): NaC1, 8.0; KCI, 0.2;Na2HPO4@ 7H20, 1.4; KH2PO4, 0.2. After 10 days theanimals were bled and the blood of similarly treated animals was pooled. The antibody titers against sheep re@cells were determined in the serum by hemagglutinationassay as described by Beckmann et a!. (5).

RESULTS

We first carried out a preliminary investigation using 2dose levels of the lipopolysaccharides, i.e., 40 and 80gig/tumor-bearing mouse. We selected these levels because,in an earlier experiment on toxicity, these doses werenonlethal for various core lipopolysaccharides and lipopolysaccharides. At a dose level of 40 pg/mouse, in the smallgroup of animals used in the 1st experiment, the mean

survival times in days for control and those treated withmR595, mR3, mR7, mRz, mR5, mR345, mR6O and mRswere, respectively: l0(control), 19, 15, 15, 14, 12.5, 13.2, 13,and I I. At a dose level of 80 ag/mouse, the mean survivaltimes in days were, respectively: 9.5 (control), 23.5, 15, 13,19.5, 15.5, 12.5, 11, and 11 days. Since there was augmentation in the survival times at the higher doses by some of thelipopolysaccharides, a 2nd experiment was carried out with20 tumor-bearing mice per group using a dose level of 80@zg/mouse. The results of this experiment are shown inTable 2. It was observed that mean survival days wereincreased nearly 2-fold by the injection of mR595, mR3,and mRz. In case of mR3 and mRz, 2 out of 20 and 3 out of20 mice, respectively, had complete regression of tumor.Core lipopolysaccharides containing short chains of sugar(mRS95 and mR3) or those containing charged groups(mRz) were more effective than were core polysaccharideswith longer sugar chains (mR345 and mR6O), even thoughthese latter contained charged groups. Lipopolysaccharidewith 0-antigen (mRS and lipopolysaccharide from S.marcescens) were relatively poor in enhancing survival time.The pathological manifestations of lipopolysaccharidesfrom various sources against transplantable tumors havebeen previously shown to be gross hemorrhagic necrosis ofthe tumors. In our experiments with core lipopolysaccharides, gross necrosis of the tumor mass was observed,especially in regressing tumors.

When core lipopolysaccharide mR595 was comparedwith alkali-treated mR595, no significant differences in thesurvival times of the tumor-bearing mice were observed(Table 3). On the other hand, when oligo- and polysaccharides obtained from core lipopolysaccharides mR595, mR5,and mR6O and lipopolysaccharide mRs were tested forantitumor activity, neither an increase in the survival timeas compared to control was observed nor was there an increase in the survival with short-chain oligosaccharide incomparison to that with a longer chain or with the polysaccharide with 0-antigen activity. Thus, the lipid-freeportion of the lipopolysaccharide molecule by itself elicits

Table 2

Effect oflipopolvsaccharides on the survival time of mice bearing Ehrlich solid tumor

a Mean ± S.D.

“p> 0.001C Surviving animals free of visible tumor.

“0.001> p > 0.01.eO.Ol> p > 0.02., 0.02 > p > 0.05.

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Material injectedSurvivaltime

(days)0.9%

NaCI solution (control)9.3 ±3.0°mR595―17.4±6.2Alkali-treatedmR59518.1

±4.70.9%

NaCI solution containing sodium8.7 ±2.9acetateOiigosaccharide

from mR5959.0 ±3.0Oligosaccharidefrom mRS8.5 ±2.5Oligosaccharidefrom mR6O8.5 ±3.2Polysaccharide

from mRs9.4 ±4.8

A ntitumor Action of Bacteria! Core Lipopo!ysaccharides

reduction in protein contamination than does the originalphenol-water method (43), without a loss in many of thebiological parameters associated with bacterial lipopolysaccharides. Further, it is reasonable to expect association of asmall amount of amino acids in the isolated bacterial corelipopolysaccharides and bacterial lipopolysaccharides because they are bacterial@ cell wall components and arepresumably built in the cell membrane with a short aminoacid chain attached to them in much the same way asmucopolysaccharides, which also contain a short aminoacid chain (29). A similar view has been expressed byNowotny (3 1). Pronase-treated lipopolysaccharide, whichcontains only a few amino acid residues, exhibits most of thebiological activities normally associated with endotoxins(22).

Since bacterial Iipopolysaccharides form aggregates andsince they contain many charged groups, it has beensuggested that antitumor activity may be associated with thesize of the molecule and/or its charge. My experimentsprovide little support for such suggestions. The treatment ofmR595 with dilute alkali, which results in the disaggregation of the molecule, does not result in a decrease in thelength of the survival time of the tumor-bearing mice.Furthermore, a look at the proposed structures (Table 1)shows chain elongation does not always result in thealteration of the charge (compare mR595 with mR7 ormR5), although it is accompanied by lowering in thesurvival time of the tumor-bearing animals (Table 2). Eventhough it may appear that the presence of phosphate andethanolamine in mRz is responsible for its greater antitumor activity over mR7, this is denied in the comparison ofmRS with mR345. In my opinion, the addition of sugarresidues alters the ratio between the hydrophilic andhydrophobic portions of the core lipopolysaccharides andmay also result in a change in the steric configuration of themolecule. This is reflected by an alteration in the antitumoractivity of the lipopolysaccharide. Insofar as the activeportion of the lipopolysaccharide molecule is concerned, itseems to be the lipid portion. The oligo- and polysaccharideportions exhibited no antitumor activity (Table 3). Duringdilute acid hydrolysis of lipopolysaccharides, the lipidportion precipitates as an aggregate because it is highlyhydrophobic. Association with hydrophilic sugars apparently keeps it in solution. Conjugate of lipid A with bovineserum albumin has been prepared and this material has beenshown to have antitumor activity (1). Lipid A solubilized in0.5% triethylamine also exhibits antitumor activity (7).

From a look at the structures of the core lipopolysaccharides and lipopolysaccharide (Table I) and the data presented in Table 2, certain conclusions can be drawn. (a)Antitumor activity seems to decrease as the carbohydratechain lengths increase; (b) the nature of the sugar addedin chain elongation is immaterial; (c) the presence of phosphate and ethanolamine has no clear effect on the antitumoractivity; and (d) lipid A region seems to be necessary in theelicitation of antitumor activity. Results similar to thosepresented in this paper were obtained by Nowotny et al.(32) when they compared tumor-hemorrhagic activity ofcore lipopolysaccharide mR595 with the activity of corelipopolysaccharide from S. marcescens.

Table 3

Survival times of Ehrlich tumor-bearing mice given injections of mR595,alkali-treated mR595, and oligosaccharides and poivsaccharide derived

f rom variouslipopolvsaccharidesDetails of the experiment are provided in “Materials and Methods.―

a Mean ±S.D.‘1Tumor regressed in I animal. It was deleted in the calculation.

no antitumor activity.In order to substantiate that the action of lipopolysaccha

rides of various chain lengths is due to their relativeadjuvancy action, serum antibody titers against sheep redcells were obtained after injection of mR6O, and mRs intorats previously treated with sheep red cells, which was usedas an antigen. The dilution of serum required for immunelysis of sheep red cells for sera obtained from control ratsand lipopolysaccharide mRS- and core- lipopolysaccharidesmR6O and mR595 injection-treated rats were as follows:control (phosphate-buffered saline injected), @/@;mRS.¼o: mR6O, %4; mR595, 1420 mR595 m@definitelysuperior to mR6O and mRS in enhancing the anti-sheep redcell antibody in the rat. Comparative increases in survivaltimes due to mR595, mR6O, and mRS (18.5, 12.5, and I 1.9)are in the same order as the antibody titers (142o,@ andlAo)

DISCUSSION

Antitumor action of endotoxins of bacterial core lipopolysaccharide and lipopolysaccharides appears to be welldocumented. Active research to determine their efficacyhas been erratic, presumably because of a wide variety ofpathophysiological manifestations that accompany their usein animals. With a better understanding of their mode ofaction and modification of their structure to reduce theirtoxicity (30, 31), it seems possible that these substancescould emerge as useful aids in cancer therapy.

There is apparently a lack of agreement regarding theportion of the endotoxin molecule that is responsible fortheir tumor-necrotizing action. The presence of peptideresidues in most endotoxin preparations clouds the role oflipid and polysaccharide units of lipopolysaccharide in themediation of tumor necrosis. In this experiment, the effectof peptide contamination is minimized because the mosteffective glycolipid, m R595, contains nondetectableamounts of amino acids, whereas the least effective lipopolysaccharide, mRS, contains up to 4% amino acid. Themethod of isolation of bacterial glycolipids from R mutantsdescribed by Galanos et a!. (17) results in considerably more

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V. N. Nigam

As regards the mode of action of bacterial core lipopolysaccharides in damaging tumors, it seems to be mediated byan increase in the immune response against the tumortransplant (28). Data given in the text indicate that antibody titers are increased against sheep red cells by lipopolysaccharides, and the order of effectiveness with the 3lipopolysaccharides used is mR595, mR6O, mRS. This isalso the order of increase in the survival times of tumorbearing mice with the same lipopolysaccharides. It is interesting to note (6) that core lipopolysaccharides from S.minnesota R mutants inhibit the growth of cultured transformed cells but not of counterpart normal cells, and thedegree of inhibition follows the order mR595, mR7, mRS.mR6O, a pattern similar to the increase in survival timesfound in my experiments. Although experiments with cultured cells may suggest core lipopolysaccharide binding bythe tumor cells (3, 4) and the inhibition of their growth (6),the amounts administered are small in animals and a singleinjection is sufficient for tumor damage, whereas culturedtransformed cells require contact with the core lipopolysaccharide at all times for their growth inhibition.

The role of antitumor antibody in eliciting immunedamage of tumor in the animals is at present unclear. Prehn(33) has observed that a high antibody titer against a tumortransplant is obtained in animals with regressing tumors,whereas a low antibody titer seems to favor the growth ofthe tumor transplant. It seems possible that the role ofantibody lies in its ability to inactivate tumor antigens shedby the tumor, so that an immune attack by the sensitizedlymphocytes can be launched. On the other hand, increasedgrowth of tumors by antibody is a more complex phenomenon and the reason for it can only be speculated about. It ispossible that antibody caps tumor cell antigens and thenextrudes them into the surrounding fluid (23), and thechange in tumor cell membrane permeability due to theremoval of antigen allows for their greater growth. In anycase, it appears that a fine balance between the humoralantibody, the size of the tumor, and its state of differentiation plays a part in the ability of a tumor to escape immuneattack by the lymphocytes.

My work and the work ofthose using lipopolysaccharidesand endotoxins for tumor regression contrast with theinvestigations in which plant polysaccharides have beendemonstrated to have antitumor activity. However, polysaccharides require higher doses (36, 42) and necrotize tumorswithout hemorrhage ( I 6). The latter effect is presumablyrelated to the exceptional sensitivity of blood vessels in tumors to the hemorrhage-inducing effect of the endotoxins(10).It is possiblethat theassociationof tumorantigenswith the blood vessels makes them a target for immuneattack by increased antitumor antibody.

ACKNOWLEDGMENTS

I wish to thank Huguette Basilières for her technical assistance. I amalso thankful to Dr. A. Nowotny for reading an earlier version ofthis paperand making helpful suggestions.

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1975;35:628-633. Cancer Res   Vijai N. Nigam  R Mutants on the Survival Times of Mice Bearing Ehrlich Tumor

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