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Cortisol metabolism in lymphocytes from cancer-bearing patients
Cortisol Metabolism in Lymphocytes From Cancer-bearing Patients Ami Klein, Haiuta
Kaufmann,
Shoshana
A study was conducted to determine the pattern of cortirol metabolism by lymphocytes obtained from four groups of subjects: 27 male and female patients suffering from various types of malignancy other than malignancy of lymphatic tissues; and 26 healthy male and female controls. Known concentrations of cells were incubated with 1 ,2-3H-cortisol and the products were isolated by thin-layer and paper chromatography. Three metabolites were found to be produced by lymphocytes from both normal and cancer-
Mannheimer,
and Henry Joshua
bearing patients: 2Oa-hydroxycortisol, 20&hydroxycortisol, and tetrahydrocortisol. Cells from the female control group were found to be more active than those from the male controls, while cells from cancer-bearing patients were markedly more active than the normal cells, regardless of sex. It is suggested that this finding of increased metabolism of cortisol by lymphocytes from patients with different types of malignancy other than lymphoma may provide the basis for a new diagnostic aid.
T
HE DESTRUCTIVE EFFECT of cortisol on lymphocytes as well as the ability of lymphocytesto effect alterations in the molecular structure of cortisol have been demonstrated by Dougherty and his associates.‘s2 These workers used murine lymphocytes and found them capable of oxidizing the 11 position, thus transforming cortisol to cortisone, and also of reducing the 20 position, thus transforming cortisol to 20a-hydroxycortisol and 20&hydroxycortisol. In contrast, the studies on human leukocytes carried out by Forker et al.3 provided partial evidence of a transformation of cortisol to tetrahydrocortisol (THF) without formation of cortisone or 20-hydroxy derivates of cortisol. Jenkins and Kemp,4 who performed experiments on human polymorphonuclear leukocytes and lymphocytes, observed that both cell groups were capable of metabolizing cortisol to THF, 20a-hydroxycortisol, and 20/S hydroxycortisol, with no production of cortisone found. Furthermore, in the above studies it was found that lymphocytes obtained from murine lymphoma’ and also from patients suffering from various forms of leukemia’S3*4were more active in metabolizing cortisol than normal cells. In the present study the in vitro metabolism of cortisol by lymphocytes
Abbreviations: Cortisol: 11/3,17a,21-trihydroxy-4-pregnene-3,20-dione. Cortisone: 17a,21-dihydroxy-4-pregnene-3,11,20-trione. I Ifi-Hydroxyandrostendione: 1l/Shydroxy-4-androstene-3,17dione. Zoo-Hydroxycortisol: 11&17~~,20a, 21-tetrahydroxy-4-pregnene-3-one. 20@-Hydroxycortisol: 11/3,17a,20@,21-tetrahydroxy-4-pregnene-3-one. 6&Hydroxycortisol: 1Ip,l7a,6&21-tetrahydroxy-4-pregnene-3,20-dione. Tetrahydrocortisol (THF): 3o,l lp,l7o,21-tetrahydroxy-5-pregnan20-one. From the Endocrinological Laboratory and the Clinical Laboratory, Beilinson Medical Center. Petah Tiqva. Israel. Received for publication August 18. 1977. Supported by the Israel Cancer Research Fund (George and Rose Blumenthal Research FellowshipJor Hodgkin’s Disease). Address reprint requests to Dr. Ami Klein, Endocrinological Laboratory, Beilinson Medical Center, Petah Tiqva,‘israel. 0 1978 by Grune & Stratton, Inc. 0026-0495/78/270&0011$01.00/0
Metabolism,
Vol. 27,
No. 6
(June) 1978
731
732
KLEIN
ET AL.
obtained from patients suffering from various forms of cancer (in almost all cases carcinoma and in no case malignancy of the lymphatic tissues) was investigated and the results were compared with the changes incurred by normal lymphocytes. MATERIALS
AND METHODS
The subjects in this study were 13 male and 14 female patients (24-75 yr old), most of whom were suffering from different forms of carcinoma. A control group consisted of 15 males and I I females (18-60 yr old) selected from among healthy blood donors. Lymphocytes from the controls were prepared from huffy coats obtained from the blood bank while those from the cancer patients were prepared from 50 ml heparinized blood drawn by venipuncture. They were isolated by centrifugation at 400 g using the Ficoll-Isopaque method, ,washed twice, and resuspended in phosphate buffered saline solution, pH 7.4, containing penicillin and streptomycin, 100 HI/ml each (preliminary investigation showed that the TC-199 medium usually used in this procedure could be substituted for by the buffered saline). The lymphocytes were incubated in a shaking bath at 37°C for I7 hr in sealed flasks containing the following: (I) I ml lymphocyte suspension; (2) an NADPH generating system composed of I.0 rmole NADPH (Sigma), 5.0 pmoles glucose-6-phosphate (Sigma), 1.0 Korenberg unit glucose-6-phosphate dehydrogenase, and 1.4 pmole MgClz, all dissolved in 0.1 ml buffered saline; (3) 5 pCi 1,2-3H-cortisol (specific activity 45.9 mCi/mmole). At the end of incubation 9505 viability was proven by the trypan blue dye method. A blank containing all components with the exception of the lymphocytes was also processed. At the end of the incubation period the contents of each flask were extracted twice with 5 ml chloroform and 50 fig of each of the following steroids were added: cortisol, 20a-hydroxycortisol, ZOO-hydroxycortisol, and THF. The chloroform extract was evaporated to dryness under nitrogen and the residue was dissolved in ethanol and applied on silica gel HF 254 (Merck) thinlayer plates. The plates were developed in chloroform: methanol (9O:lO V/V). Following chromatography the plates were viewed in ultraviolet (uv) light at 254 nm and scanned using the Berthold LB 2723 radioactivity scanner. The product and substrate spots as well as the remain,der of the plate (for recovery calculations) were scraped off and extracted twice with ethanol (final volume 5.0 ml). Samples of 0.1 ml were transferred into scintillation vials and the radioactivity was read in a liquid scintillation spectrometer (Packard model 3390). Twenty vials, five from each group of subjects (male and female patients, and male and female controls), containing the steroid products were dried off and the residues, dissolved in ethanol, were transferred to the starting line of a Whatman No. I chromatography paper. After equilibration the strips were chromatographed in the Bush B-5 system (benzene:methanol:water, 100:50:50) for I6 hr.5 A control strip contained 6@-hydroxycortisol, ZOa-hydroxycortisol, 20&hydroxycortisol, and THF for purposes of reference. Following chromatography the paper was scanned and viewed under a uv lamp. The control, in addition, was sprayed with a blue tetrazolium solution in order to detect those steroids with a reduced A ring and the presence of l7-hydroxy, 2Oa-keto groups (THF). The radioactive spots were cut out, extracted with ethanol (10.0 ml), and counted in the scintillation counter in O.l-ml aliquots. Chromatography of acetylated compounds was performed using the Bush B-3 system.’ Nonlabeled cortisol (160 pg) was also incubated with lymphocyte suspension, and the extracted metabolites were subjected to gas chromatography using an SE-30 column. The data were analyzed in accordance with sex since the types of tumors found in the males and females differed. The cortisol-converting activity of the various lymphocyte cultures was expressed as the percentage of converted cortisol in relation to the total amount added. Furthermore, since the amount of lymphocytes in each culture varied considerably, it was considered appropriate that the comparison of cortisol-converting activity be expressed in relation to a constant number of cells. A regression line was therefore drawn by plotting the percentage conversion of control lymphocytes against the number of lymphocytes present in each culture; this resulted in perfect linearity (Fig. I) and allowed us to calculate the conversion activity on the basis of a constant number of lymphocytes (5 x IO’).
CORTISOL
METABOLISM
IN
733
CANCER
Fig. 1. Regression curve for the values for percentage conversion of 1 ,2-3H-cortisol to the product versus the lymphocyte count in the culture medium.
i__-__ O
Cell
~ 50 Number
-.. ~-I100 10’
RESULTS
All of the extracts of lymphocyte suspensions preincubated with labeled cortisol, run on thin-layer plates, and then scanned showed two radioactive peaks--a major, less polar peak corresponding to cortisol itself, and a minor, more polar peak representing the total amount of metabolized cortisol. No steroid compounds less polar than cortisol were visualized. Paper chromatography of eluates from the minor peak resulted in the appearance of three steroid metabolites identified with the appropriate reference materials as 20~ hydroxycortisol, ZO&hydroxycortisol. and THF. Upon chromatography of the acetylated metabolites the radioactive spots detected with the scanner were found to correspond with the reference acetylated materials run in parallel. Gas chromatography of the metabolites of the nonlabeled cortisol yielded Table 1. Cortisol Metabolism Cancer Patients
(N =
by lymphocytes
From Female
14) and Female Controls (N = 11) No. of Cells
% Conversion/
107Cells
Subject
Age
Y.G
50
Mammary
carcinoma
5.5
9.34
N.V
46
Mammary
carcinoma
4.2
1 1.94
K.R
64
Mammary
carcinoma
5.0
11.75
B.S
63
Mammary
carcinoma
5.1
10.99
P.E
63
Endometriol
K.R
49
Mammary
carcinoma
G.E
56
Mammary
carcinoma
7.0
6.71
SE
69
Mammary
carcinoma
4.5
10.19
carcinoma
2.0
17.85
3.1
13.27
Diagnosis
P.G
55
Mammary
s.0
61
Cecum
carcinoma
carcinoma
x 10’
5 x
4.3
11.71
2.9
13.45
H.H
54
Mammary
carcinoma
2.0
15.65
A.A
61
Mammary
carcinoma
3.7
1 1.09
H.T
29
Mammary
carcinoma
2.4
5.38
S.G
53
M&IlOm~
7.0
14.58
Meoll Controls *Difference
1 B-60 wets statistically
4.0-5.1 significant
(p
< 0.001)
11.71
f
3.29*
7.27
f
2.2+
734
KLEIN
Table 2. Cortisol Metabolism
ET Al.
by Lymphocytes From Male
Cancer patients (N = 13) and Male Controls (N = 15) No. of Cells Subject
Diagnosis
Age
x 107
% Conversion/ 5 x 10' Cellr
65
Lung carcinoma
6.6
10.43
60
Lung carcinoma
4.8
9.92
E.I
24
Seminoma
4.9
T.A
71
Lung carcinoma
3.8
13.03
G.R
59
Carcinoma
of kidney
4.6
12.21
K.M
51
Carcinoma
of larynx
3.6
14.58
R.Z
73
Carcinoma
of thyroid
3.2
7.81
S.L
72
Lung carcinoma
4.7
11.70
Z.Y
74
Sweat
3.7
11.08
B.A
75
Mammary
R.M
55
R.Y T.Y
0.M
W.A
gland
carcinoma
carcinoma
3.7
7.45
Lung carcinoma
2.0
15.25
66
Carcinoma
of intestine
4.5
17.59
62
Carcinoma
of rectum
9.0
Mean
6.53 11.18+3.35*
Controls *Difference
7.74
was
18-60 statistically
2.0-10.0 significant
(p
5.55
f
0.7*
< 0.001).
peaks corresponding to ZO&hydrocortisol and THF. 20a-Hydroxycortisol could not be detected because of its low rate of production. The percentages of cortisol metabolized by the lymphocytes from all four groups of subjects are shown in Tables 1 and 2. The mean cortisol-converting activity of lymphocytes from healthy males was significantly lower than that found in those of healthy females (p < 0.03). Comparison of the lymphocytes of control subjects to those of the cancer patients showed that the converting activity of both cancer-bearing groups was higher (twice as high in the males, p < 0.001; and 1.6 times as high in the females, p < 0.001). The difference between the male and female cancer patients,was not significant (Fig. 2). Table 3 shows the percentage distribution of each of the metabolites obtained: 20a-hydroxycortisol, 20/3-hydroxycortisol, and THF. There was no significant difference in this distribution among the four groups of subjects. DISCUSSION
As noted before, earlier studies performed with lymphocytes obtained from both murine and human lymphomas showed that these lymphocytes have an increased capacity to metabolize cortisol.14 Dougherty et al.’ postulated that the ability of lymphocytes to effect changes in cortisol constitutes an important homeostatic mechanism in the regulation of the lymphocyte population. They further suggested that one of the features of malignancy is the ability of the cell to metabolize biologically active compounds that normally control its proliferation, resulting in the production of metabolites that have no such function. The comparative study of the pattern of cortisol metabolism by human lymphocytes reported here clearly demonstrates that the conversion rate of lymphocytes obtained from cancer-bearing patients is significantly higher than that of lymphocytes obtained from healthy donors, regardless of sex. It further shows that this increased conversion rate is present not only in cases of malignancy of the lymphatic system but also in patients bearing other forms of cancer. It
CORTISOL
METABOLISM
IN CANCER
>ontrol
Control
Cancer
Zancer
d
Q
d .
9.
. . . .. ..
. . 0. L. . . .
. . . ... **
.
5
more polar products as performed by lymphocyter from patients with the indicated clinical conditions.
*
l
l
l
.
Means and distributions of the Fig. 2. values for conversion of 1 ,2-3H-cortisol to the
*.
l
.
.
Mean
3
S.0
5.5 5
227
11.18
0.70
2.20
3.35
11.71 3.2 9
may be speculated that if this phenomenon is found to be characteristic of general malignancy, it could provide us with a new diagnostic tool. Qualitatively, the findings of this study confirm those obtained by Jenkins and Kemp4 and Forker et a1.,3 who showed that human polymorphonuclear leukocytes and lymphocytes are able to transform cortisol to more polar steroids. The results of paper chromatography, which showed the production of the three metabolites 20a-hydroxycortisol, 20/3-hydroxycortisol, and THF, are in agreement with those reported by Jenkins et a1.4 These results differ from the findings in murine lymphocytes’*2 as well as in other extrahepatic human organs, 6-‘4in which there was a production of both less and more polar steroids (cortisone, 1l&hydroxyandrostendione, and C-6 reduced cortisol, respectively). From the work done by Jenkins et a1.4 as well as the present study, it would appear that lymphocytes and polymorphonuclear leukocytes are the only extrahepatic cells capable of transforming cortisol to THF. The similar Table 3. Percentage (f
SD) of Cortisol Metaboliteb
Calculated From Total Production Peak 20wOH
Cortisol
20&OH
Cortirol
THF
Controls Mole
6.9
f
1.76
39.78
*
5.71
53.32
f
7.94
f
2.41
34.52
+
6.18
57.54
zt 6.16
Mole
3.91
f
0.57
44.36zt
11.11
51.74*
Female
6.64
f
2.91
43.16
f
4.34
50.19
Female Cancer
Production were
obtained
ferences
were
6.12
patients
peak by
was
obtained
paper
not statistically
by
thin-layer
chromotogrophy. significant.
chromatography
Values
ore
means
and f
SD
the of
separations 5
patients
11.40 * of
in
4.97 the
each
metobolites group.
Dif-
736
KLEIN
ET Al.
pattern of cortisol metabolism found in these two types of cells may be related to their common origin. Our study showed that the increased capacity for cortisol metabolism found in lymphocytes from cancer patients resulted from a virtually equal rise in production of all three metabolites. These findings largely agree with those of Jenkins et a1.,4although those authors found that the increased production of metabolites by lymphocytes from cases of acute and chronic lymphatic leukemia was not equal for the three metabolites. In none of the above cited works was an attempt made to find sex-dependent differences in the metabolism of cortisol by lymphocytes. In our study it was of interest to find that the lymphocytes obtained from normal healthy females had a higher rate of activity than those from males, although these sex differences disappeared in the cancer-bearing patients. In conclusion, we may state that the present study has clearly demonstrated that the rise in the metabolism of cortisol by the lymphocytes of cancer-bearing patients is associated not only with malignancy of the lymphatic tissues but evidently may also occur in patients with other forms of malignancy, notably carcinoma. ACKNOWLEDGMENT The authors are grateful to Dr. J. Malchi and his staff at the Beilinson Hospital Blood Bank for supplying us with huffy coat. We also thank Dr. S. Bernstein, Lederle Laboratories, Pearl River, N.Y., who provided us with 6fi-hydrocortisol. In addition we would like to thank R. Fradkin and S. Albukrek for revising and typing the paper.
REFERENCES I. Dougherty TF, Berliner DL, Berliner ML: Corticoid-tissue interactions. Metabolism 10: 966-987, 1961 2. Berliner DL, Dougherty TF: Hepatic and extrahepatic regulation of corticosteroids. Pharmacol Rev 13:329-359, 1961 3. Forker AD, Bolinger RE, Morris JH, et al: Metabolism of cortisol-‘4C by human peripheral leukocytes cultures from leukemic patients. Metabolism 12:751-759, 1963 4. Jenkins JS, Kemp NH: Metabolism of cortisol by human leukemic cells. J Clin Endocrinol29: 1217-1221, 1969 5. Bush IE: Methods of paper chromatography of steroids applicable to study of steroids in mammalian blood and tissues. Biochem J 50: 370-378, 1952 6. Miabo S, Kishida S, Hisada T: Metabolism and conjugation of cortisol by various dog tissues in vitro. J Steroid Biochem 4: 567-576, 1973 7. Mahesh VB, Ulrich F: Metabolism of cortisol and cortisone by various tissues and subcellular particles. J Biol Chem 235:356-360, 1960 8. Jenkins JS: The metabolism of cortisol by human extrahepatic tissues. J Endocrinol 34: 51-56, 1966
9. Hudson RW, Schachter H, Killinger DW: The conversion of cortisol to ll&hydroxyandrostenedione by beef adrenal tissues. Endocrinology 95:38-47, 1974 10. Klein A, Siebenmann R, Curtius HCH, et al: Steroid 1I@-hydroxylase activity in the microsomal fraction of human adrenals. J Steroid Biochem 7:283-287, 1976
II. Sweat ML, Grosser BI, Berliner DL, et al: The metabolism of cortisol and progesterone by cultures of uterine fibroblasts, strain U12-705. Biochim Biophys Acta 28:591-596, 1958 12. El Attar TMA, Murray WR, Anderson CE: In vivo catabolism of cortisol in synovial fluid of the rheumatoid and osteoarthritic knee. Arthritis Rheum I1:178-183, 1968 13. El studies gingiva 355-364,
Attar TMA: In vitro metabolism of (1,2,6,7-‘H)-cortisol in human in health and disease. Steroids 25: 1975
14. El Attar TMA, Gustafson GM, Wong DG: Comparative metabolism in vivo of (4-14C) cortisol in synovial fluid in the normal, rheumatoid and osteoarthritic knee. Biochim Biophys Acta 137:375-387, 1967
Kaufmann,
Shoshana
A study was conducted to determine the pattern of cortirol metabolism by lymphocytes obtained from four groups of subjects: 27 male and female patients suffering from various types of malignancy other than malignancy of lymphatic tissues; and 26 healthy male and female controls. Known concentrations of cells were incubated with 1 ,2-3H-cortisol and the products were isolated by thin-layer and paper chromatography. Three metabolites were found to be produced by lymphocytes from both normal and cancer-
Mannheimer,
and Henry Joshua
bearing patients: 2Oa-hydroxycortisol, 20&hydroxycortisol, and tetrahydrocortisol. Cells from the female control group were found to be more active than those from the male controls, while cells from cancer-bearing patients were markedly more active than the normal cells, regardless of sex. It is suggested that this finding of increased metabolism of cortisol by lymphocytes from patients with different types of malignancy other than lymphoma may provide the basis for a new diagnostic aid.
T
HE DESTRUCTIVE EFFECT of cortisol on lymphocytes as well as the ability of lymphocytesto effect alterations in the molecular structure of cortisol have been demonstrated by Dougherty and his associates.‘s2 These workers used murine lymphocytes and found them capable of oxidizing the 11 position, thus transforming cortisol to cortisone, and also of reducing the 20 position, thus transforming cortisol to 20a-hydroxycortisol and 20&hydroxycortisol. In contrast, the studies on human leukocytes carried out by Forker et al.3 provided partial evidence of a transformation of cortisol to tetrahydrocortisol (THF) without formation of cortisone or 20-hydroxy derivates of cortisol. Jenkins and Kemp,4 who performed experiments on human polymorphonuclear leukocytes and lymphocytes, observed that both cell groups were capable of metabolizing cortisol to THF, 20a-hydroxycortisol, and 20/S hydroxycortisol, with no production of cortisone found. Furthermore, in the above studies it was found that lymphocytes obtained from murine lymphoma’ and also from patients suffering from various forms of leukemia’S3*4were more active in metabolizing cortisol than normal cells. In the present study the in vitro metabolism of cortisol by lymphocytes
Abbreviations: Cortisol: 11/3,17a,21-trihydroxy-4-pregnene-3,20-dione. Cortisone: 17a,21-dihydroxy-4-pregnene-3,11,20-trione. I Ifi-Hydroxyandrostendione: 1l/Shydroxy-4-androstene-3,17dione. Zoo-Hydroxycortisol: 11&17~~,20a, 21-tetrahydroxy-4-pregnene-3-one. 20@-Hydroxycortisol: 11/3,17a,20@,21-tetrahydroxy-4-pregnene-3-one. 6&Hydroxycortisol: 1Ip,l7a,6&21-tetrahydroxy-4-pregnene-3,20-dione. Tetrahydrocortisol (THF): 3o,l lp,l7o,21-tetrahydroxy-5-pregnan20-one. From the Endocrinological Laboratory and the Clinical Laboratory, Beilinson Medical Center. Petah Tiqva. Israel. Received for publication August 18. 1977. Supported by the Israel Cancer Research Fund (George and Rose Blumenthal Research FellowshipJor Hodgkin’s Disease). Address reprint requests to Dr. Ami Klein, Endocrinological Laboratory, Beilinson Medical Center, Petah Tiqva,‘israel. 0 1978 by Grune & Stratton, Inc. 0026-0495/78/270&0011$01.00/0
Metabolism,
Vol. 27,
No. 6
(June) 1978
731
732
KLEIN
ET AL.
obtained from patients suffering from various forms of cancer (in almost all cases carcinoma and in no case malignancy of the lymphatic tissues) was investigated and the results were compared with the changes incurred by normal lymphocytes. MATERIALS
AND METHODS
The subjects in this study were 13 male and 14 female patients (24-75 yr old), most of whom were suffering from different forms of carcinoma. A control group consisted of 15 males and I I females (18-60 yr old) selected from among healthy blood donors. Lymphocytes from the controls were prepared from huffy coats obtained from the blood bank while those from the cancer patients were prepared from 50 ml heparinized blood drawn by venipuncture. They were isolated by centrifugation at 400 g using the Ficoll-Isopaque method, ,washed twice, and resuspended in phosphate buffered saline solution, pH 7.4, containing penicillin and streptomycin, 100 HI/ml each (preliminary investigation showed that the TC-199 medium usually used in this procedure could be substituted for by the buffered saline). The lymphocytes were incubated in a shaking bath at 37°C for I7 hr in sealed flasks containing the following: (I) I ml lymphocyte suspension; (2) an NADPH generating system composed of I.0 rmole NADPH (Sigma), 5.0 pmoles glucose-6-phosphate (Sigma), 1.0 Korenberg unit glucose-6-phosphate dehydrogenase, and 1.4 pmole MgClz, all dissolved in 0.1 ml buffered saline; (3) 5 pCi 1,2-3H-cortisol (specific activity 45.9 mCi/mmole). At the end of incubation 9505 viability was proven by the trypan blue dye method. A blank containing all components with the exception of the lymphocytes was also processed. At the end of the incubation period the contents of each flask were extracted twice with 5 ml chloroform and 50 fig of each of the following steroids were added: cortisol, 20a-hydroxycortisol, ZOO-hydroxycortisol, and THF. The chloroform extract was evaporated to dryness under nitrogen and the residue was dissolved in ethanol and applied on silica gel HF 254 (Merck) thinlayer plates. The plates were developed in chloroform: methanol (9O:lO V/V). Following chromatography the plates were viewed in ultraviolet (uv) light at 254 nm and scanned using the Berthold LB 2723 radioactivity scanner. The product and substrate spots as well as the remain,der of the plate (for recovery calculations) were scraped off and extracted twice with ethanol (final volume 5.0 ml). Samples of 0.1 ml were transferred into scintillation vials and the radioactivity was read in a liquid scintillation spectrometer (Packard model 3390). Twenty vials, five from each group of subjects (male and female patients, and male and female controls), containing the steroid products were dried off and the residues, dissolved in ethanol, were transferred to the starting line of a Whatman No. I chromatography paper. After equilibration the strips were chromatographed in the Bush B-5 system (benzene:methanol:water, 100:50:50) for I6 hr.5 A control strip contained 6@-hydroxycortisol, ZOa-hydroxycortisol, 20&hydroxycortisol, and THF for purposes of reference. Following chromatography the paper was scanned and viewed under a uv lamp. The control, in addition, was sprayed with a blue tetrazolium solution in order to detect those steroids with a reduced A ring and the presence of l7-hydroxy, 2Oa-keto groups (THF). The radioactive spots were cut out, extracted with ethanol (10.0 ml), and counted in the scintillation counter in O.l-ml aliquots. Chromatography of acetylated compounds was performed using the Bush B-3 system.’ Nonlabeled cortisol (160 pg) was also incubated with lymphocyte suspension, and the extracted metabolites were subjected to gas chromatography using an SE-30 column. The data were analyzed in accordance with sex since the types of tumors found in the males and females differed. The cortisol-converting activity of the various lymphocyte cultures was expressed as the percentage of converted cortisol in relation to the total amount added. Furthermore, since the amount of lymphocytes in each culture varied considerably, it was considered appropriate that the comparison of cortisol-converting activity be expressed in relation to a constant number of cells. A regression line was therefore drawn by plotting the percentage conversion of control lymphocytes against the number of lymphocytes present in each culture; this resulted in perfect linearity (Fig. I) and allowed us to calculate the conversion activity on the basis of a constant number of lymphocytes (5 x IO’).
CORTISOL
METABOLISM
IN
733
CANCER
Fig. 1. Regression curve for the values for percentage conversion of 1 ,2-3H-cortisol to the product versus the lymphocyte count in the culture medium.
i__-__ O
Cell
~ 50 Number
-.. ~-I100 10’
RESULTS
All of the extracts of lymphocyte suspensions preincubated with labeled cortisol, run on thin-layer plates, and then scanned showed two radioactive peaks--a major, less polar peak corresponding to cortisol itself, and a minor, more polar peak representing the total amount of metabolized cortisol. No steroid compounds less polar than cortisol were visualized. Paper chromatography of eluates from the minor peak resulted in the appearance of three steroid metabolites identified with the appropriate reference materials as 20~ hydroxycortisol, ZO&hydroxycortisol. and THF. Upon chromatography of the acetylated metabolites the radioactive spots detected with the scanner were found to correspond with the reference acetylated materials run in parallel. Gas chromatography of the metabolites of the nonlabeled cortisol yielded Table 1. Cortisol Metabolism Cancer Patients
(N =
by lymphocytes
From Female
14) and Female Controls (N = 11) No. of Cells
% Conversion/
107Cells
Subject
Age
Y.G
50
Mammary
carcinoma
5.5
9.34
N.V
46
Mammary
carcinoma
4.2
1 1.94
K.R
64
Mammary
carcinoma
5.0
11.75
B.S
63
Mammary
carcinoma
5.1
10.99
P.E
63
Endometriol
K.R
49
Mammary
carcinoma
G.E
56
Mammary
carcinoma
7.0
6.71
SE
69
Mammary
carcinoma
4.5
10.19
carcinoma
2.0
17.85
3.1
13.27
Diagnosis
P.G
55
Mammary
s.0
61
Cecum
carcinoma
carcinoma
x 10’
5 x
4.3
11.71
2.9
13.45
H.H
54
Mammary
carcinoma
2.0
15.65
A.A
61
Mammary
carcinoma
3.7
1 1.09
H.T
29
Mammary
carcinoma
2.4
5.38
S.G
53
M&IlOm~
7.0
14.58
Meoll Controls *Difference
1 B-60 wets statistically
4.0-5.1 significant
(p
< 0.001)
11.71
f
3.29*
7.27
f
2.2+
734
KLEIN
Table 2. Cortisol Metabolism
ET Al.
by Lymphocytes From Male
Cancer patients (N = 13) and Male Controls (N = 15) No. of Cells Subject
Diagnosis
Age
x 107
% Conversion/ 5 x 10' Cellr
65
Lung carcinoma
6.6
10.43
60
Lung carcinoma
4.8
9.92
E.I
24
Seminoma
4.9
T.A
71
Lung carcinoma
3.8
13.03
G.R
59
Carcinoma
of kidney
4.6
12.21
K.M
51
Carcinoma
of larynx
3.6
14.58
R.Z
73
Carcinoma
of thyroid
3.2
7.81
S.L
72
Lung carcinoma
4.7
11.70
Z.Y
74
Sweat
3.7
11.08
B.A
75
Mammary
R.M
55
R.Y T.Y
0.M
W.A
gland
carcinoma
carcinoma
3.7
7.45
Lung carcinoma
2.0
15.25
66
Carcinoma
of intestine
4.5
17.59
62
Carcinoma
of rectum
9.0
Mean
6.53 11.18+3.35*
Controls *Difference
7.74
was
18-60 statistically
2.0-10.0 significant
(p
5.55
f
0.7*
< 0.001).
peaks corresponding to ZO&hydrocortisol and THF. 20a-Hydroxycortisol could not be detected because of its low rate of production. The percentages of cortisol metabolized by the lymphocytes from all four groups of subjects are shown in Tables 1 and 2. The mean cortisol-converting activity of lymphocytes from healthy males was significantly lower than that found in those of healthy females (p < 0.03). Comparison of the lymphocytes of control subjects to those of the cancer patients showed that the converting activity of both cancer-bearing groups was higher (twice as high in the males, p < 0.001; and 1.6 times as high in the females, p < 0.001). The difference between the male and female cancer patients,was not significant (Fig. 2). Table 3 shows the percentage distribution of each of the metabolites obtained: 20a-hydroxycortisol, 20/3-hydroxycortisol, and THF. There was no significant difference in this distribution among the four groups of subjects. DISCUSSION
As noted before, earlier studies performed with lymphocytes obtained from both murine and human lymphomas showed that these lymphocytes have an increased capacity to metabolize cortisol.14 Dougherty et al.’ postulated that the ability of lymphocytes to effect changes in cortisol constitutes an important homeostatic mechanism in the regulation of the lymphocyte population. They further suggested that one of the features of malignancy is the ability of the cell to metabolize biologically active compounds that normally control its proliferation, resulting in the production of metabolites that have no such function. The comparative study of the pattern of cortisol metabolism by human lymphocytes reported here clearly demonstrates that the conversion rate of lymphocytes obtained from cancer-bearing patients is significantly higher than that of lymphocytes obtained from healthy donors, regardless of sex. It further shows that this increased conversion rate is present not only in cases of malignancy of the lymphatic system but also in patients bearing other forms of cancer. It
CORTISOL
METABOLISM
IN CANCER
>ontrol
Control
Cancer
Zancer
d
Q
d .
9.
. . . .. ..
. . 0. L. . . .
. . . ... **
.
5
more polar products as performed by lymphocyter from patients with the indicated clinical conditions.
*
l
l
l
.
Means and distributions of the Fig. 2. values for conversion of 1 ,2-3H-cortisol to the
*.
l
.
.
Mean
3
S.0
5.5 5
227
11.18
0.70
2.20
3.35
11.71 3.2 9
may be speculated that if this phenomenon is found to be characteristic of general malignancy, it could provide us with a new diagnostic tool. Qualitatively, the findings of this study confirm those obtained by Jenkins and Kemp4 and Forker et a1.,3 who showed that human polymorphonuclear leukocytes and lymphocytes are able to transform cortisol to more polar steroids. The results of paper chromatography, which showed the production of the three metabolites 20a-hydroxycortisol, 20/3-hydroxycortisol, and THF, are in agreement with those reported by Jenkins et a1.4 These results differ from the findings in murine lymphocytes’*2 as well as in other extrahepatic human organs, 6-‘4in which there was a production of both less and more polar steroids (cortisone, 1l&hydroxyandrostendione, and C-6 reduced cortisol, respectively). From the work done by Jenkins et a1.4 as well as the present study, it would appear that lymphocytes and polymorphonuclear leukocytes are the only extrahepatic cells capable of transforming cortisol to THF. The similar Table 3. Percentage (f
SD) of Cortisol Metaboliteb
Calculated From Total Production Peak 20wOH
Cortisol
20&OH
Cortirol
THF
Controls Mole
6.9
f
1.76
39.78
*
5.71
53.32
f
7.94
f
2.41
34.52
+
6.18
57.54
zt 6.16
Mole
3.91
f
0.57
44.36zt
11.11
51.74*
Female
6.64
f
2.91
43.16
f
4.34
50.19
Female Cancer
Production were
obtained
ferences
were
6.12
patients
peak by
was
obtained
paper
not statistically
by
thin-layer
chromotogrophy. significant.
chromatography
Values
ore
means
and f
SD
the of
separations 5
patients
11.40 * of
in
4.97 the
each
metobolites group.
Dif-
736
KLEIN
ET Al.
pattern of cortisol metabolism found in these two types of cells may be related to their common origin. Our study showed that the increased capacity for cortisol metabolism found in lymphocytes from cancer patients resulted from a virtually equal rise in production of all three metabolites. These findings largely agree with those of Jenkins et a1.,4although those authors found that the increased production of metabolites by lymphocytes from cases of acute and chronic lymphatic leukemia was not equal for the three metabolites. In none of the above cited works was an attempt made to find sex-dependent differences in the metabolism of cortisol by lymphocytes. In our study it was of interest to find that the lymphocytes obtained from normal healthy females had a higher rate of activity than those from males, although these sex differences disappeared in the cancer-bearing patients. In conclusion, we may state that the present study has clearly demonstrated that the rise in the metabolism of cortisol by the lymphocytes of cancer-bearing patients is associated not only with malignancy of the lymphatic tissues but evidently may also occur in patients with other forms of malignancy, notably carcinoma. ACKNOWLEDGMENT The authors are grateful to Dr. J. Malchi and his staff at the Beilinson Hospital Blood Bank for supplying us with huffy coat. We also thank Dr. S. Bernstein, Lederle Laboratories, Pearl River, N.Y., who provided us with 6fi-hydrocortisol. In addition we would like to thank R. Fradkin and S. Albukrek for revising and typing the paper.
REFERENCES I. Dougherty TF, Berliner DL, Berliner ML: Corticoid-tissue interactions. Metabolism 10: 966-987, 1961 2. Berliner DL, Dougherty TF: Hepatic and extrahepatic regulation of corticosteroids. Pharmacol Rev 13:329-359, 1961 3. Forker AD, Bolinger RE, Morris JH, et al: Metabolism of cortisol-‘4C by human peripheral leukocytes cultures from leukemic patients. Metabolism 12:751-759, 1963 4. Jenkins JS, Kemp NH: Metabolism of cortisol by human leukemic cells. J Clin Endocrinol29: 1217-1221, 1969 5. Bush IE: Methods of paper chromatography of steroids applicable to study of steroids in mammalian blood and tissues. Biochem J 50: 370-378, 1952 6. Miabo S, Kishida S, Hisada T: Metabolism and conjugation of cortisol by various dog tissues in vitro. J Steroid Biochem 4: 567-576, 1973 7. Mahesh VB, Ulrich F: Metabolism of cortisol and cortisone by various tissues and subcellular particles. J Biol Chem 235:356-360, 1960 8. Jenkins JS: The metabolism of cortisol by human extrahepatic tissues. J Endocrinol 34: 51-56, 1966
9. Hudson RW, Schachter H, Killinger DW: The conversion of cortisol to ll&hydroxyandrostenedione by beef adrenal tissues. Endocrinology 95:38-47, 1974 10. Klein A, Siebenmann R, Curtius HCH, et al: Steroid 1I@-hydroxylase activity in the microsomal fraction of human adrenals. J Steroid Biochem 7:283-287, 1976
II. Sweat ML, Grosser BI, Berliner DL, et al: The metabolism of cortisol and progesterone by cultures of uterine fibroblasts, strain U12-705. Biochim Biophys Acta 28:591-596, 1958 12. El Attar TMA, Murray WR, Anderson CE: In vivo catabolism of cortisol in synovial fluid of the rheumatoid and osteoarthritic knee. Arthritis Rheum I1:178-183, 1968 13. El studies gingiva 355-364,
Attar TMA: In vitro metabolism of (1,2,6,7-‘H)-cortisol in human in health and disease. Steroids 25: 1975
14. El Attar TMA, Gustafson GM, Wong DG: Comparative metabolism in vivo of (4-14C) cortisol in synovial fluid in the normal, rheumatoid and osteoarthritic knee. Biochim Biophys Acta 137:375-387, 1967