[CANCERRESEARCH39, 1807-1813, May 1979]0008-5472/79/0039-0000502.00

Growthand Treatment of EhrlichTumor in Mice with Alloxan-inducedDiabetes

Kreèimir Paveliô,1 Milivoje Slijeptevié, Jasminka Pavelié,Jasna lyle, Silvana Audy-Jurkovió, Zlatko P.Pavelic,2 and Milivoj Boranió

Department of Experimental Biology and Medicine, Rudjer Bo@kovicInstitute (K. P., M. S., J. P., M. B.), and Department of Gynecology and Obstetrics, CytologicalLaboratory. Medical Faculty, University of Zagreb, Zagreb, Croatia, Yugoslavia(J. I., S. A-J.j

ABSTRACT

Hypoglycemia and hypoinsulinemia accompanied i.p. or i.m.growth of the Ehrlich tumor in CBA/H and BALB/c mice.Simultaneously, insulin accumulated in the ascitic fluid of tumor-bearing mice. in hosts rendered diabetic by means ofalioxan, the tumor decreased the blood glucose almost to thelevel seen in nondiabetic mice.

Tumor growth was retarded in diabetic hosts, but cells fromsuch tumors, transplanted into secondary diabetic recipients,grew faster than in their primary diabetic hosts, similarly toâ€â€˜nondiabetic' †tumor cells growing in nondiabetic hosts. This

phenomenon of ‘‘adaptation'‘of the tumor to the diabetic statewas prevented if diabetic tumor-bearing mice were daily treatedwith insulin.

The tumor did not grow in all diabetic recipients; the frequency of takes correlated with severity of the diabetes, i.e.,with the dose of alloxan given to induce it. The greater thedose, the less mice accepted the tumor. Insulin injection intodiabetic tumor-bearing mice promoted the tumor growth. Simuitaneous treatment of diabetes and the tumor afforded thebest antitumor effect.

INTRODUCTION

The metabolism of glucose in tumor cells differs from that innormal cells (6, 8). Due to intensive glycolysis, tumor cells(e.g. , Ehrlich tumor) consume more glucose than do cells ofnormal tissue (5, 6, 8, 17, 22). This excessive glycolysis(anaerobic and aerobic) has been seen in many tumors otherthan Ehrlich (10, 11, 26), and it can lower the level of the bloodsugar (18). Also, this hypoglycemic tendency seems to bedirectly related to the total burden of tumor cells in the organism(23). Another reason for the hypoglycemia of tumor-bearinghosts can be the ability of some tumors to produce insulin orinsulin-like substances (4, 13). The greater permeability of themembrane of some tumor cells (often virus induced) for glucoseor an enhanced transport of glucose into tumor cells, mightalso be a factor in inducing hypoglycemia in cancer (12, 30).

Some tumors, Novikoff hepatoma, Walker 256 carcinosarcoma, Ehriich tumor, thymoma and a few others, grow moreslowly in diabetic than in normal animals (10, 11, 24, 26, 28).Hypomnsulinemiamight be an important reason for this impaired

I To whom requests for reprints should be addressed, at Department of

Experimental Biology and Medicine, P. 0. Box 1016, Bljeni@ka54, 41001Zagreb, Croatia, Yugoslavia.

2 Present address: Department of Experimental Therapeutics and Grace

Cancer Drug Center, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, N. Y. 14263.

Received June 19, 1978; accepted January 22, 1979.

growth, since insulin is required for mitosis, growth, and activation of glucose phosphorylation at the glucokinase enzymereaction, and in this way, it can accelerate tumor glycolysis(1 8, 30). indeed, insulin therapy of diabetic mice bearingthymoma or of diabetic rats bearing mesothelioma causedfaster development of the tumors (24); however, poorer growthof tumors after insulin treatment has also been described (27).Surgical extirpation of tumor, on the other hand, has beenreported to return the blood sugar from tumor-induced hypoglycemia back to the normal range. Consequently, therapy ofdiabetic tumor hosts should take care of metabolic and endocrinological disorders in addition to antitumor effects. We werenot able to find reports describing simultaneous treatment ofdiabetes and tumor.

The aim of these experiments was first to follow the growthof ascitic or solid Ehrlich tumor in relation to the number oftransplanted tumor cells and to the dose of alloxan injected toinduce diabetes before the tumor. Second, by transplantingtumor cells from primary diabetic recipients into secondarydiabetic or nondiabetic recipients, it was also tested whetherthe tumor habituates itself to the diabetic condition. Third, wehave investigated how therapy of the diabetic situation or ofboth diabetes and tumor affects the tumor development.

MATERIALS AND METHODS

Mice. We used4-month-oldfemale CBA/H mice bred in theRudjer Bo@kovióInstitute and 6-month-old female BALB/cmice from the Faculty of Natural Sciences (Department ofAnimal Physiology). The mice were kept in plastic cages (3 to5/cage) and were provided with food (Pliva, Zagreb, Yugoslavia) and tap water ad ilbitum.

Tumor. The Ehrlichtumor was used in both the ascitic andthe i.m. forms (32). After i.p. transplantation, it grows in asciticform, and after injection of the cells i.m., it grows as a solidnode. The tumor was propagated in C57BL/H x A/H F1mice.The suspension of tumor cells was prepared from the ascitesof mice with ascitic tumor. The fluid was centrifuged for 10 mmat 720 g, and the cells were then resuspended, counted, anddiluted by the standard methods by Hr@akand Marotti (15).The numbers of tumor cells injected in particular experimentsare indicated in Tables 1 to 7.

Experimental DIabetes. Alloxan (Merck AG, Darmstadt,West Germany) was injected i.v. as a 0.5% solution in Hank'sbalanced salt solution. The doses were 75, 100, and 120 mg/kg of bodyweight.Inallexperimentalgroups,the micereceivedailoxan 3 days before tumor cells.

Insulin. Crystalline insulin (Pliva, Zagreb, Yugoslavia)was

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Dependence of the growth0of

Ehrlich ascites or solid tumor in alloxan-diabetifl the number of injected tumor cellsc

CBA/HmiceType

of tumorNo.

of tumorcells inoculated on Day

0No.

Alloxan No. of cells inof (100 mg/ ascites (x 106)

mice kg) on Day 22Tumorwt (g)

on Day 17Survivalof

mice(days)Ehrlich

ascitictumor1

x i0@

5 x 1O@

2 x iO@5

No 45.0 ±12.1a6 Yes 23.0 ±9,9b

@ No 55.3 ±18.84 Yes 25.5 ±i0,i@6 No 78.0 ±13.18 Yes 30.1 ±12.8―30.5

±4.245.0 ±6,2b28.3 ±4.439.0 ±2.2―24.0 ±1.920.0 ±4.4Ehrlich

solidtumor1 x iO°

5 x 1O@

1 x iO@5

No5 Yes6 No6 Yes

@ No5 Yes6.3

±1.52.9 ±0,4b7.2 ±2.24.2 ±1,9―9.3 ±1.95.2 ±2.2―33.0

±2.945.2 ±4,5b23.5 ±4.430.0 ±51―19.1 ±4.325.0 ±36―

K. Pavelid et al.

Table 1

a Mean ±S.D.S@ < 0.05 (or less) compared to the corresponding control group not receiving alloxan.

injected i.m. in the dose of 8 lU/mouse/day, starting on theday of tumor cell transplantation.

Cyclophosphamide. On the thirdday after tumortranspiantation, tumor-bearing mice received Endoxan (Bosnalijek, Sarajevo, Yugoslavia), 200 mg/kg of body weight i.p.

Blood Glucose. The level of blood glucose was determinedby the method of Hyvarmnenand Nikkila (16). For each determination, 2 samples of 0.025 ml of blood from the tail vein fromeach mouse were obtained. The results represent the mean ofboth samples. The level of glucose was expressed as mg/i 00ml of the blood.

Tumor Growth. The basiccriterionfor bothtumorformswasthe survival of the recipients. In the ascitic form, the cells in theperitoneal cavity were also counted; most of these tumor cellsand the rest (approximately 5 to 8%) are macrophages andpolymorphonuclear leukocytes. In mice with the solid tumor,we measuredthe diameteror the weightof the tumornode.

The number of cells in the peritoneal cavity of killed micewas counted as follows: 3-mi of sterile 0.9% NaCI solutionwere injected i.p. The abdomenwas massagedfor 1 mm, and5 drops of the ascitic fluid were taken by a needle into a tube.The number of cells was expressed per ml of this abdominalwashing without attempting to calculate the total cell populationin the abdominal cavity. (This method resembles the commonpractice of expressing the number of leukocytes per @tIofblood).

The weight of the solid tumor was determined on a torsionbalance (acuity, 0.1 mg) after a careful dissection of the tumornode from tissues of killed mice. Two perpendicular diametersof the tumor in situ were measured by caliper.

Tissue Extract. Excised solid tumor or muscle tissue washomogenized in 0.9% NaCI solution (50 mg tissue per ml ofmedium) using an all-glass Potter-Eivehjem homogenizer. Thehomogenate was centrifuged at 75,000 x g for 1 hr at 4°(9).The supernatant was decanted and used for insulin determination.

Biochemical Analysis. Immunologicallyreactive insulin inthe sera and in extracts of the tumor or muscle was determinedby the method of Morgan and Lazarow (21), using 1251-insulinand the crystalline rat insulin standards (Sorin, Saluggia, Italy).

Cytological and Cytochemical Procedures. Cytomorphological and cytochemical characteristics of the cells were analyzed in sediments of the ascites fluid punctioned 10 or 18days after tumor inoculation. Sedimentation ‘‘smears'‘(2) werestained after air-drying by the method of May-GrünwaldGiemsa. PAS3-positive substances were revealed by themethod of Hotchkiss (14) and Bauer-Si@(3). Glycogen wasdetermined after reduction with diastase (20).

Statistical Analysis. The data are presented as the arithmetical mean ±S.D., and the significance between the resultswas checked by the Student t test. The level of significancewas put at the level of p < 0.05.

RESULTS

Growth of EhrIlch Tumor in Diabetic Mice. Table 1 showsthe influence of the number of injected tumor cells on thelength of survival of diabetic mice and on the rate of tumorgrowth. Diabetic mice lived, on the average, between 7 and 14days longer than did nondiabetic mice given injections of equalnumbers of tumor cells. In the group of mice that received thelargest i.p. dose of tumor cells (2 x 10w), these differenceswere obscured by rapid growth of the tumor. The number ofcells in the abdominal washing of diabetic mice with the ascitictumors and the weight of tumor nodes in mice with solid tumorswere significantly smaller than in the appropriate groups ofnondiabetic mice.

Table 2 shows the dependence of the growth rate of theEhriich tumor on the dose of alloxan which caused the diabetes.Mice with diabetes elicited by alioxan (75, 100, or 120 mg/kg)had fewer tumor cells in the abdominal washings, but therewas no clear correlationbetween the drug dose and the cellnumber. Diabetic mice always lived longer than did nondiabeticanimals (Tables 1 and 2), but the differences abated withâ€â€˜severity' ‘of the diabetes, probably due to an excessively

weaker condition of the more diabetic animals. A generalconclusion from this experiment is that diabetes hinders thegrowth of this tumor.

3 The abbreviation used is: PAS, periodic acid-Schiff.

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Dependence of the growth of Ehrlich ascitic or solid tumor in alloxandiabeticCBA/H miceon thediabetogenicdoseofaioxanAllmice (5 to 6/group) received 1 x 106 tumor cells i.p. or i.m.onDay

0. Alloxanwasinjected3 daysbeforethat.No.of cellsinType

of tu- Alloxan ascites (x 10@) Tumor wt (g) Survivalofmor(mg/kg) on Day 25 on Day 30 mice(days)Ehrlich

ascitic 0 55.0 ±13.0k 29.0 ±3.8tumor75 23.0 ±8.3― 47.0 ±6.6―100

20.0±11.5―39.1±4.9―12025.0 ±9.1― 34.0 ±8.8Ehrlich

solid 0 6.5 ±1.9 35.0 ±8.7tumor75 4,O±l.7b463±62b100 3.3 ±1.2― 45.0 ±3.9―120

1.0±0.3―49.0±2.8―

Type of tumorExperimentNo.

of tumorcells inocu

latedAlloxan (mg/kg@No.

of micewith tumor/total no. ofmiceEhrlich

ascitictumor1iO@0 10020/2027/3025x10°0

10016/1614/143iO@0

1006/66/64i00

7510012020/20

15/1527/30

14/18Ehrlich

solidtumor51

O@010018/1820/2265x1060

10015/1512/127iO@0

10010/1010/108iO°0

7510012018/18

9/1020/2221/25

Growth @ndTreatment of Ehrilch Tumor in Diabetic Mice

and nondiabetic mice with ascitic or solid tumor. As expected,alioxan causes hyperglycemia, and the effect is clearly dosedependent; larger doses of the drug cause more severe diabetes. Tumor cells, on the contrary, lower the blood glucose,both in nondiabetic mice and in mice with alloxan-induceddiabetes. This effect is also dose dependent, and it reflects thetumor cell load; the greater the number of tumor cells inoculated into diabetic or normal mice, the lower the glucose levelin their blood. Inoculation of many tumor cells into diabetic

mice even abolishes the alloxan-induced hyperglycemia andkeeps the blood glucose level within the normal range.

The ‘‘antihypergiycemic', effect of Ehrlich tumor is evidentfrom data in Chart 3. In mice with ailoxan-induced diabetes,both the ascitic and the solid form of the tumor decreased theblood sugar, so that in mice with terminal tumors the glucoselevels approached the normal range. in nondiabetic animals,terminal tumors caused hypoglycemia.

The blood sugar decreasing activity was significantly higherin the tumor extract (corresponding to the insulin level of 22.5±9.1 @iunits/mI) than in the muscle extract (8.3 ± 2.6 @&units/

ml). The level of plasma insulin, on the other hand, was lower

I

Dose of Tumor CellsChart 1. Effect of the initial tumor cell load on blood glucose levels In alloxan

diabetic and nondiabetic CBA/H mice with Ehrlich tumor. Glucose was determined 18 days after inoculation of tumor cells and 21 days after injection ofalloxan (100 mg/kg). columns, means of 6 mice/group; bars, S.D.: ‘,significantdifference from the control group of nondiabetic mice without tumor (shadedarea).

6

8

4)

3C,

Chart 2. Effect of alloxan dose on blood glucose levels in CBA/H micesubsequently Inoculated with Ehrllch tumor. Glucose was determined 18 daysafter Inoculationof 10' tumor cells and 21 days after injectionof alloxan.Columns, means of 6 mice/group; bars, S.D.; ‘, significant difference from thecontrol group of nondiabetic mice without tumor (shaded area).

Table2

aMean±S.D.b@ < 0.05 compared to the group of mice with ascitic or solid tumor

withoutalloxan.

Table3Dependence of the takes of Ehrlich ascites and solid tumor in

aioxan-diabetic CBA/H mice on the number of transplanted cells andon the dose of alloxan

=

—@

10' 5z10 2*l0@ los 5s10 10'

a Alloxan was injected 3 days before tumor cells.

Neither the ascitic nor the solid form of the tumor developedin all diabetic mice, although they did in all nondiabetic mice(Table 3). The rate of tumor takes depended both on thenumber of inoculated tumor cells and on the dose of alioxanused to induce diabetes. If the diabetes was caused by alioxan(1 00 mg/kg), tumor grew in all mice only after inoculation of 5x 106 or 1 x 107 tumor cells, whereas 1 x 106 cells failed inestablishing the tumor in 3 of 30 mice inoculated i.p. (ascitictumor) and in 2 of 22 mice inoculated i.m. (solid tumor) (Table3, Experiments 1 and 5). The larger the alloxan dose whichcaused the diabetes, the smaller the number of mice acceptingthe tumor after inoculation of 106 cells (Table 3, Experiments4 and 8).

Charts 1 and 2 show glucose levels in the blood of diabetic

Doss of Allozan (rr@q/kg)

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K. Paveliö et al.

in mice with ascitic or solid Ehrlich tumor than in healthy controlmice. In animals with tumors that have grown for 28 days, theplasma insulin level was approximately two-thirds of the levelin healthy control mice. Mice with diabetes induced by ailoxan(100 mg/kg) given 8 days before measurement of the insulinlevel, had their plasma insulin approximately at one-third of theconcentration in healthy control mice. Presence of the tumor(ascitic or solid) did not change this low level of insulin (Chart4),

Cytology and Cytochemistry. The morphology oftumor cellsgrown for 10 or for 18 days in diabetic animals did not differfrom the morphology of cells grown for the same time in normalenvironment. More mitoses and degenerated cells were foundon Day 18 than on Day 10 in both groups.

Cytochemical analysis also revealed no differences in typeor intensity of PAS reaction in ascites smears from diabetic andnondiabetic mice. In all smears, the PAS reaction was ofmedium intensity. Digestion by diastase did not demonstrateglycogen in any sample.

Adaptation of Ehrllch Tumor to Diabetic Environment.Since diabetic environment only retarded growth of the EhrIichtumor but did not prevent death caused by it, we conclude thattumor cells eventually adapt themselves to hyperglycemia,shortage of insulin, ketonemia, acidosis, and/or other metabolic alterations of the diabetes situation.

indeed, ‘‘diabetic'‘tumor cells proliferated at approximately

Table4

Habituation of Ehrlich ascitic tumor on diabetic conditions in BALB/cmice

Experiment@

2

No. of micewith tumor/

Donor Host di- total no. ofdiabetic abetic mice

— — 5/5— + 6/7+ — 5/5+ + 6/6

— — 9/9— + 15/15+ — 15/15+ + 15/15

No. of cells inascites (x 1O@Y'

71.0 ±10.3c40.1±6.3―68.5 ±11.465.3 ±5.8

59.0 ±12.130.0 ±8.1―58.5 ±12.256.5 ±9.9

Survival of mice(days)

22.2 ±3.440.1 ±63d25.0 ±4.824.3 ±5.2

17.2 ±3.319.7 ±2.113.0 ±4.318.0 ±5.1

20.0 ±3.838.0±5.6―22.0 ±3.2

25.0 ±2.938.0 ±5.7―23.0 ±4.8

3 — — 5/5 66.0 ±12.8— + 5/7 38.0 ±5.8―+ + 6/6 78.2±21.5

4 — — 8/8 68.3±6.1— + 7/9 41.5±6.2―

+ + 6/6 77.3 ±10.9

a Experiment 1 , 2.5 x 1 o@ tumor cells, alloxan (1 00 mg/kg); Ex

periment2, 2.5 x 1O@tumorcells, alloxan(100 mg/kg); Experiment3, 2.5 x 10°tumor cells, alloxan (75 mg/kg); Experiment 4, 2.5 x106tumorcells,alloxan(120 mg/kg). Alloxanwasalwaysgiven3 daysbeforetumorcells.Dayof tumorinoculationis Day0.

b Counted on the 1 7th day after inoculation of the tumor in Experi

ments1, 3, and4 andon the 12th day in Experiment2.C Mean ± S.D.

d ,@< 0.05 (or less) compared to the control group (donor and host

withoutdiabetes).

the same rate in the abdominal cavities of diabetic hosts as didâ€â€˜nondiabetic' ‘tumor cells in nondiabetic hosts and caused

death approximately at the same time (Table 4). There was noclear evidence that diabetic tumor cells became either less ormore virulent when returned into nondiabetic environment. Asexpected from previous experiments, nondiabetic tumor cellsgrewpoorlyindiabetichosts.

Similar experiments were done with the solid Ehrlich tumor(Table 5). Diabetic tumor cells grew at a normal rate in diabetichosts. in accordance with previous experience, not all diabeticmice given injections of tumor cells acquired the tumor. Thisresistance was overcome by increasing the number of tumorcells in the inoculum. If, however, tumor cells from diabeticmice were transplanted into diabetic hosts, the tumors grew inall recipients, regardless of the number of injected tumor cellsand the dose of alloxan used to cause diabetes.

Effect of insulin. Habituation of the tumor to growth indiabetic conditions was abolished by daily injections of insulin.A group of diabetic mice, prospective donors of tumor cells forsecondary recipients, received insulin (8 units/day) for 15days after tumor inoculation. Tumor cells were then transplanted to nondiabetic recipients and to diabetic recipientstreated or not treated with insulin (Table 6). Tumor cells thathad adapted themselves to the diabetic environment grew wellin new diabetic hosts.

Therapy of DiabetIc Mice Bearing the Ehrllch Tumor. Theabove-mentioned experiments demonstrate that diabetes retards tumor growth (Tables 1 to 3) and that tumor growthcounteracts hyperglycemia (Chart 1). A simplistic point of viewwould be then thattreatment of alloxan-diabetes would promotethe growth of the tumor and that the control of tumor growth

450

350

250

ISO

50

S II 6 19 22Days After Tumor Inoculation

6

8.5

64)4)03

CD

Chart 3. Blood glucose levels (mg/i 00 ml) in alloxan-diabetic or nondlabeticCBA/H mice with or without Ehrllch ascitic or solid tumor. Means from 6 miceare indicated. Bars, S.D.; ‘, significant difference from the control group ofnondiabetic mice without tumor (lower shaded area).

25@ -@@25

@20L@ • 20; I5@ • i15@

@IOflEfli@@@( ASCITIC SOLID ALLOX*Id AL.LOXAN £LLOXAPd

TUMOR TUMOR 4 4ASCIT@ SOLIDTUMOR TUMOR

Chart 4. Plasma insulin levels (@tunlts/ml)in CBA/H mice treated with alloxan(100 mg/kg) 3 days before and inoculated with 1o@cells of Ehrlich ascitic orsolid tumor. Points, mean from 4 mice; bars, S.D.; ‘, significant difference fromthe control group of mice without alloxan and tumor (white column).

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Habituationof Ehrlichsolid tumor ondiabetic conditions in BALB/cmiceExperl

menrDonordi

abeticHostdia

beticNo.

of micewith tumor/total no. of miceTumor

diameter(mm) on Day 19Tumor

wt (g)on Day 19Survival

of mice(days)1—

—

+—++5/5

9/106/610.5

±3.3―5.5 ±2.8c

13.0 ±3.57.3

±2.23.0 ±1.8c8.0 ±2.128.2

±6.6395 ±3,2c29.4 ±5.62—

—

+—

++6/6

5/55/515.3

±6.68.1 ±33C

14.4 ±6.89.1

±2.92.5 ±33C9.9 ±3.424.5

±5.632.5 ±2.8c22.1 ±0.93—

—

+—+

+8/89/10

15/159.7

±2.46.3 ±2,5c

12.1 ±2.16.7

±2.83.1 ±0.9c8.1 ±2.527.8

±2.337.1 ±0.5c29.5 ±0.34—

—

+—++8/8

6/710/1010.8

±4.78.8 ±3.89.2 ±7.49.1

±1.36.2 ±1.8c7.7 ±3.430.5

±2.935.0 ±2.9c31.5 ±8.8

All micereceivedalloxan(100 mg/kg) and3 daysbefore106Ehrlichtumor cells i.p. (ascitictumor)ori.m. (solidtumor).Dayof tumorinoculationis Day0.No.

ofmicewith No. of cells in as

Type of tu- Donor dia- tumor/total cites (x 1O@)onTumor wt (g) onSurvival ofmicemorbetic Host diabetic no. of mice Day 28Day26(days)Ehrlich

— — 6/6 38.0 ±5,6a36.0 ±5.1ascitic— + 5/6 20.0 ±6.6―43.0 ±5.2―tumor-F -F 6/6 35.5 ±6.334.0 ±1.0+

(insulin)C + 7/7 23.5 ±8.6―50.0 ±44b+(insulin) + (insulin) 6/6 40.0 ±10.135.5 ±5.7Ehrlich

— — 5/55.2 ±2.933.0 ±4.4solid— + 4/42.2 ±0,9b40.0 ±15btumor+ + 6/64.9 ±1.932.0 ±2.2+

(insulin) + 7/72.1 ±0,7b43.1 ±5.1―+(insulin) + (insulin) 7/76.2 ±2.134.5 ±5.5

Growth and Treatment of Ehrlich Tumor in Diabetic Mice

Table5

a Experiment 1 , 1 O@ tumor cells, alloxan (1 00 mg/kg); Experiment 2, 1 oT tumor cells,

alloxan (1 00 mg/kg); Experiment 3, 1O@tumor cells, alloxan (75 mg/kg); Experiment 4, 106tumor cells, alloxan (120 mg/kg). Alloxan was always given 3 days before tumor cells. Day oftumorinoculationis Day0.

b Mean ± S.D.

C@ < 0.05 (or less) compared to the control group (donor and host without diabetes).

Table6Abrogation of the habituation of tumor cells on diabetes in alloxan-diabetic CBA/H mice after

treatment with insulin

a Mean ± S.D.

b ,@,< 0.05 compared to the control (donor and host without diabetes) group.

C Treated with insulin. Insulin (8 lU/mouse/day) was injected daily for 1 5 days after tumor inoculation.

On the 15th day, miceweresacrificed,and their tumorcells weretransplantedinto hostwith diabetesinducedby alloxan6 daysbefore.One-halfof thesereceivedinsulindaily(8 IU)until thedayof killingordeath.

would enhance the diabetes; hence, the host would benefitfrom neither. We have, however, demonstrated that tumoradjusts itself to diabetic conditions (Tables 4 and 5) and thatinsulin reverts it to ‘‘normal'â€metabolism (Table 6). What therapy, then, would be optimal for diabetic mice with Ehrlichtumors?

Diabetic mice with the tumors were treated for the diabetes,for the tumor, or for both (Table 7). if the tumor was caused bya small inocuium (106 cells), insulin abrogated the inhibitoryeffect of diabetes on the tumor, and the tumor behaved as innondiabetic hosts. Treatment of the tumor with cyclophosphamide was equally effective in diabetic and nondiabetic mice.Simultaneous therapy of diabetes with insulin and of the tumorwith cyciophosphamide slowed down the tumor the most, as

judged by the tumor cell count and by host survival. In Experiment 2, the tumor was induced by a larger inoculum, 1O@tumor cells. Although it grew more slowly in diabetic mice(fewer cells in the abdominal cavity), survival of the mice wasnot significantly longer than was the survival of nondiabeticmice. The antidiabetic therapy accelerated the growth of thetumor to the rate seen in nondiabetic mice. Treatment ofdiabetic mice with cyciophosphamide slowed down the tumorsignificantly and postponed deaths. Similar data were obtainedafter simultaneous therapy of diabetes and of the tumor.

Glucose level in the blood(Chart 5) was followedin diabeticmice after therapy against diabetes, the tumor, or both. Towardthe end of life, hypoglycemiabecame manifest in mice withascitic tumor, as already described in Chart 3. Therapy of the

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Effect of cyclophosphamide on Ehrlich ascitic tumor in alloor untreated with insulinxan-diabetic

CBA/H micetreatedNo.

ofNo. of cells in as Survival ofmiceTumorinoculumTreatment― micecites Cx 1Os)―(days)Ehrlich

asciticNone 560.0 ±11.2c32.5 ±4.6tumor(106Cyclophosphamide 628.5 ±12. 1d40.0 ±5,5dcells)Alloxan

7Alloxan + insulin 7Alloxan + cyclophosphamide 6Alloxan + cyclophospha- 7

mide + insulin39.5

±9.2―57.3 ±10.333.0 ±9.8―22.5 ±6.8―45.3

±5.2d31 .0 ±8.642.0 ±6.2―53.0 ±6.6―Ehrlich

asciticNone 579.0 ± 1 5. 122.5 ±6.4tumor(1 o@Cyclophosphamide 554.0 ±7,9d29.0 ±4,3dcells)Alloxan

5Alloxan + insulin 6Alloxan + cyclophospha- 7

mideAlloxan + cyclophospha- 6

mide + insulin55,5

±11,1d81 .6 ±20.544.0 ±9.1'

56.0 ±11 9―25.3

±5.520.3 ±3.129.5 ±3.2―

32.0 ±5.8―

K. Pavelid et al.

Table7

a Alloxan (1 00 mg/kg) given 3 days before tumor inoculation; cyclophosphamide (200 mg/

kg) on Day 3 after tumor inoculation; insulin (8 lU) daily starting on Day 1 after tumorinoculation. Day of tumor inoculation is Day 0.

b Counted 25 days after tumor inoculation in Experiment 1 and after 1 4 days in Experiment

2.C Mean ± S.D.

d@ < 0.05 (or less) compared to the control group (received tumor cells only).

that hypoinsulinemicdiabetes and its secondary ketonemia@ and/or acidosis provide no suitable environment for the tumor.

Acidosis per se is a well-known factor in retarding tumor growth(1 ), and this is probably related to the inhibitory effects ofacidosis on tumor glycolysis. Retardation of growth of thetumor did not depend on the number of injected tumors cells,the routeof their inoculation,or the dose of ailoxan.

Othershavealsoobservedslowergrowthof EhrlichtumorinN@@―oI@ alloxan-diabetic mice and in mice with hereditary hypoglycemic

C,,@ syndrome (i 8). Many other experimental tumors grow slowly

in diabetic animals (10, 11, 24, 26). Direct effect of alloxan ontumor cells does not account for the growth retardation sincethe half-lifeof thiscompoundis only about 1 hr (25).

Ehrlich tumor adjusts itself, however, to adverse metabolicconditions.As soon as 2 weeks after transplantationintodiabetic hosts,tumorcells develop‘‘resistance'‘towarddiabetesand grow at normal (unabated) rate in secondary diabetichosts.Insulinabrogatedthe processof adjustment.A previouspaper from our group dealing with the growth of leukemia indiabetic mice (29), has discussed the phenomenon of habituation. A hypothesis was put forward that leukemia cells acquirethe ability to transportand utilize glucose in spite of diabeticconditionsand shortage of insulin, but this compensationisnever complete.

We showedthat both formsof the Ehriichtumorcaused fallof the blood glucose in the host. Hypoglycemia was proportional to the number of tumor cells inoculated into the host.One reason for hypoglycemiacould be an augmented consumptionof glucoseby the cells of the tumor(5, 22). Indeed,hypoglycemia was most expressed in animals with large (terminal) tumors, i.e. , with the highest tumor cell load. A fastertransport of glucose through the membrane of tumor cellscouldalso be an importantfactor sincetransportof 3-O-methylglucose is faster in Rous sarcoma cells than in fibroblasts(30). Facilitated transport of glucose is attributed to the

_fF@@@ I @-@@__I_ @@t_@

5 II 4 18 21 24Days After Tumor Inoculation

6

8

I,0

CD

00

Chart 5. Blood glucose levels(mg/iOO ml) in alloxan-diabetic or nondiabeticCBA/H micewithor withoutEhrllchascitictumortreatedby cyclophosphamide,Insulin, or both. Points, meanfrom 5 to 7 mice; bars, S.D.; , significant differencefrom the control group of nondiabetic mice without tumor (lower shaded area).

tumor with cyclophosphamide postponed the terminal fail ofblood glucose in nondiabetic mice. Diabetic mice with theEhiiich tumors became almost normoglycemic as the tumorprogressed, even without insulin treatment, in spite of theinitially high glucose levels in the blood. Tumor therapy withcyclophosphamidepreventedthe ‘‘normalization'‘of the bloodglucose. Insulin helped to keep the blood sugar of alloxantreated mice close to the normalrange or withinit. In diabeticmice receiving the combined treatment (cyclophosphamide andinsulin), the blood glucose was maintained just above thenormalrange except for the terminalfall.

Our findings have shown that growth of the Ehrlich tumordepends on insulin and on glucose metabolism. The growthwas inhibited in diabetic mice, whereas daily injections ofinsulinintodiabetic hostsaccelerated the growth.This means

I 8i 2 CANCER RESEARCH VOL. 39

DISCUSSION

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Growth and Treatment of Ehrlich Tumor in Diabetic Mice

10. Goranson, E. S., Botham, F., and Willms, M. Inhibition of growth of trans.planted hepatomas in alloxanized Wistar rats. Cancer Res., 14: 730-733,1954.

11. Goranson, E. S., and Tilser, G. J. Studies on the relationship of alloxandiabetes and tumor growth. Cancer Res., 15: 626-631 , 1955.

12. Hatanaka, M., and Hanafusa, H. Analysis of a functional change in membranein the process of cell transformation by Rous sarcoma virus; alteration In thecharacteristics of sugar transport. Virology, 4 1: 647—652,1970.

13. Hobs, C. B., and Miller, A. C. Review of endocrine syndromes associatedwith tumors of non-endocrine origin. J. Clin. Pathol (Lond.), 19: 119—123,1966.

14. Hotchkiss, R. D. A microchemical reaction resulting in the staining ofpolysaccharide structures in fixed tissue preparations. Arch. Biochem. Blophys.,16:131-141,1948.

15. Hr@ak,I., and Marotti, T. Immunosuppression mediated by Ehrlich ascitesfluid. Eur. J. Cancer, 9: 717—724,1973.

16. Hyvarlnen, A., and Nikkila, E. A. Specific determination of blood glucosewith 4,-toiuidine. Clin. Chim. Acts, 7: 140—143, 1962.

17. Ibsen, K. H., Coe, E. L., and Mckee, R. W. Some factors influencingrespiration and glycolysis in Ehrlich ascites tumor cells. Cancer Res., 20:1399-1407, 1960.

18. JehI, J. A., Mayer, J., and Mc Kee, R. W. Influence of the hereditary obesehyperglycemic syndrome and of alloxan diabetes on the survival of mice withEhrlich ascites carcinoma. Cancer Res., 15: 341—343,1955.

19. Uppold, 0. C. J.. and Winton, F. R. Human Physiology, pp. 521-522.London: J. & A. Churchill, Ltd., 1968.

20. Luna, L. G. Methods for carbohydratesand mucoproteins.In: Manual ofHistologic Staining Methods of the Armed Forces Institute of Pathology, Ed.3, p. 171. New York: McGraw-Hill Book Company, Inc., 1968.

21 . Morgan, C. R., and Lazarow, A. Immunoassay of Insulin; two antibodysystem. Plasma insulin levels of normal, subdiabetic and diabetic rats.Diabetes. 12: 115—121,1963.

22. Nakamura,W.,andHosoda,S. Theabsenceof glucosein Ehrlichascitestumorcellsandfluid.Biochim.Biophys.Acta, 158:212—218,1968.

23. Papaioannou, A. N. Tumors other than Insulinomas associated with hypoglycemia. Surg. Gynecol. Obst., 123: 1093-1 109, 1966. .

24. Paveli& K., and Slljepôevl@M. Growth of a thymoma In diabetic mice treatedwith insulin. Eur. J. Cancer, 14: 675—679,1978.

25. Rerup, C. C. Spontaneous remission of alloxan diabetes in mice. Diabetologia, 4: 312-318, 1968.

26. Sablina, I. I., Zilbere, A. M., and Zidermane, A. A. The effect of alloxandiabetes on the growth of transplantable tumors. Latv. PSR Zinat. Akad.Vestis, 1: 53-58, 1975.

27. Salter, J. M., and De Meyer, R. Effect of insulin and glucagon on tumorgrowth. Br. Med. J.. 5: 5-7, 1958.

28. Shapot, V. S. Some biochemical aspects of the relationship between thetumor and the host. Adv. Cancer Res., 15: 253-286, 1972.

29. Slijep@evic,M., and Radaôi&M. The growth of a transplantable leukemia innormal and alloxan treated mice. Period. Biol., 78(Supp. 1): 130-132,1976.

30. Weber, M. J. Hexose transport in normal and in Rous sarcoma virus.transformed cells. J. Blol. Chem., 248: 2978-2983, 1973.

31 . Woods, M. Hormonal control of metabolism in cancer. In: E. Wright (ed),Control Mechanisms in Respiration and Fermentation, pp. 253-264. NewYork: Roland Press, Co., 1963.

32. Yoshida, T. Comparative studies of ascites hepatomas. Methods CancerRes.,6:97—157,1971.

MAY 1979 1813

changes of the membraneof virus-transformedcells (12).Higher levelsof insulin-like(glucose-lowering)substancesin

the cell-free extract from the tumor than in the muscleextractindicate that the tumor either uses up host's insulin(togetherwith glucose), or produces an insulin-like(glucose-lowering)principleitself.Several authorshavedescribedhigherconcentration of insulin-like substances in the plasma of mice withsome tumors (7, 28), and there is evidence that tumors otherthan insulinomascan produce insulin-likesubstances(4, 13).However, we have found a decrease of insulinactivity in theplasmaof tumorhosts.

Diabetes interfered with the tumor take and with its growth,but after the ‘‘adjustment'‘to diabeticconditionthe tumorcellsregained their ability to grow well in all secondary diabeticrecipients.

Simultaneous therapy of both the tumor and diabetes wasoptimal. Administration of cyciophosphamide without concomitant administration of insulin diminished the antihypergiycemiceffect of the tumor.

ACKNOWLEDGMENTS

We are indebted to Zlata Jagodiéfor excellent technical assistance, and toProfessor Dr. NikáaAllegretti and Professor Dr. Vlatko Silobr@iófor critique ofthepaper.

REFERENCES

1. Anghelerl, L. G. Tumor growth inhibition by ammonium chloride-inducedacidosis. Int. J. Pharmacol., 12: 320—326,1975.

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olism, 22: 663-674, 1973.5. Coe, E. L. Correlation of glycolytic and respiratory events after addition of

a smallamountof glucoseto Ehrlichascitescarcinoma.CancerRes., 26:269-275, 1966.

6. Daehnfeldt,J. L., andWinge,P. Isotopeinequilibriumof glucosemetabolitesIn intact cells and particle-free supernatants of Ehrlich ascites tumor(38671). Proc. Soc. Exp. Biol. Med., 148: 974-980, 1975.

7. Dunbar, J. C., Walsh, M. F., and Foa, P. P. SecretIonof immunoreactiveinsulin and glucagon in hamsters bearing a transplantable insulinoma. DiabeteMetab.,2:165—169,1976.

8. Fountain, J. A., Parks, M. E., Dickey, A., and McKee, R. W. Lactatedehydrogenase isoenzymes in tissues of normal and Ehrlich-Letréascitestumor-bearing Swiss mice. Cancer Res., 30: 998-1 002, 1970.

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1979;39:1807-1813. Cancer Res   Kresimir Pavelic, Milivoje Slijepcevic, Jasminka Pavelic, et al.   Alloxan-induced DiabetesGrowth and Treatment of Ehrlich Tumor in Mice with

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