Effect of combined lignan phytoestrogen and melatonin treatment on secretion of steroid hormones by adrenal carcinoma cells
Kellie A. Fecteau, PhD; Hugo Eiler, DVM, PhD; Jack W. Oliver, DVM, PhD
Objective-To investigate the in vitro effect of the combination of lignan enterolactone (ENL) or lignan enterodiol (END) with melatonin on steroid hormone secretion and cellular aromatase content in human adrenal carcinoma cells.
Sample-Human adrenocortical carcinoma cells.
Procedures-Melatonin plus ENL or END was added to cell culture medium along with CAMP (100µM); control cells received cAMP alone. Medium and cell lysates were collected after 24 and 48 hours of cultivation. Samples of medium were analyzed for progesterone, 17-hydroxyprogesterone, androstenedione, aldosterone, estradiol, and cortisol concentra- tion by use of radioimmunoassays. Cell lysates were used for western blot analysis of aromatase content.
Results-The addition of ENL or END with melatonin to cAMP-stimulated cells (treated cells) resulted in significant decreases in estradiol, androstenedione, and cortisol concen- trations at 24 and 48 hours, compared with concentrations in cells stimulated with cAMP alone (cAMP control cells). The addition of these compounds to cAMP-stimulated cells also resulted in higher progesterone and 17-hydroxyprogesterone concentrations than in cAMP control cells; aldosterone concentration was not affected by treatments. Compared with the content in cAMP control cells, aromatase content in treated cells was significantly lower.
Conclusions and Clinical Relevance-The combination of lignan and melatonin affected steroid hormone secretion by acting directly on adrenal tumor cells. Results supported the concept that this combination may yield similar effects on steroid hormone secretion by the adrenal glands in dogs with typical and atypical hyperadrenocorticism. (Am J Vet Res 2011;72:675-680)
T he adrenal gland cortex secretes glucocorticoids, intermediate steroid hormones (sex steroid hor- mones), and mineralocorticoids. It is well known that clinical signs associated with hyperadrenocorticism in dogs result from excessive adrenal secretion of cortisol. What is not well known is that a variant of hyperad- renocorticism exists, referred to as atypical hyperadre- nocorticism, in which cortisol concentration is within reference limits.1 However, in animals with atypical dis- ease, there is excessive secretion of adrenal intermedi- ate steroid hormones,1 some of which have been shown to induce clinical signs that mimic those caused by ex- cess cortisol secretion.1,2 Treatment of atypical hyper- adrenocorticism with mitotane, a drug commonly used to treat dogs with naturally occurring hyperadrenocor- ticism,3 is effective in lessening the secretion of most
| ENL | ABBREVIATIONS |
|---|---|
| Enterolactone | |
| END | Enterodiol |
adrenal intermediate steroid hormones, although it has a variable effect on estradiol secretion because sources of estradiol may include the adrenal glands,4 gonads, and peripheral tissues such as adipose tissues and skin.5
A treatment that may lower concentrations of es- tradiol as well as concentrations of other intermediate steroid hormones is the combination of phytoestro- gen6-9 and melatonin.10-15 It is our experience that the combination of lignan phytoestrogen and melatonin is effective in controlling atypical hyperadrenocorticism in some dogs. Phytoestrogens are plant-derived com- pounds with weak estrogenic activity and are classified as isoflavones, lignans, and coumestans. Lignans are found in whole grains, seeds, nuts, legumes, and veg- etables; however, the most abundant source is flaxseed and, more specifically, flaxhulls.16
The main lignans in serum and urine of humans and other animals are the mammalian lignans ENL and END.17 These compounds are referred to as mamma- lian lignans because they are formed by bacteria in the intestinal tract from the plant lignans matairesinol and
From the Department of Comparative Medicine, College of Veteri- nary Medicine, University of Tennessee, Knoxville, TN 37996.
Supported by the American Kennel Club Canine Health Foundation. The contents of this publication are solely the responsibility of the au- thors and do not necessarily represent the views of the Foundation.
The authors thank DeAnne Gibbs for assistance with radioimmunoas- says and Ann Reed for assistance with statistical analysis.
Address correspondence to Dr. Fecteau (kfecteau@utk.edu).
secoisolariciresinol.17 Several in vitro studies6-8 have revealed that treatment of certain cells with ENL and END decreases estrogen production and aromatase activity (an enzyme that converts androgens to estro- gens). Furthermore, it has been suggested that lignans may affect uptake and metabolism of sex hormones by participating in the regulation of plasma sex hormone- binding globulin and may compete with estradiol for estrogen binding sites.9
Melatonin is a pineal gland hormone, the synthe- sis of which is influenced by photoperiod, with light suppressing its synthesis and darkness increasing its synthesis in mammals.18 Melatonin has various physi- ologic functions,18 including regulation of sexual repro- duction in seasonal breeders.10,11 Melatonin inhibits re- production (antigonadotropic effect) during the winter months in long-day breeding animals12 and reportedly inhibits ovarian activity in cats10 and androgen produc- tion in isolated hamster Leydig cells.11 In addition to decreasing sex hormone secretion, melatonin inhibits ACTH-stimulated cortisol production in primate adre- nal glands13 and modulates aromatase activity in vari- ous cell types.14,15
The purpose of the study reported here was to de- termine whether the combination of lignan and melato- nin had an effect on adrenal steroid hormone secretion. Specifically, we sought to evaluate the effectiveness of the lignan-melatonin combination in blocking adre- nal steroid hormone secretion at the cellular level and whether this combination would affect protein content of the steroidogenic enzyme aromatase in adrenal cells. Because many forms of human and canine disease have pathophysiologic similarities,19 commercially available human adrenal carcinoma cells were chosen as a substi- tute for canine adrenal gland cells to evaluate adreno- cortical function in the treatment conditions.
Materials and Methods
Cell culture-Human NCI-H295R adrenocortical carcinoma cellsª were maintained in Dulbecco modified Eagle medium with nutrients,b 2.5% growth medium supplement,“ and universal culture supplementd and culti- vated at 37℃. The lignan phytoestrogens ENLe and ENDe were dissolved in dimethyl sulfoxide to a concentration of 10mM to create stock solutions. Aliquots of the stock so- lutions were frozen at -20℃. Melatonine and cAMPf solu- tions at a concentration of 1mM were newly prepared for each experiment in sterile water. Cellular steroidogenesis was stimulated with cAMP.20 Cells were seeded at a density of 3 X 106 cells/100-mm cell culture plate and separated into the following groups: untreated control, 100M cAMP (cAMP control), 100uM cAMP plus ENL plus melatonin (ENL treated), and 100uM cAMP plus END plus melato- nin (END treated). Cells were treated for 24 and 48 hours. Cell culture medium was collected, and cells from each group were harvested at the 2 time points and frozen at -80℃ until analyzed. This experiment was repeated 9 times.
Radioimmunoassay-Steroid hormones included in a canine adrenal profile test offered by the Clini- cal Endocrinology Service at the University of Tennes- see were analyzed. Concentrations of progesterone,g
17-hydroxyprogesterone,h androstenedione,h aldosterone,g estradiol,h and cortisol& were determined in the cell cul- ture medium by use of a radioimmunoassay method. As- says were performed in accordance with manufacturers’ instructions.
Performance characteristics for each radioim- munoassay were determined for cell culture medium. Intra-assay and interassay coefficients of variation for the various radioimmunoassays were, respectively, as follows: progesterone, 4.1% and 7.0%; 17-hydroxypro- gesterone, 6.7% and 17.2%; androstenedione, 5.5% and 7.9%; aldosterone, 5.5% and 6.6%; estradiol, 10.4% and 10.6%; and cortisol, 10.4% and 6.4%. Mean percent- age recoveries of known amounts of hormones added to the medium were as follows: progesterone, 104.0%; 17-hydroxyprogesterone, 115.0%; androstenedione, 101.0%; aldosterone, 104.4%; estradiol, 97.0%; and cortisol, 102.0%. Serial dilution of cell culture medium yielded the following percentages of expected values: progesterone, 81.0%, 70.0%, and 71.0%; 17-hydroxy- progesterone, 111.0%, 99.0%, and 117.0%; andro- stenedione, 103.0%, 107.0%, and 125.0%; aldoste- rone, 108.0%, 113.0%, and 128.0%; estradiol, 106.0%, 113.0%, and 104.0%; and cortisol, 92.0%, 86.0%, and 83.0%.
Western immunoblot analysis-Cell pellets were incubated in lysis buffer’ supplemented with protease inhibitors on ice for 10 minutes. Supernatants were iso- lated from cell lysates after centrifugation of crude ly- sates at 15,000 X g for 20 minutes. After protein quanti- fication, 20 µg of proteins from each lysate was resolved via electrophoresis in 10% SDS-polyacrylamide gels and transferred to a nitrocellulose filter. Nonspecific protein sites on the filter were blocked by incubation of the fil- ter with 3% nonfat milk in NaCl-Tris-Tween 20 buffer (10mM Tris-HCl, pH 7.2; 150mM NaCl; and 0.05% Tween 20) at ambient temperature (approx 23℃) for 30 minutes, then the filter was incubated with an an- tibody specific for aromatasej (1:250) for 16 hours
Progesterone (ng/ml)
140
C
120
C
100
C
c
80
60
40
20
a
b
b
a
0
24
48
Time (h)
at 4℃. The filter was washed and incubated with a horseradish peroxidase-conjugated antibodyk at ambi- ent temperature for 45 minutes. The antigen-antibody complex on the filter was detected via chemilumines- cence and visualized by means of autoradiography. Sig- nals from the immunoblot were assessed by means of densitometry.
Statistical analysis-Differences in hormone concentrations based on treatment (untreated, cAMP control, ENL-treated, and END-treated cells) and time point (24 and 48 hours of cultivation) were evaluat- ed by use of ANOVA with a randomized block design and least significant difference post hoc test. Statisti- cal analysis was performed by use of commercially available software.1 A value of P < 0.05 was considered significant.
Results
Secretion of 4 of 6 steroid hormones was stimulated by the addition of cAMP to culture medium containing
17-hydroxyprogesterone (ng/ml)
80
b
70
60
b
b
b
50
40
30
20
a
a
a
10
a
0
24
48
Time (h)
27
a
24-
a
Androstenedione (ng/ml)
21
a
18
a
15
12
b
b
9
6
b
b
3
0
24
48
Time (h)
human adrenal carcinoma cells. Progesterone concen- trations increased with addition of cAMP to the culture
2,250
b
b
b
2,000
Aldosterone (pg/mL)
1,750
1,500
1,250
b
1,000
b
b
750
500
250
a
a
T
0
24
48
Time (h)
1,750
b
Estradiol (pg/mL)
1,500-
1,250
1,000
b
750
a
a
500-
a
a
a
a
250
0
24
48
Time (h)
300-
b
Cortisol (ng/ml)
250-
200-
150-
b
a
a
100-
a
50-
a
a
a
0
24
Time (h)
48
Aromatase 24 hours
Aromatase 48 hours
Untreated
CAMP
CAMP+ENL CAMP+END
+melatonin +melatonin
medium; however, there were no observable increases in 17-hydroxyprogesterone and androstenedione con- centrations (Figures 1-3). In addition to progesterone, aldosterone, estradiol, and cortisol concentrations in- creased with cAMP stimulation (Figures 4-6).
The combination of ENL or END with melatonin in the culture medium of cAMP-stimulated cells re- sulted in significant increase in progesterone concen- trations 24 and 48 hours after cultivation, compared with results for the cAMP-stimulated control cells. The cells responded similarly with increased 17-hy- droxyprogesterone concentrations at 24 and 48 hours in cAMP-stimulated treatment groups, compared with concentrations in the cAMP-stimulated control cells. The combinations of ENL and END with melatonin in culture medium resulted in a significant decrease in androstenedione, estradiol, and cortisol concentrations in cAMP-stimulated cells; however, neither treatment combination had a significant effect on aldosterone concentrations, compared with the cAMP-stimulated control cells.
Addition of cAMP to the culture medium increased aromatase content by approximately 400%, compared with the content in untreated control cells (Figure 7). When lignan, melatonin, and cAMP were combined in the culture medium, there was a mean decrease in aromatase content to approximately 60% of the cAMP- stimulated value at 24 hours and to approximately 40% of the cAMP-stimulated value at 48 hours.
Discussion
Traditionally, cortisol is the only steroid hormone that has been measured in dogs suspected of having hyperadrenocorticism. However, dogs with hyperad- renocorticism, whether pituitary- or adrenal-depen- dent, may also have high amounts of adrenal sex hor- mones.21 In dogs22,23 and cats2,24 with adrenal-dependent hyperadrenocorticism, blood concentrations of sex steroid hormones secreted by the adrenal glands are report- edly higher than reference limits; however, cortisol con- centrations can be within or lower than reference limits.
In the present study, because of a lack of commercial availability of canine adre- nal tumor cells, human H295R adrenocor- -55 kDa tical carcinoma cells were used to investi- gate the effect of lignans and melatonin on adrenocortical function. The H295R cells are capable of producing mineralocorti- coids, corticosteroids, androgens, and es- trogens and possess the enzymes involved -55 kDa in steroid hormone formation.25,26 Basal cortisol concentration in the present study comprised 48.0% of steroid hormone pro- duction by H295R cells, followed by an- drostenedione at 26.6%, 17-hydroxypro- gesterone at 14.7%, progesterone at 9.6%, estradiol at 0.4%, and aldosterone at 0.3%. The relative proportion of basal steroid hormone concentrations in the present study with H295R cells is similar to that found in initial blood samples from dogs with adrenal-dependent hyperadrenocorti- cism,21 which suggests similarities in the steroid bio- synthetic pathways of dogs and human H295R adreno- cortical carcinoma cells. Furthermore, as in dogs and cats with adrenal tumors, humans with adrenal tumors can have high sex steroid hormone concentrations,27,28 with cortisol concentrations within reference limits.27 Given the similarities in adrenal steroid biosynthetic pathways and in adrenal tumor steroid hormone secre- tions, human H295R adrenocortical carcinoma cells appear to be an acceptable in vitro model for canine adrenal-dependent hyperadrenocorticism for the pur- pose of studying the effects of various treatments on adrenal steroidogenesis.
To the authors’ knowledge, our study is the first to address the effects of a combination of lignan phytoes- trogen and melatonin on cell secretion of multiple ad- renal steroid hormones. Stimulation of cells with cAMP allowed evaluation of this combination on exaggerated hormone concentrations representative of the disease condition. Interestingly, 17-hydroxyprogesterone and androstenedione secretion did not increase with the ad- dition of cAMP to the cell culture medium. The reason for this lack of stimulation is not known; however, it may have been attributable to increased conversion of 17-hydroxyprogesterone and androstenedione to corti- sol and estradiol, respectively. Our results indicated the lignan-melatonin combination was effective in decreas- ing concentrations of androstenedione, estradiol, and cortisol in cAMP-stimulated adrenal tumor cells. Cell treatment with the combination decreased cAMP-stim- ulated estradiol and cortisol secretion to basal (control) values and androstenedione to less than basal values. In addition, in vitro treatment with the lignan-melatonin combination reduced the amount of aromatase protein, a pivotal enzyme in the conversion of androstenedione or testosterone to estradiol, in concert with a decrease in estradiol concentration.
Estradiol is one of the hormones commonly de- tected in high blood concentrations in dogs with atypical hyperadrenocorticism, and because it can be synthesized in various tissues,4,5 treatment of affected dogs can be challenging. The suppression of aroma-
tase activity by lignan or melatonin is not specific to the adrenal gland because an inhibitory effect has also been detected in other cells and tissues such as MCF-7 breast cancer cells,8 mammary tumors,29,30 granulosa- luteal cells,7 and preadipocytes.31 Therefore, it is ex- pected that the lignan-melatonin combination would be effective in decreasing estradiol secretion regardless of the tissue source. Interestingly, treatment with the lignan-melatonin combination increased progesterone and 17-hydroxyprogesterone concentrations in the cAMP-stimulated cells which, as with estradiol and an- drostenedione, are commonly high in dogs with atypi- cal hyperadrenocorticism. This increase in progestin concentration has been documented by other research- ers, who evaluated phytoestrogens in rat adrenocorti- cal cell cultures32 or melatonin in primate adrenal cell cultures.13 The increase is reportedly due to a reduction in expression of cytochrome P450 c21-hydroxylase.32 It is not known whether the combination of lignans ENL and END with melatonin inhibit the 21-hydroxy- lase enzyme in H295R cells; however, inhibition of this enzyme would be expected to increase progestins and decrease cortisol concentrations, as was observed in the present study. Inhibition of cytochrome P450 c21- hydroxylase would also be expected to cause a decrease in aldosterone concentration, which was not observed, and the reason for this is not clear.
Given in vitro results, it does not appear that the lignan-melatonin combination would be an ap- propriate treatment for dogs with atypical hyperad- renocorticism involving high blood progesterone or 17-hydroxyprogesterone concentrations. However, it is our experience that in dogs treated with a combina- tion of lignan and melatonin, the combination does not cause an increase in progestins concentrations in all dogs and even decreases high concentrations in some. The response differences to lignan and melato- nin among dogs is not unexpected given individual dog variability, whereas cell cultures provide a more homo- geneous population that responds similarly over time without the influence of body tissue factors. It is also possible that the lignan-melatonin combination treat- ment affects steroid hormone secretion differently in dogs with pituitary-dependent hyperadrenocorticism versus dogs with adrenal-dependent hyperadrenocorti- cism; however, this possibility has not been evaluated clinically. Furthermore, it is not known whether treat- ment with the lignan-melatonin combination directly affects any steroidogenic enzymes or whether it may af- fect steroidogenesis through modulation of 1 or more signaling pathways, such as the extracellular signal- regulated kinase pathway. Others have reported that ACTH induces activation of Erk1/2 in human H295R adrenal cells33 and that activation of the ERK cascade is involved in steroid hormone production by Y1 mouse adrenocortical cells.34 In addition, several in vitro stud- ies have shown that melatonin inhibits steroidogenesis by reducing the secreted or intracellular concentration of cAMP35-37 and can affect both basal and stimulated cAMP production.11,36
We do not know whether the decrease in hormone concentrations in the present study can be attributed, in part, to melatonin inhibiting the stimulatory effect
of the added cAMP. However, this possibility is unlikely because decreases were not detected in all hormones by the lignan-melatonin combination. The mechanism or mechanisms by which that combination modulates adrenal steroidogenesis remains to be clarified. The fact that treatment of human adrenal carcinoma cells with a lignan-melatonin combination resulted in a signifi- cant lowering of androstenedione, estradiol, and cor- tisol concentrations in cell cultures suggested that this combination may yield similar effects on adrenal ste- roid hormone secretion in dogs with atypical or typical hyperadrenocorticism.
a. American Type Culture Collection, Manassas, Va.
b. DMEM/F-12, American Type Culture Collection, Manassas, Va.
c. NuSerum, BD Biosciences, Bedford, Mass.
d. ITS Premix, BD Biosciences, Bedford, Mass.
e. Sigma-Aldrich Inc, St Louis, Mo.
f. N6,2’-O-Dibutyryladenosine 3’,5’-cyclic monophosphate so- dium salt, Sigma-Aldrich Inc, St Louis, Mo.
g. Coat-a-Count, Siemens Medical Solutions Diagnostics, Los An- geles, Calif.
h. ImmunChem Double Antibody, MP Biomedicals, Solon, Ohio.
i. Cell lysis buffer (10X), Cell Signaling Technology, Beverly, Mass.
j. Mouse Anti-Human Cytochrome P450 Aromatase, Serotec, Ox- ford, England.
k. Rabbit Anti-Mouse IgG:HRP, Serotec, Oxford, England.
l. SAS, version 9.1, SAS Institute Inc, Cary, NC.
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