StatisticalAnalysisofHormonalEffectsontheSteroid...

[CANCER RESEARCH 30, 221-227, January 1970] .

Statistical Analysis of Hormonal Effects on the SteroidResponsiveness of Solid Ehrlich Tumors

Mitsuo Kodama and Tosh Â¡koKodama

Laboratory of Chemotherapy, Aichi Cancer Center Research Institute, Nagoya, Japan

SUMMARY

The influence of the gonads and adrenals on tumor growthwas studied with SMA mice bearing solid Ehrlich tumorswhich have been produced by subcutaneous injection ofascitic Ehrlich tumor cells. It was found that the tumorgrowth was stimulated by the testes and inhibited by theovaries and also by the administration of hydrocortisoneacetate. The inhibitive effect of the adrenocortical hormonefor tumor growth was observed only in male mice. Theadministration of testosterone and its two derivativesenhanced the tumor growth in castrated male and intactfemale mice, and estradici suppressed it in intact male mice.A positive correlation for testosterone and a negativecorrelation for estradici in the tumor response versushormone dose were statistically significant. The diploidEhrlich tumor was less responsive than the tetraploid Ehrlichtumor with respect to the effects of testosterone andhydrocortisone.

In the statistical calculation with the tumor tissue, thelogarithm of the tumor weight (growth index of tumor) wasused in place of the tumor weight itself on the basis of thefact that the distribution of the former with 99 mice wasnormal, whereas the distribution of the latter with the samematerial showed a great deviation from the normaldistribution.

INTRODUCTION

The previous studies from our laboratory presentedevidence that the development of Ehrlich ascites tumor isclosely related to function of the adrenals and the gonads ofthe host. The castration experiment indicated that thegrowth of solid Ehrlich tumor was stimulated by thepresence of the testes and inhibited by the presence of theovaries (16, 17). In the hormone administration experimentshowever testosterone failed to give an appreciable effect onthat tumor (17).

This study was initiated to investigate the action of varioussteroid hormones in greater detail, and thus to substantiatethe hormone responsiveness of Ehrlich tumor.

MATERIALS AND METHODS

All animals were SMA mice, propagated at the animalsupply center of the Nagoya University School of Medicineby random mating. The hyperdiploid Ehrlich/2N ascitestumors and the hypotetraploid Ehrlich/4N ascites tumorswere supplied in January 1963 by Dr. K. Kaziwara of theTakeda Research Laboratories, Osaka, Japan. These tumorswere maintained in female mice by i.p. inoculation of 1 XIO7 cells/mouse at 10-day intervals. Solid Ehrlich tumors

were produced in mice by s.c. injection of ascitic Ehrlichtumor cells of the 8th inoculation day, 5 X IO6/mouse, into

the right shoulder region. The animals received a standardpellet diet (Oriental Yeast Mfg. Ltd., Tokyo, Japan), withdrinking water ad libitum.

The following hormones were used: testosterone, SigmaChemical Company, St. Louis, Mo.; 2-hydrooxymethylene-17a-methyldihydfotestosterone and 2a-methyl-3-oxo-5-ot-androstane-170-ol-propionate, Syntex S. A., Mexico City,Mexico; estradiol, N. V. Organon; hydrocortisone acetate,Merck, Sharp and Dohme, West Point, Pa. With theexception of S-3900,1 the hormones were suspended in 0.9%

NaCl solution before use. S-3900 was dissolved in sesame oil.They were all injected i.m. into the hind legs of theexperimental mice. Control mice received the vehicles only.Castration was performed 1 month before tumor inoculation.Control animals underwent sham surgery. Data obtainedwere analyzed by Student's t test. For tumor weight, the

deviation of the observed values from the normal distribution curve was estimated by the chi square test in anattempt to check the validity of the routine statisticalprocedures for an experiment, the data of which showed agreat variability. Some of the statistical calculations wereperformed with a HITAC-201 computer.

RESULTS

The Deviation of the Distribution of Tumor Weight fromthe Normal Distribution. The early reports by Kodama (16,17) showed that the variability of tumor weight wasrelatively large, and it was asked whether the distribution of

Received April 11, 1968; accepted April 21, 1969.

'The abbreviations used are: S-3900, 2a-methyl-3-oxo-5a-andro-stane-170-ol-propionate; HCA, hydrocortisone acetate; HMD,2-hydroxymethylene-17a-methyldihydrotestoste^one.

JANUARY 1970 221

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Mitsuo Kodama and Toshiko Kodama

the data is sufficiently like a normal distribution to applythe ordinary statistical analysis for this parameter. A total of99 SMA male mice were inoculated s.c. with ascitic Ehrlichtumor cells, and the animals were sacrificed 12 days later.The weight of the excised tumors varied within the range of151 to 6127 mg. The growth index of each tumor wasestimated by converting the weight (mg) into a commonlogarithm which may represent the rate of cell proliferationof the corresponding tumor providing that the cell divisionproceeds geometrically. Table 1 shows the distribution of 99tumor-bearing mice according to the tumor weight at right,and also according to the logarithmic value of tumor weightat left. The expected values are computed by determiningthe proportion of the area of the normal distribution curveat the corresponding class intervals. The chi square test wasapplied for estimating the deviation of the above 2 sets ofdistribution from the normal curve. The value of p for thetumor weight is less than 0.0005, and the one for thelogarithm of tumor weight falls between 0.416 and 0.549.These p values for the 2 sets of distribution were littleaffected by a change in the magnitude of the classificationintervals. The conclusion is that the distribution of thelogarithm of tumor weight is essentially normal but that ofthe tumor weight itself is not. On the contrary it was foundthat the weight of normal tissues (including body weight)with the same 99 mice was distributed normally. Hence thestatistical significance (p) and the correlation coefficient (r)hereafter will be calculated in terms of the logarithm for thetumor weight.

The Relationship between the Growth of Solid EhrlichTumor and the Gonads. Ascitic tumor cells were inoculateds.c. on Day 0, and the animals were sacrificed on Day 15 fortumor weighing. Marked sex difference was observed in thegrowth of solid Ehrlich/4N as well as Ehrlich/2N tumors,and the above difference was reversed by castration (Table2). Body weight showed a similar sex dependency with theexception of female mice bearing Ehrlich/4N tumors.

Effect of HCA Administration on Tumor Growth. Thetreatment with HCA, 1 mg/day/mouse, was started on Day 7and ended on Day 13, a total of 7 injections. All animalswere killed on Day 14 and the solid tumors were excisedand weighed. HCA administration significantly suppressedthe growth of Ehrlich/4N tumor in male mice (Table 3).Ehrlich/2N tumor was less responsive to HCA. Both tumorswere little affected in female mice by the same treatment.

Effect of Estradici Administration on Tumor Growth.Estradiol, 0.2 mg and 1.0 mg/day/mouse, was administeredon Days 1, 3, 5, 7, 9, 11, and 13, tumor inoculation beingdone on Day 0. The solid tumors were excised and weighedon Day 14. With the increase of hormone dose, the meantumor weight in male mice decreased progressively (Table 4).There is no essential difference between Ehrlich/2N andEhrlich/4N tumors concerning the effect of estradiol.

Effect of Androgen Administration on Tumor Growth. Theeffect of male hormone was first investigated in castratedmale mice. Testosterone, 0.05 mg and 0.25 mg/day/mouse,was administered on Days â€”11,-9, -7, -5, -3, â€”1,1, 3,

5, 7, 9, and 11. Ehrlich/4N tumors were inoculated on Day0. The animals were sacrificed on Day 14 for tumorweighing. The mean tumor weight in castrated male miceincreased parallel with the increase of hormone dose (Table5)-

The hormone responsiveness of Ehrlich/2N and Ehrlich/4Ntumors was next compared in female mice. Testosterone, 0.2mg and 1.0 mg/day/mouse, was administered on Days 1, 3,5, 7, 9, 11, and 13. The tumors were weighed on Day 14.Ehrlich/4N tumor in female mice was significantly stimulatedby the male hormone (Table 6). The body weight was alsoincreased by the same treatment, but no stimulative effectwas detected with Ehrlich/2N tumor in female mice. ThusEhrlich/2N tumor seems to be less hormone responsive thanEhrlich/4N tumor with respect to HCA and testosterone.

In another experiment HMD, 0.5 mg and 2.0 mg/day/mouse, and S-3900, 0.2 mg and 1.0 mg/day/mouse, were

Table 1Distribution of 99 mice with solid Ehrlich tumors according to tumor weight (mg) and also to the

logarithmic value of each tumor weight

Range of tumor weight"No. of mice

observed/expected6Range of log!o

(tumor weight, mg)aNo. of mice

observed/expected*

0-500501-10001001-15001501-20002001-25002501-30003001-35003501-8000Total13/9.224/12.924/15.415/15.712/13.62/10.03/6.36/5.799/88.8C2.1000-2.50002.5001-2.70002.7001-2.90002.9001-3.10003.1001-3.30003.3001-3.50003.5001-3.70003.7001-4.1000Total4/3.09/7.813/16.825/24.126/23.313/15.15/6.64/2.399/99.0

"The mean values Â±S.D. aie 1554 Â±1235 for the set of 99 tumor weights and 3.0826 Â±0.3118 forthe logarithms of the weights of the same tumors.

6The expected values are computed by determining the proportion of the area of the normaldistribution curve for the indicated class intervals.

The normal distribution curve for the given mean Â±S.D. should have had a long tail on the negativeside of tumor weight so that the sum of the above 8 expected values on the positive side falls farbehind the observed value of 99.

222 CANCER RESEARCH VOL. 30



Steroid Responsiveness of Solid Ehrlich Tumors

Table 2Influence of the gonadi for the growth of solid Ehrlich/4N and Ehrlich/2N tumors

GroupNo.Ehrlich/4N12r2'Ehrlich/2N12r2'HostGonadectomyMaleMale

+FemaleFemale

+MaleMale

+FemaleFemale

+Mean

body wt Â±S.D.(g), based on (n)mice28.9

Â±2.1(10)6 \)27.0

Â±2.0(10)\(24.9

Â±1.6(10) 1,21.

5 Â±2.8(9)'33.7+2.7(19)

"i)(278+2.7(18)

\\24.5

+ 2.8 (20))25.7

Â±2.2(19)Pn.s.cp

<0.0010.001<p<0.01p<0.001p

<0.001n.s.Mean

logio (tumor wt,mg) Â±S.D., based on (n) micep"2.8353

Â±0.4312 (10)\2.3729

Â±0.4476 (10)\Ã•2.1922

Â±0.4212 (10)J/2.6841

Â±0.2869(9)>.2.9943+0.2538(19)

J(,2.7975+0.2708(18)

/2.4331+0.2438(20))|2.4380

Â±0.2725 (19) '>â€¢

0.02 <p<0.05k0.001<p<0.010.001<p<0.010.02

<p<0.05p

<0.001n.s.

ap values are calculated from the logarithm of each tumor weight.^Numbers in parentheses represent no. of mice.cNot significant.

Table 3Effect of hydrocortisone acetate on solid Ehrlich/4N and Ehrlich/2N tumors

GroupNo.Ehrlich/4N1

21'21Ehrlich/2N12r2'Daily

dose ofHost HCA(mg)Male

MaleFemaleFemaleMaleMaleFemaleFemale01.001.001.001.0Mean

body wt Â±S.D.(g), based on

(n)mice29.3

Â±1.9(13)Â«Â»^ )

28.0 Â±1.5(9)\]22.5

Â±2.0(15) ))>22.0

Â±2.2(10))30.3

+ 1.1 (14)\ÃŒ(f26.8

Â±3.4(9)\]23.4

Â±2.5(15)))t20.4

Â±1.5(9) }Pn.s.cp<0.001&JS,0.001

<p<0.01p

<0.0010.001<p<0.01Mean

logio (tumorwt,mg)Â± S.D. based

on (n)mice2.8550

Â±0.2932 (13)^ |

2.2838+0.2023(9)\\2.1074

Â±0.2020 (15)))f2.1614

Â±0.1715 (10)}2.7875+0.1937(14)

^/([2.5834+0.2487(9)

M2.3517+0.3168(15)

)ÃŒ[2.3147

Â±0.2156 (9) )pÂ°p

<0.001p

<0.001n.s.0.02<p<0.05p

<0.001n.s.


given on Days -11, -9, -7, -5, -3, -1, 1, 3, 5, 7, 9, and

11. Ehrlich/4N tumor was inoculated on Day 0 and weighedon Day 14. As with testosterone, a significant stimulation oftumor growth was induced in female mice by thesetestosterone derivatives but not in male mice.

Correlation between the Hormone Dose and the Responsefrom the Side of the Tumor and Host. Table 7 summarizesthe correlation coefficients r and the p values for theresponse of the tumor and host versus the hormone doseswhich are calculated from the preceding data. There is ahighly significant negative correlation between the dose of

estradiol and the mean tumor weight in Ehrlich/2N andEhrlich/4N tumors as well. A highly positive correlation wasshown between the dose of testosterone and the meanweight of Ehrlich/4N tumors in the castrated male as well asin the intact female mice. However, this correlation was notsignificant for Ehrlich/2N tumor Â¡nfemale mice. The meanbody weight and the mean uterine weight of female micewere also positively correlated to the dose of testosterone.The positive correlation between the dose of HMD and themean weight of Ehrlich/4N tumor in female mice wasstatistically significant. Similarly, the correlation between the

JANUARY 1970 223




Table 4Effect of estradiol administration on solid Ehrlich/4N and Ehrlich/2N tumors

GroupNo.Ehrlich

4/N1

234Ehrlich/2N1

2

34Daily

Mean body wt Â±S.D.dose of (g), based on

Host estradiol (mg) (n) micepMale

MaleMaleFemaleMale

Male

MaleFemale00.21.000

0.2

1.0028.2

Â±2.6 (19)*>\\)j 1 l 0.02 <p< 0.05

26.0 Â±3.8(19) fU}('28.9

Â±3.2(20) (\\/p<0.00120.3

Â±2.5 (20)/23.4

Â±3.4 (21) \ \)23.6+2.6(10)

fU} ( n.s.

21.7+2.5(10) t 1l7n.s.22.5

Â±1.2(10) /Mean

Iogj0 (tumorwt,mg)Â±S.D., based

on (n) micep"3.1336

Â±0.3918 (19)

2.9410 Â±0.4218(19)2.7318+0.5012(20)2.4922

Â±0.3846 (20),\\

"</'0.001<p<0.011'

p<0.0012.5508+0.3730(21)

2.5357 Â±0.1422 (10)

2. 1834 Â±0.1767(10)2.2937

Â±0.3681 (10)/'

0.001<p<0.01

' n.s.


Table 5Effect of testosterone administration on solid Ehrlich/4N tumor in castrated male mice

Gonad- Daily Mean body wt Â±S.Group ecto- dose of (g), based on

No. Host my testosterone (mg) (n)mice1

Male2

Female-3

Male +

4 Male+5

Male +0000.050.25312528

29324

Â±1.6.2

Â±1.9.4

+2.2.9

Â±1.6(IO)"(10)(10)

(10)(11)D.

Pi

Ifyp<o.ooi>

\i0.001<p<0.01

) |n.s.t

n.s.Mean

lugj 0 (tumorwt,mg)Â± S.D., based

on (n)mice2112.8978+0.4515.9256Â±0.4321.8404Â±0.2429

.2467Â±0.35592.4435Â±

0.4849(10)}(10))L(10v)(10)

>(11)

)pap

<0.001p

< 0.001

>0.001<p<0.010.001<p<0.01


dose of S-3900 and the mean weight of Ehrlich/4N tumor infemale mice was significant at the 5% level.

DISCUSSION

Beatson's discovery in 1896 (1) of the suppressive effect of

ovariectomy on human breast cancer was followed by manyinvestigations on the experimental mammary cancer.Lacassagne (18) was the first to succeed in induction ofbreast cancer in male mice by weekly injections of 30 ÃŸgestrone benzoate, but, as MÃ¼hlbock(21) stated, most of themammary tumors in mice with and without the tumor agentbecome hormone independent when they reached a palpablesize.

The previous studies by Kodama (16) showed that thegrowth of solid Ehrlich tumor, a mammary cancer in origin,is closely related to the function of the gonads and theadrenals, but there remained some doubt as to theidentification of hormones which are responsible for thestimulated growth of tumor in male mice (17). Also worthinvestigating is the significance of United States strains in theabove experiments.

The present study with Japanese strains of mice andtumors reconfirmed the old findings about the suppressiveeffect of the corticosteroid hormone for the growth of solidEhrlich tumor in male mice as well as the antagonisticrelation between the male and female gonads in the sameprocess (16, 17). Furthermore, the stimulative factor of the





Table 6Effect of testosterone administration on solid Ehrlich/4N and Ehrlich/2N tumors in intact female mice

GroupNo.Ehilich/4N1234Ehrlich/2N1234Daily

Mean body wt Â±S.D.dose of (g), based on

Host testosterone (mg) (n)miceFemaleFemaleFemaleMaleFemaleFemaleFemaleMale00.21.0000.21.0020.622.7242821.2.7.822.62327.3.5Â±1.4Â±2.9Â±1.4Â±2.5Â±1.5Â±1.6Â±1.5.â€”,(20)

\)(19)

f>(20)

Ã•\P\i(l

n-s-CH)

p<0.001p

<0.001JIV0.01<p<0.02n0.01<p<0.027p<0.001Â±3.0

(20) /Mean

Iog10 (tumorwt, mg)Â±S.D., based

on (n)mice2.59852.87223.11553.21332.37432.31502.43452.9967Â±0.3804Â±0.4963Â±0.2617Â±0.2784Â±0.3021Â±0.3388Â±0.3487Â±0.2877(20)

'(19)(19)(20),(20)(19)(20)(20)Pa!Jiln.s.Ul

p <0.001'p

<0.001\\

1H

n.s.M('n.s.p

< 0.001

ap values are calculated from the logarithm of each tumor weight.^Numbers in parentheses indicate no. of mice.cNot significant.

Table 7Correlation between the hormone dose (x) and the response (y) of the tumor and host

XEstradici(mg/day/mouse)Testosterone(mg/day/mouse)HMD(mg/day/mouse)S-3900(mg/day/mouse)"For

tumortissue,0Notsignificant.yMean

weightofEhrlich/4Ntumor

(mg)MeanweightofEhrlich/2Ntumor

(mg)Mean

bodyweight(g)

Mean weightofEhrlich/4Ntumor


(mg)Meanbodyweight

(g)Meanuterineweight

(mg)MeanweightofEhilich/2Ntumor

(mg)Mean



(mg)Mean



(mg)HostMale

miceMale

miceCastratedmale

miceCastratedmalemiceFemale

miceFemale

miceFemale

miceFemale

miceMale

miceFemale

miceMale

miceFemale

micethe

r and p values are calculated from ther"-0.34-0.500.250.500.480.440.290.110.11t0.39,i

â€¢0.250.43logarithm

of eachPa0.009

<p< 0.0130.001<p<

0.002n.s.60.005

<p<0.0070.002

<p<0.004p

=0.0010.027

<p<0.036n.s.n.s.0.021<p<

0.028n.s.'0.021<p<0.028tumor

weight.

JANUARY 1970 225




testes for the tumor growth was identified as testosteronethrough the hormone administration experiment. The totalbody weight showed a relatively small deviation, whereas agreat fluctuation was observed in the weight of neoplastictissue which is beyond the homeostatic control of the host.Experimental data with a greater standard deviation mayrender some difficulty in applying the t test for statisticalevaluation. The logarithm of each measurement could besometimes more appropriate for that purpose, as suggestedby Bross (3). In our case, the logarithm of tumor weightmay serve as a growth index of the corresponding tumorprovided that the tumor growth proceeds as an exponentialfunction of inoculation day. Our old findings on the growthof solid Ehrlich tumor showed a very slight deviation fromthe exponential increase (17). The chi square test wasapplied for these data to see whether or not they aredistributed normally enough to permit the use of the mean,standard deviation, and table of areas of the normal curve indescribing a set of measurements. It is clear that thedistribution of logarithmic values of tumor weight isessentially normal, making a sharp contrast to thedistribution of tumor weight itself which shows a remarkabledeviation from the normal curve. By the same test thedistributions of the weight of the liver, the kidney, and thetotal body were normal.

There are several reports that the male hormone maystimulate the growth of mammary cancer (5, 10, 25, 26).Mineshita and Yamaguchi (20) reported on an androgen-dependent mammary tumor of mouse which failed to growin the absence of the male hormone. Furth (8) also statedthat a "male type" of mammary tumor first induced in a

male rat preferred the male host in successive transplantation. These findings indicate that the hormonedependency of cancer cells can be produced under theinfluence of hormonal milieu intÃ©rieur.The stimulative effectof testosterone in this case was observed with the Ehrlichtumors which had been maintained in female mice over 3years.

The hypotetraploid tumor seems to be more responsivethan the hyperdiploid tumor with regard to the effect oftestosterone and hydrocortisone. The same differencebetween the above 2 tumors has also been noted in theirsusceptibility to 6-mercaptopurine (22). Some discrepanciesbetween our data and those of others (9, 24) might beexplained by the difference in the tumors or in theexperimental procedures. The inhibitive effect of hydro-cortisone is observed only in male mice bearing solidEhrlich/4N tumors. The most probable explanation for ourfindings is that the growth of solid Ehrlich/4N tumorconsists of 2 portions; one portion is steroid insensitive andthe other is steroid sensitive. In female mice the growth ofsolid Ehrlich tumor proceeds only through the formerprocess in the absence of stimulative action of testosterone.In male mice the growth of solid Ehrlich tumor is asummation of the first and the second processes, the latterbeing stimulated by the male hormone of the host. Ifhydrocortisone is administered to male mice, the secondportion of tumor growth is extinguished by an inhibitiveaction of the hormone. In female mice no inhibition is

observed in the tumor growth, which is entirely steroidindependent. There is no information available as to whetherthe presence of steroid-sensitive and -insensitive growth isrelated to the inhomogeneity of the cell population of theEhrlich tumor.

The protein-anabolic action of testosterone was noted byKochakian et al. (14, 15) who found that testosteroneincreased the carcass weight of the rodents with nosignificant change in the proportion of protein, fat, or water.In the radioisÃ³topo experiment, the incorporation of labeledamino acids into proteins of various organs was increased bythe pretreatment with testosterone (4, 6, 7). Evidenceindicates that the most probable action of testosterone is toinfluence the concentration of template (messenger) RNA ofribosomes(19, 27).

Kim et al. (11) emphasized the significance of mammo-tropic hormone in the growth of estrogen-responsivemammary tumor; they showed that estradici failed tostimulate the growth of mammary tumor in hypophys-ectomized rats, while mammotropin was highly effective instimulating tumor growth. It was concluded that thestimulatory action of estradiol for mammary tumor resultsfrom stimulation of pituitary mammotropes. For Ehrlichtumor, the possible participation of the pituitary in thesteroid hormone assays is not excluded, but there areindications that some of the androgen actions areindependent of the pituitary (12, 13, 15, 23).

Blum (2) stated that the growth of cancer might be eitheran expression of the overall growth of the animal or afunction of the specific factor which governs only thegrowth of the target tissue. The action of hormones asmeasured in terms of tumor weight was not specificallydifferentiated in this respect. The present investigationindicated a stimulative effect of testosterone and aninhibitive effect of hydrocortisone and estradiol for thegrowth of solid Ehrlich tumor. There is a possibility that theadministration of a hormone will affect the food intake ofthe host animal which may in turn influence the tumorgrowth. The significance of host factors which might beinvolved in the expression of hormonal action is to beelucidated in future. Some of the metabolic changes inhormone-treated tumor cells will be described in asubsequent publication.

ACKNOWLEDGMENTS

We are grateful to Professor Susumu Hibino for his continuedencouragement, to Dr. Kyo Kaziwaia for his supply of Ehrlich ascitestumors, and to Dr. I. D. J. Bross for his valuable information anddiscussion about the statistical procedures.

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JANUARY 1970 227



1970;30:221-227. Cancer Res Mitsuo Kodama and Toshiko Kodama Responsiveness of Solid Ehrlich TumorsStatistical Analysis of Hormonal Effects on the Steroid

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