Tumors Esters of Chlorohydroxyacetone in ...

Esters of Chlorohydroxyacetone in Chemotherapy of Murine Tumors Paula Babiarz-Tracy, Dennis McCarthy, Paul Simon, et al. Cancer Res 1980;40:3274-3280.

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[CANCER RESEARCH 40, 3274-3280, September 1980] 0008-5472/80/0040-0000$02.00

Esters of Chlorohydroxyacetone in Chemotherapy of Murine Tumors1 Paula Babiarz-Tracy,2 Dennis McCarthy, Paul Simon,3 William J. Burlingham,2and Thomas P. Fondy4 Department of Biology, Syracuse University, Syracuse, New York 13210

ABSTRACT

INTRODUCTION

1-Deoxyhalo analogs of dihydroxyacetone and of DL-glycerol The plasma membrane of the cell governs biological func were tested for cytostatic effects in vitro against dividing cell tions important in neoplasia, including transport of ions and lines of murine Li 210 leukemia and Ehrlich carcinoma, against metabolites, immunogenicity of surface antigens, cell-cell in primary stationary Ehrlich cells in vitro, and for in vivo chemo teractions, tissue and organ morphology, and recognition of therapeutic effects against a number of murine tumors. Ben molecular control signals that regulate proliferation and differ zoate, p-nitrobenzoate, and 3,5-dinitrobenzoate esters of 1- entiation. Modifications of cell surface groups by chemical and chloro-3-hydroxyacetone inhibited proliferation in log-phase biological agents have produced effects on membrane-me cultured cells by 50% at concentrations between 2 and 4 zM diated phenomena in both normal and neoplastic cells (1 , 7, and inhibited thymidine incorporation by 50% into stationary 1 0, 1 2, 1 4, 1 5). This suggests that drug-induced alteration of cells at 60 to 150 jzM. Comparable in vitro activities were membrane structures may provide new approaches to both obtained with the acetate ester and with chloroacetone. Cor chemotherapy and immunotherapy. Such therapeutic ap responding glycerol analogs and the fluoro analogs in either proaches based on drug-induced alteration of plasma mem the glycerol or dihydroxyacetone series were inactive. Chlo brane structures have the added potential advantage that drug rohydroxyacetone benzoate, chlorohydroxyacetone p-nitro resistance based on failure of drug transport or resistance benzoate, and chlorohydroxyacetone 3,5-dinitrobenzoate pro arising from drug deactivation by intracellular enzyme systems duced 50 to 100% cure of 2.5 x 1O@Ehrlich tumor cell may not be a significant limitation. challenges in C57B1/6 x DBA/2F1 (hereafter called B6D2F1) We have observed that murine DBA/2-derived Friend eryth hosts with only a single drug injection, but only if the hosts roleukemia cells treated in culture with CLHAB5 or with related were immunocompetent. The 3,5-dinitrobenzoate ester was analogs and implanted into syngeneic hosts generate altered the most effective, while the acetate ester was only weakly host-tumor interaction of protective benefit to the host. Molec active and neither the phosphate ester nor chloroacetone was ular features of the active analogs suggest that they function active in vivo. Chlorohydroxyacetone 3,5-dinitrobenzoate pro as lipophilic alkylating agents and thus may exert effects at duced significant increases in life span and a number of long least in part by reaction with electron donors in lipophilic cell term cures of P815 mastocytoma in semisyngeneic B6D2F1 compartments, including the plasma membrane (6). hosts, but had no effect in syngeneic DBA/2 hosts. The analog Alteration of host-tumor interaction by prior treatment of the was inactive against Li 210 leukemia, EL4 lymphoma, and tumor cells in culture has a number of attractive features with CaD2 mammary adenocarcinoma in B6D2F1 semisyngeneic respect to characterization of biochemical and immunological hosts. Tumor-bearing treated animals were able completely to events. However, it is also important to determine whether resist an added normally lethal tumor challenge given as soon direct treatment in vivo is capable of altering host-tumor inter as 24 hr after single-drug injection chemotherapy. After initial action in a manner that is empirically beneficial to the host. We tumor challenge and single-injection treatment, viable tumor have therefore examined the in vivo chemotherapeutic effects cells were recovered from treated animals that were lethal to of esters of chlorohydroxyacetone and related compounds in naive passive recipients, even though cagemates of the treated allogeneic and syngeneic tumor model systems. The work was tumor cell donors were cured. These results show that chlo designed to determine (a) whether there are significant in vivo rohydroxyacetone benzoate esters are chemotherapeutically chemotherapeutic effects with CLHAB and related benzoate active in vivo under conditions that suggest drug-induced al esters and (b) whether any such observed effects reflect altered teration of host-tumor interaction after initial direct cytotoxicity. host-tumor interaction beyond direct cytotoxicity, possibly par Such results are consistent with previously obtained altered alleling the in vitro alteration effects observed in the Friend host response to murine Friend erythroleukemia cells treated erythroleukemia studies. in vitro with these same agents. It is not yet known whether the MATERIALSAND METHODS mechanisms responsible for the in vitro alteration of erythroleu kemia cells are related to in vivo chemotherapy obtained in this Halo Analogs. The agents used in this work are shown in work with Ehrlich tumor cells. Chart 1. The DL-i -deoxyhaloglycerols, their 3-phosphate es ters, and the halohydroxyacetone phosphates were prepared and characterized as previously described (3, 5, 9). The other halohydroxyacetone analogs and hydroxyacetone benzoate National Cancer Institute. were prepared and characterized as detailed elsewhere (13, 1 This

work

was

supported

by

2 Present

address:

Mayo

3 Present

address:

Department

Clinic,

USPHS

Research

Rochester,

of

Minn.

Microbiology,

Grant

CA-i

0250

from

the

55901.

University

of

California,

Los

Angeles, Calif. 90024. 4 Recipient

of

USPHS

Career

Development

Award

CA-70332

from

the

National

Cancer Institute. To whom requests for reprints should be addressed, at Depart ment of Biology, Syracuse University, 108 College Place, Syracuse. N.Y. 13210. Received March 6, 1978; accepted June 11, 1980.

3274

5 The

abbreviations

used

are:

CLHAB.

chlorohydroxyacetone

benzoate;

PFC.

plaque-forming cell; lC@, concentration inhibiting cell proliferation by 50%; CLHA-pNB, chlorohydroxyacetone p-nitrobenzoate; CLHA-3,5-DNB, chlorohy droxyacetone 3,5-dinitrobenzoate; PMN. polymorphonuclear leukocyte.

CANCERRESEARCHVOL. 40


Chemotherapy with Esters of Chlorohydroxyacetone i 6). Chloroacetone

and i ,3-dichloroacetone

were obtained

from Eastman Kodak Co., Rochester, N. V. Animals and Tumors. The maintenanceand in vivotesting of Li 210 leukemia has been detailed previously (4). P8i 5 mastocytoma of DBA/2 origin, EL-4 lymphoma of C57B1/6 origin, and CaD2 mammary adenocarcinoma of DBA/2 origin were supplied by Dr. Bertie Argynis and Dr. Russell Toman, Upstate Medical Center, Syracuse, N. V. These tumors were maintained by weekly i.p. injection of i 06 cells into syngeneic female hosts. Ehnlich ascites carcinoma cells were obtained from the source cited in our earlier work and were maintained as set forth there (1 7). All experiments were performed using C57BI/6 x DBA/2 F1 mice (hereafter called B6D2F1) of either sex, obtained from Dr. W. T. Bradnen, Bristol Laboratories, Syracuse, N. V. Che motherapy testing was conducted in mice weighing between i 8 and 24 9 with 6 to 8 mice/group.

Dose schedules for each

compound were determined from previously established 50% lethal dose values (13). None of the drug schedules listed caused a weight loss greater than 5%. Waten-soluble corn pounds were administered i.p. in sterile 0.85% NaCI solution. Water-insoluble compounds were suspended in i 0% Tween 80 in sterile 0.85% NaCI solution. Cell Cultures. All cell culture Supplieswere purchasedfrom Grand Island Biological Company, Grand Island, N. Y. Dividing Li 210 leukemia and Ehnlich carcinoma cell lines were main tamed and used as previously described (i 7). Ehnlich tumor cells obtained directly from the peritoneal cavities of passage mice were treated in vitro with different concentrations of the test agents for 1 hr in stationary suspension. Treated cells were washed twice with sterile 0.85% NaCI: 0.02% KCI:8 mM Na2HPO4:2m@KH2PO4,pH 7.4; suspended in Roswell Park Memorial Institute Medium i 640: i 0% fetal calf senum:2% penicillin-stneptomycin:25 mM4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid; and added to micnotitenwells at 2 x i 0@cells/well. Tnitium-labeled thymidine (i MCi/well; ICN Chemical Company, Irvine, Calif.) was added, and incorpora tion was permitted to proceed at 37Â°in 5% CO2 for either 10 or 18 hr. Control cells incorporated 2.2 x i o5 cpm/i 06 cells into macromoleculan components in the i 0-hr incubation and 3.9 x i o@cpm/i 06 cells in the 18-hr incubation. Immunosuppressionof Mice. Mice were subjectedto whole body X-irnadiation at 500 rads from a General Electric Maximar 250 type III X-ray therapy unit (2i 5 kV, i 5 ma) 24 hr prior to tumor transplantation. Leukocyte Influx. Mice were given injections of the normally curative doses of each agent and sacrificed i , 2, 3, and 5 days after drug treatment. Penitoneal cells were quantitatively han vested, and differential staining was performed as detailed previously (17). Primary Humoral Immune Response.The lgG PFC response against sheep RBC was determined by a modification of the Jerne assay (1 1). The 1gM response was determined by he molysis in aganose gel by the method of Fuji (8). RESULTS

In Vitro Cytostatic Activity against Proliferating Ehrlich Carcinoma and L1210 Leukemia Cell Lines. 1C50'swere de tenmined for each of the compounds shown in Chart 1. The results were essentially the same as those previously reported SEPTEMBER

GLYCEROL

ANALOGS:

CH2CI (F)

.8i.

B2@

COMPOUNDS

CHR1

-OH

-P@((,@JNH'@

CldG-3-P

CH2R2

(DICYCLOHEXYL

AMMONIUM

SALT)

0 -OH

-O'C-@

CldG-3-BENZOATE

â€˜OH

0 -O'C-@-NO2

CIdG-3-p-NI

9

0

-O-C-CH3 DIHYDROXYACETONE

CH2CI (F)

TRO-BENZOATE

CIdG-2,3 - DIACE TATE

-O-C-CH'@ ANALOGS:

B@

C=O

-Pc@(Na1@Hr

I

9

CH2R

CIHAP(SODIUM

-O-C-CH3

C HA-ACETATE

0 -o-@: -@

CIHA- BENZOATE

SALT)

0

-O-C-@'NO2

CIHA-p -NITRO- BENZOATE

9 .NO2 â€˜NO2

CIHA-3,5-

DINITRO - BENZOATE

CONTROL COMPOUNDS: 0 CH3 C CH2CI

CHLOROACE TONE

0 CI CH2C CH2CI

DICHLOROACE TONE

90 CH3CCH2OC

HYDROXYACETONE-BENZOATE

Â©

Chart 1. Structures of analogs. cIdG-3-P, chlorodeoxyglycerol 3-phosphate; cldG-3-benzoate, chlorodeoxyglycerol 3-benzoate; cldG-3-p-nitro-benzoate. chlorodeoxyglycerol 3-p-nitrobenzoate; cIdG-2,3-diacetate, chlorodeoxygly cerol

2,3-diacetate;

chlorohydroxyacetone

CIHAP, chlorohydroxyacetone

phosphate;

CIHA-acetate,

acetate.

for dividing Friend erythroleukemia cells (6), with the exception of the benzoate and p-nitrobenzoate esters of DL-i -chlono-i deoxyglycerol, which gave lCse's in the range of 20 to 30 @zM in the Ehrlich and Li 210 systems compared to 500 @tM ongreater for the benzoate ester in the Friend erythroleukemia system. CLHAB, CLHA-pNB, CLHA-3,5-DNB, and the acetate esters of chlorohydroxyacetone, as well as chlonoacetone and i ,3-di chloroacetone, all gave lC50's between 2 and 4 @tM, whereas the corresponding glycerol analogs, the fluoro analogs in either the glycerol on the halohydnoxyacetone series, and the hydro philic phosphate ester of chlorohydroxyacetone were less cy tostatic by factors ranging from 5- to 250-fold or more. As with the Friend erythroleukemia system, these results show that the presence of an a-chloroketone group and a relatively lipophilic molecule are required for activity.

In Vitro Cytotoxicity against in Vivo-derivedEhrlich Tumor Cells. Chlonohydroxyacetone esters, chloroacetone,and chlo roacetate were tested against Ehnlich tumor cells derived di rectly from passage animals. Cytotoxicity was measured by inhibition of tnitiated thymidine incorporation and by in vivo bioassay for tumonigenicity of treated cells. Chart 2 shows that the benzoate and acetate esters of chlonohydroxyacetone in hibited thymidine incorporation more strongly than did chlo roacetone. CLHA-3,5-DNB was markedly less cytotoxic than

1980


3275

P. Babiarz-Tracy et a!.

-J

mice, as shown in Table 2. The same dose regimen was ineffective against a challenge of 5 x 1O@P815 cells in syn geneic DBA/2 hosts and was also ineffective against chal lenges of 1 x i 06 EL4 lymphoma cells (C57BL/6 origin) or 1 x 106 CaD2 mammary adenocarcinoma cells (DBA/2 origin) in B6D2F1 semisyngeneic hosts. Increases in median life spans of 20 to 40% were obtained against Li 210 leukemia (DBA/2 origin) in B6D2F1 hosts when high cell challenges (1o@cells i.p.) were treated beginning within 3 or 6 hr after implantation using multiple-dose injections

100

F-

z

8 l0

Ui

(CLHAB, >.

@

Cl-HAS

__..-0@, _â€˜,-- â€˜.

-... .@â€”

100

200

300

400

500

pM

Chart 2. In vitro cytotoxiÃ§ityagainst Ehrlich tumor cells. In vivo-derived Ehrlich ascites tumor cells were washed with sterile 0.85% NaCI solution, resuspended at 10@cells/mi in Earles balanced salt solution, and treated for 1 hr in stationary culture with agents dissolved in ethanol. Final ethanol concentration did not exceed 1%. Control cells were treated with 1% ethanol. Cells were washed twice with sterile 0.85% NaCI:O.02% KCI:8 mM Na2HPO4:2mM KH2P04, pH 7.4; suspended in Roswell Park Memorial Institute Medium 1640: 10% fetal bovine serum:2% penicillin-streptomycin:25 m@4-(2-hydroxyethyl)-1 -piperazineethane sulfonic acid; and added to microtiter wells at 2 x 1o@cells/well. Tritium-labeled thymidine (1 @Ci/weII) was added, and incorporation was permitted to proceed at 37' in 5% CO2 for either 10 or 18 hr. Control cells incorporated 2.2 x 1O@ cpm/106 cells into macromolecular components in the 10-hr incubation and 3.9 x 1o@cpm 10@cells in the 18-hr incubation.

the other agents, and chloroacetate exerted no measurable cytotoxic effect. The relationship between the thymidmneincorporation assay and proliferative viability was established by in vivo reimplan tation of analog-treated tumor cells and determination of tu morgenicity. Chart 3 shows that the in vivo bioassay directly paralleled the in vitro thymidine incorporation assay. Cells treated with 500 @tM CLHA-3,5-DNB remained tumorigenic to some of the animals, while cells treated with 300 jzMCLHAB or CLHA-pNB were completely nontumorigenic. In Vivo Chemotherapy against Ehrlich Ascites Tumor. CLHAB, CLHA-pNB, and CLHA-3,5-DNB given in multiple-dose regimens produced 50 to 80% long-term survivors in repeated experiments and were able to cure 40 to 50% of the animals, even when given as a single-drug injection (Table 1). The acetate ester of chlorohydroxyacetone under comparable con ditions was very weakly active at best. Chloroacetone, despite its structural relationship to the active compounds and its cytotoxicity in vitro, showed no chemotherapeutic activity, even at cumulative doses exceeding those used for the benzoate esters, either in survival assays (Table 1) or in the determination of total packed cell volume 8 days after tumor challenge and drug treatment (data not shown). Neither dichloroacetone, hydroxyacetone benzoate, 1-fluoro-3-hydroxyacetone ben zoate, the phosphate esters of either chlorohydroxyacetone or fluorohydroxyacetone, nor any of the compounds in the glyc erol series (see Chart 1) produced significant effects in survival assays when administered at the highest tolerated in vivo doses. Chemotherapy of DBA/2 and C57BL/6 Tumors in Synge neic and Semisyngeneic Hosts. CLHA-3,5-DNB administered i.p. to animals bearing various i.p. challenge doses of P8i 5 mastocytoma cells produced significant increases in median and mean survival and a number of long-term cures in B6D2F1 3276

50 mg/kg/day,

Days

1 to 4; CLHA-pNB,

50 mg/kg/

day, Days 1, 3, and 5; or CLHA-3,5-DNB, 100 mg/kg/day, Days 1, 3, and 5). These effects were not observed at chal

I F-

lenges

of

1 06

cells

the 1 0@ cell challenge

or

less

or

if

was delayed

the

beginning

of

treatment

until 1 2 or 24 hr after

of

tumor

implantation. Role of Host Immunocompetencein Cure of Ehrlich Tumor with Chlorohydroxyacetone Esters. It was apparent that some features of host, tumor, and drug interaction other than direct cytotoxicity were in part responsible for the cures obtained in the Ehrlich tumor system, since the cytotoxic properties of the chlorohydroxyacetone esters were only weakly effective or completely ineffective in other tumor systems. Moreover, the most effective of the analogs with respect to chemotherapy in vivo, CLHA-3,5-DNB, was the least cytotoxic of the active analogs tested in vitro against Ehrlich cells. In order to test for the extent of host participation in the cures obtained with the chlorohydroxyacetone esters, the esters were used for che motherapy either in single-dose or in multiple-dose regimens in animals that had been partially immunosuppressed by expo sure to 500 rads of whole-body X-irradiation prior to tumor challenge and drug treatment. The results of multiple-dose treatments in normal and immunosuppressed tumor-bearing hosts are shown in Chart 4A, and the single-dose results are shown in Chart 4B. Immunocompetent control animals re sponded with the proportion of survivors expected from earlier experiments shown in Table 1. For all of the treated immuno suppressed animals, there was an increase in median life span compared to untreated suppressed controls, as might be ex pected due to direct cytotoxicity of the agents, but chemo therapeutic benefit was transient, and none of the animals were long-term survivors. Only immunocompetent hosts could be cured of Ehrlich tumor challenge by either single- or multiple dose treatment.

100pM

300pM

500ijM

Chart 3. In vivo bioassay for in vitro cytotoxicity. Ehrlich cells treated as detailed in Chart 2 were washed twice with sterile 0.85% NaCI:0.02% KCI:8 mt@i Na2HPO4:2mMKH2P04,pH 7.4, and 2.5 x 10@cells/mouse were implanted i.p. into groups of 10 B6D2F, hosts. The percentage of deaths was determined 60 days after implantation with treated tumor cells.

CANCERRESEARCHVOL. 40 Downloaded from cancerres.aacrjournals.org on June 4, 2013. © 1980 American Association for Cancer Research.

Chemotherapy with Esters of Chiorohydroxyacetone Table 1 chemotherapy

of Ehrlich ascites tumor with chiorohydroxyacetone

esters

Survival assays Cumula tive dose schedule (mmol/kg)

Dose schedule (mg/kg/day)

Compound

25(Days 1,3,5,7)

CLHAB

50(Day

1); 25(Day

3)

50 (Day 1)

60-day survivors/ no. of mice treated

Survi vors (%)

No. of experi ments

0.47 0.36 0.24

15/29 7/10

52 70

5

16/39

41

7

76 44

5 7

CLHA-pNB

50(Days 1,3,5,7) 100 (Day 1)

0.77 0.39

22/29 17/39

CLHA-3,5-DNB

75(Days 1,3,@,7) 100 (Day 1); 50 (Day 3)

0.99 0.50 0.50

24/29 8/8 20/39

83 100 51

5 1 7

25(Days 1,3,5,7) 36 (Day 1); 18 (Day 3) 50 (Day 1)

0.66 0.36 0.33

1/20 0/10

5 0

2 1

20

2

25(Days 1,3,5,7)

1.08 0.90

0/6 0/10

0 0

1 1

3/224

1

36

150(Day Chlorohydroxyacetone

acetate Chloroacetone

55(Day

1)

1); 27(Day

vehicle controls

3)

4/20

Table 2 Chemotherapy against P815 mastocytoma with CLHA-3,5-DNB Treated mice received 100 mg/kg (0.33 mmol/kg) p. on Day 1 and 50 mg/ kg (0.1 7 mmol/kg) i.p. on Day 3. No. of sur MedianMeanvivors/no.P81 5 cells i.p.survivalsurvivalaof mice(Day

treatment (days)(days)treatedFemale

0)Drug hosts5x105Treated 0/101

B6D2F

Controls

42 3444Â±8'@ 34 Â±22/11

Controls

80 2756

Â±9 36 Â±74/8

25 Controls 0/5Female 2233

Â±9 24 Â±21/6

x 1o@Treated

1/72 x I o7Treated

hosts5 x io@Treated

DBA/2 21 Controls

a Animals

surviving

beyond

80

2420 days

were

entered

Â±2 23 Â±20/5 into

the

calculation

0/5 of mean

survival as if they had died on Day 80. b Mean Â±S.E.

Tumorigenicity of Ehrlich Cells in Hosts Immunosup pressed by X-lrradiation. Evaluation of the results shown in Chart 4 requires a quantitative determination of the tumori genicity of the Ehrlich tumor cells in immunosuppressed hosts. Animals partially immunosuppressed by whole-body X-irradia tion under the conditions used in Chart 4 were given graded doses of Ehrlich cells ranging from 1 x 1O@ to 2.5 x 10@cells, and survival results were compared with those of matched immunocompetent controls. Inocula of 1 x 106 cells were lethal in 80 to 100% of the mice, whether the animals had been immunosuppressed or not, and median survival times were 20 (normal hosts) to 23 (suppressed hosts) days. Inocula of 2.5

DAY

DAY

Chart 4. Effect of host immunosuppression on chemotherapeutic activity of esters of chlorohydroxyacetone. Ehrlich tumor cells (2.5 x 10@)were given p. on Day 0, and either multiple doses of the esters were given p. on Days 1, 3, 5, and 7 (A) or a single dose was given p. on Day 1 (B). A, results are pooled from triplicate determinations for a total of 15 animals for each line shown. Cumulative doses: CLHAB, 0.47 mmol/kg; CLHA-pNB, 0.77 mmol/kg; CLHA-3,5-DNB, 0.99 mmol/kg. B, results are the average of duplicate experiments for a total of 10 animals for each line shown. CLHAB, 0.24 mmol/kg; CLHA-pNB, 0.38 mmol/ kg; CLHA-3,5-DNB, 0.50 mmol/kg. . control suppressed animals; treated suppressed animals; â€” â€” â€” â€”, treated immunocompetent

animals.

cells were essentially nontumorigenic in X-ray-treated hosts. These results establish that somewhere between 2.5 x 1O@ and 1 x 106 viable Ehrlich cells are required to kill immuno x 1o@cells were nontumorigenicin normalanimalsbut fully suppressed B@D2F1hosts within 24 days of tumor challenge. tumongenic in X-ray-treated hosts, with median survival times Since the median survival times shown for the various X-ray of 24 days in one experiment and 36 days in another. An treated groups in Chart 4B are 25 days or less, these results inoculum of I x 1o@was also fully tumorigenic in suppressed indicate that approximately 1 x 106 Ehrlich cells have survived animals and gave a median survival of 39 days, while 1 x 1O@ the cytotoxic action of the analogs. SEPTEMBER

1980

3277


P. Babiarz-Tracy et a!. Resistance to Additional Viable Tumor Cell Challenge in Drug-treated Animals. Since the number of viable Ehrlich cells surviving drug treatment is in the range of i x 106 and this number is above, but close to, the tumorigenic threshold in immunocompetent hosts, additional evidence is required to confirm that treated hosts are capable of rejecting a normally lethal surviving fraction of tumor cells. To determine whether drug-treated immunocompetent animals were capable of re jecting an otherwise lethal tumor cell challenge, animals treated with a curative dose of CLHA-3,5-DNB 1 day after challenge with 2.5 x 10@Ehrlich cells were given a second challenge with graded doses of Ehrlich cells 24 hr after drug treatment. We have separately determined (data not shown) that CLHAB loses its reactive chloride rapidly under physiological condi tions and has essentially disappeared after 1 hr. Table 3 shows that animals subjected to an additional tumor burden of be tween i x 106 and 2.5 x 106 Ehrlich cells, doses that were lethal to 100% of control mice, were at least partially protected. These animals may have been completely protected, since the primary treatment used is capable of curing only about 50% of the challenged animals, even without an additional cell burden administered on Day 2 (see Table 1). Survival of Viable Tumor Cells In Drug-treated Animals. It is apparent that drug-treated animals are capable of rejecting an additional otherwise lethal tumor challenge given shortly after initial challenge and curative drug treatment. It is not necessarily true that the initial cure itself depends on augmen tation of host resistance to an otherwise lethal surviving tumor cell fraction. In order to determine whether the hosts were in fact resisting a lethal tumor challenge as part of the curative effects of the chlorohydroxyacetone esters, we transferred surviving Ehrlich tumor cells from treated hosts to matched naive control animals. On Day 8 following challenge with 2.5

x 10@Ehrlichcells and cure withCLHA-3,5-DNB, peritoneal cells from each of 9 donor mice were separately collected, washed, and transferred to each of 9 recipient mice. Eleven other drug-treated tumor-bearing animals were retained as survival controls. Of the 9 recipients of the peritoneal contents remaining in the 9 donor mice after a single injection of CLHA 3,5-DNB, 7 mice died, demonstrating that at least 1 x 106 viable Ehrlich cells had remained in the original treated donors. In the survival assay conducted in parallel with the primary treated animals, 8 of the 11 mice were cured, a proportion consistent with that expected from the results previously shown inTable 1. Effects of Drug Pretreatment and Antigenic Load on Tu morigenicity of Cells. Host participation in drug-induced cure could arise from indirect effects of the drug on the host or from Table 3 Ability of drug-treated

hosts to reject additional cell burden

Mice received 25 x 106 Ehrlich cells. On Day 1, CLHA-3,5-DNB was admin istered (0.5 mmol/kg). On Day 2, an additional cell burden was given to the mice as listed in the table. These challenges were 100% tumorigenic in control mice (5 to 10 mice/group).No. of survivors/no. of mice Additional treated1.0x106 cell burden 6/10

3278

i.5x106

3/5

2.5 x 106 10 x 106 20 x 106

3/10 0/5 0/5

indirect effects of tumor cell cytotoxicity on the host and might not involve simultaneous effects on drug-tumor-host interac tions. In chemotherapy of Ehrlich tumor with N-haloacetylhex osamine tetra-O-acetates (17), we have observed that i.p. injection of the chemotherapeutic agents prior to i.p. tumor challenge enhanced host resistance. In order to determine whether the chlorohydroxyacetone esters exhibit a similar ef fect, we administered a normally curative dose of CLHA-3,5DNB i.p. 24 hr before i.p. challenge with tumor cells. There was no distinctionin proportionof survivorsor in median day of death between animals receiving drug pretreatment and those receiving no drug treatment. It is conceivable that primary treated hosts are able to reject an otherwise lethal surviving tumor cell fraction because of the presence of a large proportion of dead tumor cells. The dead tumor cell fraction could generate either a specific host anti tumor response by virtue of the relatively high antigenic load, or it could generate a nonspecific influx of peritoneal cells augmenting host resistance to the surviving viable cells. To examine this alternative, Ehrlich ascites cells, rendered non tumorigenic by 4000 rads of X-irradiation, were mixed with viable Ehrlich cells. The proportion of killed cells ranged from 4 to 96%

in a total

inoculum

of 25

x

1 06 cells.

All the

mice

died from the viable cells present in the inoculum. As judged by the median day of death for all groups, it appeared that death was directly related only to the number of viable cells injected. Effect on PMN Influx and Comparison with N-Bromoace tylhexosamine Tetra-O-acetates. In the chemotherapy of Ehr lich tumor with N-bromoacetylhexosamine tetra-O-acetates, we observed that drug-induced cure as associated with a signifi cant increase in the percentage of PMN's in the total WBC population in the peritoneal cavity of treated animals compared to untreated tumor-bearing controls. There was also an in crease in the absolute numbers of PMN's in the peritoneal cavities of treated mice (17). We suggested that the carbohy drate analogs were enhancing host inflammatory processes at the site of tumor injection, accounting for the ability of the drug pretreated hosts to resist subsequent lethal tumor challenge as noted in the previous section and possibly contributing to the drug-induced cure of tumor-bearing hosts. Since pretreatment with CLHA-3,5-DNB prior to tumor challenge did not augment host resistance to challenge with 106 Ehrlich cells, we com pared the effects of i.p. treatment with CLHA-3,5-DNB to treatment with N-bromoacetyl-fl-D-glucosamine tetra-O-ace tate in animals in the absence of any tumor challenge. Mice were given injections of the normally curative doses of each agent and sacrificed 1, 2, 3, or 5 days after treatment. Perito neal cells were quantitatively harvested, and differential stain ing was performed as detailed previously (17). Chart 5 shows the pattern of host cell infiltration obtained in response to each agent in the absence of Ehrlich cell challenge. N-Bromoacetyl $-D-glucosamine tetra-O-acetate caused a rapid influx of PMN's and lymphocytes into the peritoneal cavities, whereas CLHA-3,5-DNB had no such effect on PMN infiltration and only marginal effect on lymphocyte infiltration. These results mdi cate thatthese 2 classes of lipophilic thiol reagents are exerting host-mediated antitumor effects by significantly different mech anisms. Effect of CLHAB on Primary Humoral Immunity. Effects on the primary IgG and 1gM responses in B6D2F1 mice to sheep CANCER

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40

Chemotherapy with Esters of Chlorohydroxyacetone erythrocytes were examined as a function of the timing of drug administration. Mice treated with a single injection of drug at chemotherapeutic dose levels 24 hr after antigen (CLHA-3,5DNB, 125 mg/kg; CLHAB, 50 mg/kg; and CLHA-pNB, 100 mg/kg) showed no significant reduction in the number of splenic lgG PFC's 6 days after antigen administration, as compared with vehicle controls. Multiple injections with CLHA 3,5-DNB (75 mg/kg/day, Days 1, 3, and 5) also failed to affect IgG PFC numbers. Complete' suppression of spleen IgG PFC response (no PFC's/spleen) versus vehicle control (8.4 Â±0.5 x 1o@PFC's/spleen) was obtained if treatment with CLHA 3,5-DNB (75 mg/kg/day) was begun 7 days before antigen administration and was continued every other day until 1 day after antigen administration. Mice treated with single injections of CLHAB on CLHA-3,5DNB (at dose levels active in chemotherapy) 1 day after antigen administration showed no reduction in Day +4 hemolytic titer (1gMresponse) compared to vehicle control mice. Suppressed 1gMresponse (â€”log2hemolysin titer = 7, compared to vehicle control titer = 10) was observed only when CLHA-3,5-DNB treatment (75 mg/kg/day) was begun 7 days before antigen administration and continued every other day until 1 day after administration. DISCUSSION Chemotherapy with Chlorohydroxyacetone Esters. The benzoate esters of chlorohydroxyacetone have potent anti Ehrlich tumor activity in vivo even with only a single-drug injection. The most effective of these esters, CLHA-3,5-DNB, also produces less marked but still significant in vivo chemo therapeutic effect against the P815 mastocytoma in semisyn geneic hosts. Antipnoliferative activity against log-phase cul tuned cell lines of Ehrlich tumor and of Li 210 leukemia parallel results observed previously against log-phase Friend erythro leukemia cells (6). When used as a measure of cytotoxic activity, inhibition of incorporation of labeled thymidine into both Li 210 and Ehrlich tumor cells derived directly from pas sage hosts parallels the dividing cell culture results and con firms that the active analogs require an a-chloroketone group and lipophilic characteristics. Further structure-activity studies are in progress, designed to determine whether these agents are monofunctional lipophilic alkylating agents or whether they require the benzoate or nitrobenzoate group as second leaving group for in vivo activity.6 At this point it is apparent that the CLHA benzoate esters are able to generate altered host-tumor interaction to DBA/2-denived Friend erythnoleukemia cells when the cells are drug treated in vitro and reimplanted into syngeneic hosts (6). The agents are also able to produce chemothenapeutic responses in vivo in other tumor model sys tems, even though they are chemotherapeutically inactive against the highly drug-resistant cultured Friend erythroleuke mia cells implanted and treated in vivo (6). There is no evidence at present that the mechanisms producing chemical alteration of Friend erythroleukemia cells in vitro are related to the mechanisms producing in vivo chemothenapeutic activity. How ever, some features of the in vivo curative responses in this work suggest that host-tumor interaction has been altered by in vivo drug treatment, producing a chemotherapeutic response a

@, McCarthy and T. P. Fondy, work in progress.

SEPTEMBER1980

Chart 5. B6D2F mice were given injections of CLHA-3,5-DNB (100 mg/kg) or N-bromoacetyl-fI-o-glucosamine tetra-O-acetat8 (50 mg/kg) on Day 0. Mice were sacrificed on Days 1, 2, 3, and 5, and their peritoneal cavities were washed with 20 ml of sterile 0.85% NaCI:0.02% KCI:8 m@Na2HPO4:2mM KH2PO4,pH 7.4, Staining

and differential cell counting

were performed

as previously

de

scribed (18). Points, mean; bars, SE.

that includes but extends beyond direct cytotoxicity. Host Participation In Cure of Ehrlich Tumor' with CLHA Esters. The inability of the CLHA esters to effect long-term cures in animals that have been immunosuppressed shows that a surviving viable cell fraction is being overcome in immuno competent mice that are cured after parallel treatment sched ules. The most potent cytotoxic analogs in vitro are not the most effective in vivo (Charts 2 and 3), and one of them chloroacetone, is not effective at all in vivo. Moreover, if direct tumor cell cytotoxicity accounted for all of the in vivo activity of these agents, one would expect a broader spectrum of at least delayed tumor growth in other tumor model systems, and some activity would be likely to persist in syngeneic hosts if activity was observed in semisyngeneic hosts. In fact, in 5 tumor model systems studied in this work and one in earlier work (6), antitumon activity is observed in 2 of the 6 systems. Treatment in vivo with CLHA esters could produce cures by reducing the surviving tumor cell population to a level below the tumorigenic threshold in the Ehnlich system in immunocom petent hosts. However, transfer to naive animals of penitoneal cells from cagemates of animals destined to survive shows that the survivors harbor a lethal tumor burden after drug treatment that kills the recipients but fails to kill the donors. In addition, lethal tumor challenges can be administered shortly after initial tumor treatment (Table 3), and animals so treated can survive the otherwise lethal challenge. A full exploration of rechallenge immunity after cure with CLHA analogs is in progress. It is important at this juncture to point out that rechallenge survival in this allogeneic tumor model system is by no means as simple to achieve as is sometimes assumed. We have shown that animals cured of primary Ehnlich tumor by methotrexate or 1$-D-arabinofuranosylcytosine are not immune to secondary tumor negrowth at other sites, whereas cure with agents such as nitrogen mustard or mitomycin C does produce rechallenge immunity(17). Arnold et al. (2) showed that animals cured of a 1O@Ehnlich cell challenge using poly-L-lysine were fully susceptible to rechallenge with 4 x 106 cells. Basis for Host Participation in Cure with CLHA Esters. Although features of altered host-tumor interaction appear to be involved in the cures obtained with the CLHA esters, it is not cleanwhat those features are or whether they are potentially significant or relatively trivial. It is clear that direct effects of antigen load are not involved, since mixtures of live and dead 3279


P. Babiarz-Tracy et a!. cells affected host animals only in proportion to the dose of live cells administered. It is also apparent that the CLHA esters produce anti-Ehrlich tumor effects by mechanisms that are at least in part distinct from those involved in cures with Nbromoacetylhexosamine tetra-O-acetates in this same system (1 7).

With

the

carbohydrate

analogs,

drug

treatment

prior

to

tumor challenge has some measurable antitumor effect (17), and, as shown in this present work, the carbohydrate analogs themselves produce altered patterns of host cell infiltration into the site of drug administration in the absence of tumor chal lenge. Neither of these properties is exhibited by the active CLHA esters. It is probable that the CLHA esters are lipophilic alkylating agents that are chemotherapeutically active under conditions that do not suppress a primary humoral immune response. Tumor-bearing animals treated with these agents in vivo exhibit increased host resistance to live tumor cells. These in vivo results are consistent with the increased host resistance to live Friend erythroleukemia cells obtained by treating Friend eryth roleukemia cells in vitro with the analogs, reimplanting the treated cells into normal recipients, and challenging the recip ients with live Friend erythroleukemia cells (6). However, in both cases, it is not yet clear whether treatment with the analogs delays primary tumor cell growth sufficiently to permit unsuppressed host response to limit the surviving cell fraction and concomitantly to produce increased host resistance to tumor rechallenge or whether increased tumor cell immuno genicity is involved. Additional experiments in the in vivo Ehrlich tumor system are in progress to test these 2 alternatives. References 1. Alley, C. D.. and Snodgrass, M. J. Effectiveness of neuraminidase in exper imental immunotherapy of two murine pulmonary carcinomas. Cancer Res., 37:95â€”101, 1977. 2. Arnold, L. J.. Jr. , Dagan, A., Gutheil, J., and Kaplan, N. 0. Anti-neoplastic activity of poly(L-lysine) with some ascites tumor cells. Proc. NatI. Acad. Sci.

3280

U. S. A., 76: 3246-3250, 1979. 3. Fondy, T. P., Ghangas, G. S., and Reza, M. J. Synthesis of 1-halo analogs of DL-glycerol-3-phosphateand their effects on glycerol phosphate dehydro genase. Biochemistry, 9: 3272-3280, 1970. 4. Fondy, T. P., Karker, K. L., Calcagnino, C., and Emlich, C. A. Effect of reserpine (NSC-59272)-induced hypothermia on lifespan in mouse Li 210 leukemia. Cancer Chemother. Rep., 58: 3i 7â€”324,1974. 5. Fondy, T. P., Pero, R. W., Karker, K. L., Ghangas, G. S., and Batzold, F. H. Synthesis of L-1-deoxyfluoroglycerol and its 3-phosphate ester. Effects of the enantiomers in BDF mice. J. Med. Chem., 17: 697â€”702,1974. 6. Fondy, T. P., Tsiftsoglou, A. S., Jacoby, R. D., and Sartorelli, A. C. Immu nogenicity of Friend erythroleukemia cells altered in culture by chemical or physical agents. Cancer Res., 39: 3583â€”3590,1979. 7. Frost, P., and Sanderson, C. J. Tumor immunoprophylaxis in mice using glutaraldehyde-treated syngeneic tumor cells. Cancer Res., 35: 2646â€” 2650, 1975. 8. Fuji, H., and Mihich, E. Selection for high immunogenicity drug-resistant sublines of murine lymphomas demonstrated by plaque assay. Cancer Res., 35: 946-952, 1975.

9. Ghangas, G. S., and Fondy, T. P. Stereospecific synthesis of o-1-fluoro deoxyglycerol 3-phosphate and its effects on glycerol 3-phosphate dehy drogenase. Biochemistry, 10: 3204â€”3210,1971. 10. Hwang, K. W., and Sartorelli, A. C. Use of plant lectin-inhibited agglutination to detect alterations in surface architecture of Sarcoma 180 caused by antineoplastic agents. Biochem. Pharmacol., 24: 1149â€”ii 52, i 975. 11. Jerne, N. K., Nordin, A. A., and Henry, 0. In: B. Amos and H. Kaprowski (eds.), Cell Bound Antibodies, pp. 109â€”1 22. Philadelphia: Wistar Institute Press,i963.

12. Meyer, A., Enker, W., Jacobitz, J., Wissler, R., and Craft, K. The tumor specific immune response of experimental active-specific immunotherapy. Cancer (Phil.), 39: 565â€”569,1977. 13. Pero, R. W., Babiarz-Tracy, P., and Fondy, T. P. 3-fluoro-1 -hydroxy-pro pane-2-one (fluorohydroxyacetone) and some esters. Syntheses and effects in BDF, mice. J. Med. Chem., 20: 644-647, 1977. 14. Prager, M. D., and Baechtel, F. A. Methods of the modification of cancer cells to enhance their antigenicity. Methods Cancer Res., 9: 339â€”400, 1974. 15. Ray, P. K., Takur, v. S., and Sundaram, K. Antitumor immunity. II. viability, tumorigenicity and immunogenicity of neuraminidase-treated cells: effective immunization of animals with a tumor vaccine. J. NatI. Cancer Inst., 56: 83â€” 87, 1976. 16. Silverman, J. B., Babiarz, P. S., Mahajan, K. P., Buschek, J., and Fondy, T. P. i-Halo analogs of dihydroxyacetone-3-phosphate. The effects of the fluoro analog on cytosolic glycerol-3-phosphate dehydrogenase and triose phosphate isomerase. Biochemistry, 14: 2252â€”2258,i975. 17. Simon, P., Burlingham, W. J., Conklin, R.. and Fondy, T. P. N-Bromoacetyl $-D-glucosamine tetra-O-acetate and N-bromoacetyl-/l-o-galactosamine te tra-O-acetate as chemotherapeutic agents with immunopotentiating effects in Ehrlich ascites tumor-bearing mice. Cancer Res., 39: 3897â€”3902.1979.

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