Role of hypothalamic-pituitarygonadal regulatory axis in growth of melanoma B16/F10 in C57/Bl6 mice

Oleg I. Kit, Valeriya A. Bandovkina*, Elena M. Frantsiyants, Irina V. Kaplieva, Natalia D. Cheryarina, Viktoria V. Pozdnyakova, Lidia K. Trepitaki, Yulia A. Pogorelova, polina S. Kachesova, Marina A. Gusareva, Ekaterina S. Bosenko, Lyudmila P. Kuchkina, Natalia S. Saforiyn, Marina S. Kuznetsova. Role of hypothalamic-pituitary-gonadal regulatory axis in growth of melanoma В16/F10 in С57/BL6 mice. Cardiometry; Issue No. 27; May 2023; p. 47-53; DOI: 10.18137/cardiometry.2023.27.4753; Available from:

Malignant melanoma is an aggressive type of skin cancer that arises from melanocytes, cells of neuroectodermal origin, which allows considering them as sensory and regulatory structures capable of detecting and converting external and internal signals to maintain homeostasis [1].

Recently, many studies have shown that the sex differences previously observed in most cancers are particularly exacerbated in melanoma, where the male sex is consistently associated with a greater risk of disease progression and a higher mortality rate [2]. The reasons for these gender differences are some biological features of the functioning of the largest neuroendocrine organ, namely, the skin, and its interaction with the regulatory axes of the body [3].

The mammalian endocrine system includes the hypothalamic-pituitary-adrenal (HPA), hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroid (HGT) regulatory axes [4]. Each axis regulates different processes via hormones: the HPA axis mainly controls the body’s response to stress via glucocorticoids, the HPG axis coordinates the reproduction with steroid hormones, and the HGT axis regulates energy metabolism and thyroid hormone production. At the same time, the endocrine axes do not function independently: there is a physiological overlap at the level of the central regulatory links such as the hypothalamus and pituitary gland, which are common organs for the biosynthesis of releasing peptides and tropic hormones. In addition, cells can respond to different hormonal signals due to cross-reactions of nuclear receptors to steroid and thyroid hormones [5].

Malignant tumors are not a local process, but they affect the entire body, exerting a mutual influence on the regulatory axes, changing biochemical reactions and metabolism in general. In addition, malignant cells, and in particular skin melanoma, are capable of producing practically biologically active substances, including steroid and thyroid hormones, their receptors, regulatory peptides, and growth factors, which allows the neoplasm to stimulate its own uncontrolled growth [6; 7].

The development of new treatment regimens requires a broader understanding of tumor biology. This can be achieved using in vivo models, which reflect the true behavior of melanoma, which exists in close relationship with the regulatory, neuroendocrine and immune systems of the body. Models should not only reproduce these features, but also reproduce the natural development of the tumor from proliferation to invasion and metastasis [8]. Mouse models are convenient due to their ease of manipulation and availability, as well as the existing knowledge base about their genetics. The most widely used cell type has become the B16 cell line, which spontaneously forms a tumor after chemical induction of melanoma in C57BL/6 mice and gives rise to a diverse spectrum of subclones with different propensities for proliferation, invasion and metastasis [9].

The aim of our research work was to study the role of the hypothalamic-pituitary-gonadal regulatory axis and its dermal analogue in C57BL/6 mice of both sexes with the growth of B16/F10 grafted melanoma.

Materials and methods

The studies were conducted in mice of both sexes of the C57BL/6 line weighing 21–24 g, delivered by the Andreevka Scientific Center for Biomedical Technologies at the Federal Medical and Biological Agency (Moscow Region). The animals were kept under the same conditions in standard plastic cages, 5 animals in each cage, under the natural light conditions, at an ambient air temperature 22–26°C, with free access to water and food. The animals were constantly monitored with regular inspection and weighing.

All studies were carried out in accordance with the requirements and conditions set forth in the International Guiding Principles for Biomedical Research Involving Animals and Order No. 267 “Approval of the rules of laboratory practice” dated June 19, 2003, issued by the Ministry of Health of Russia. Work with the animals was completed in compliance with the rules of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Directive 86/609/EEC).

We used murine melanoma B16/F10 metastasizing to the lungs, supplied by the Russian Cancer Research Center named after N.N.Blokhin at the RAMS (Moscow). Tumor strains were inoculated into females and males by subcutaneous injection of 0.5 ml of a suspension of tumor tissue in Hank’s solution or medium 199 (1:10).

The animals were divided into groups as follows: intact males and females of the C57BL/6 line (n=14); mice of both sexes with standard subcutaneous inoculation of B16/F10 melanoma (n=14). The animals were decapitated 14 days after the B16/F10 melanoma inoculation, at the stage of the logarithmic growth of the tumor. 16 animals of both sexes with transplanted B16/F10 melanoma were assigned in the groups to determine the average life spans. The gonadal releasing hormone (GnRH), LH and FSH were determined by ELISA and RIA using standard kits in 1% homogenates of the hypothalamic and pituitary tissues; we determined the contents of estradiol (E2), estrone (E1), total testosterone (T) and free testosterone (Tfr), progesterone (P4), as well as steroid hormone receptors: estrogen receptors ER ɑ and ERβ, progesterone receptor (RP4) and androgen receptor (RA) in 10% homogenates of the ovaries and the testes, the tumors and the intact skin, as well as in the blood serum.

Statistical processing of the obtained results was carried out using the parametric Student’s test with a personal computer using the STATISTICA 10.0 software and the nonparametric Wilcoxon-Mann-Whitney test. All results obtained were checked for their compliance with the law of normal distribution. Some indicators corresponded to the law, some did not. For those indicators which corresponded to a normal distribution, we utilized parametric statistics, for the outliers we applied non-parametric statistics. Differences between the two samples were considered statistically significant at p<0.05.

Results

In all animals, the melanoma growth was found in 100% of the cases after cell suspension inoculation according to the standard method. In the male mice, the tumor was palpable on days 5-7, the average life span was 22.1 ± 0.82 days, the first death of the animal was recorded on day 17, and the maximum life span reached 29 days. In the female mice, the tumor was palpable on day 10-12, and their average life span was 30.25±1.67 days; the first death was recorded on day 24, and the maximum life span reached 36 days. Up to 28 days (4 weeks) among the females we recorded survival of 62.5% of animals and only 18.8% in the male cohort. Tumor volumes were measured every week (see Table 1 herein).

The tumor volume was calculated by the formula

• V = а х b х с ,

where a is the length of the tumor, b is the width of the tumor, c is the height of the tumor.

We assumed that the sex specifics of tumor growth dynamics, namely, smaller volumes of melanoma in the females and longer life spans, that indicates the possibility of the presence of some factors of the neuroendocrine origin, having an inhibitory or stimulat- ing effect on the growth and development of a neoplasm, might be influenced by various links of the hypothalamic pituitary-gonadal regulatory axis.

It turned out that 2 weeks after the melanoma inoculation in the males (see Table 2 herein), the hypothalamus samples showed a 2.1-fold decrease in the GnRH levels compared with the intact animals, but an increase in LH concentration in pituitary samples by 2.7 times without changing the content of FSH. As a result, the ratio of LH/FSH in the pituitary gland tissues of the males 2 weeks after the tumor transplantation was 3.3 times higher compared with the norm.

• 2 weeks after the tumor growth in the females, in the samples of the hypothalamus, the content of GnRH increased by 1.4 times (p<0.05) compared with the intact animals. In the females, after 2 weeks, the connection between the hypothalamus and the pituitary gland remained available in response to an increase in GnRH in the pituitary gland, and the concentration of LH and FSH exceeded the norm by 3.4 times and 2.7 times, respectively, while the LH/FSH ratio did not have significant differences from those in the intact animals.

In the gonads of the male mice 2 weeks after the melanoma transplantation, the level of estradiol and

Table 1

Average volumes of tumors in female and male mice in the dynamics of growth of transplanted melanoma B16/F10

Groups	V (cm3) of tumor after 1 week	V (cm3) of tumor after 2 weeks	V (cm3) of tumors after 3 weeks	V(cm3) of tumors after 4 weeks
Females	------	0,85±0,121	2,75±0,731	4,69±0,861
Males	0,125±0,012	1,27±0,37	5,91±1,48	7,94±2,1

Note. 1 statistically significant differences compared with males (p<0.05)

Table 2

The level of releasing hormones in the hypothalamus and tropic hormones in the pituitary gland in females and males with transplanted B16/F10 melanoma

Groups	Hypothalamus	Pituitary gland
	GnRH ng/g tissue	LH (IU/g tissue)	FSH (IU/g tissue)	LH/FSH
MALES
Intact animals	3,5±0,31	16,7±1,4	24,6±1,98	0,68±0,05
Melanoma	1,7±0,141	44,98±3,91	20,2±1,58	2,23±0,211
FEMALES
Intact animals	2,7±0,21	19,2±1,5	23,5±1,8	0,82±0,07
Melanoma	3,8±0,281	65,1±5,41	62,75±5,21	1,04±0,09

Note: 1statistically significant differences compared with intact mice (p<0.05)

free form of testosterone decreased by an average of 1.6 times (p<0.05), without changing the content of total testosterone and progesterone (see Table 3 herein). In the females, after 2 weeks of the experiment, in the ovarian samples, the level of progesterone increased by 2.8 times, and the content of the free form of testosterone decreased by 2.8 times, without changes in the level of E2 and total T.

Our analysis of the content of tropic hormones of the pituitary gland in the blood serum of the male and female mice in the dynamics of melanoma growth showed a decrease in the concentration of FSH 2 weeks after the transplantation by 1.6 2.5 times, and LH by 1.5 – 2.3 times, respectively (see Table 4 herein). It turned out that the change in the content of LH in the pituitary gland and the blood serum in response to the growth of B16/F10 melanoma in the mice of both sexes was multidirectional: it decreased in blood, despite the increase in the pituitary gland. A discrepancy between the direction of changes in the level of FSH in blood and the pituitary gland was also revealed.

Despite the decrease in the level of E2 and the absence of changes in the level of total T in the testes, in the blood serum of the males with B16 / F10 melanoma against the background of the tumor growth, the level of estradiol increased by 3.9 times, that of total T decreased by 6.2 times and its free form Tfr by 1.6 times (p<0.05), without significant changes in the concentration of progesterone.

In the females with melanoma, the content of E2 and T in the blood serum did not have significant differences from those recorded in the intact animals, but a decrease in the amount of free T by 3.5 times and an increase in P4 by 1.9 times were found. It should be noted that only an increase in the level of P4 in the blood serum was accompanied by an increase in its content in the ovarian samples.

Next, we were interested in the change in steroidogenesis in the organ directly affected by melanoma, that is, in the skin and then in the tumor developing there (see Table 5 herein).

After 2 weeks of the tumor growth in the male mice in samples of their unaffected skin, the concentrations

Table 3

Level of sex steroids in gonads with B16/F10 melanoma in male and female mice

	Е2 pg/g tissue	Т ng/g tissue	Тfr. pg/g tissue	Р4 ng/g tissue
MALES, testes
Intact mice	0,61±0,03	97,3±3,7	308,45±0,01	1,3±0,10
Melanoma	0,38±0,031	89,7±7,2	187,84±341	1,32±0,10
FEMALES, ovaries
Intact mice	0,94±0,06	26,3±2,1	43,3±3,8	51,6±4,9
Melanoma	0,82±0,06	25,6±2,2	15,3±1,41	145,9±11,81

Note: 1statistically significant differences compared with intact mice (p<0.05)

Table 4

В16/F10 Serum hormone levels in female mice with transplanted B16/F10 melanoma

	Estradiol pg/mL	Т total ng/mL	Тfr pg/mL	P4 ng/mL	LH mIU/mL	FSH mIU/mL
Males
Intact mice	777,6±56,9	0,28±0,019	63,15±5,1	0,85±0,07	0,2±0,018	0,25±0,021
Melanoma	3026,2±236	0,045±0,0031	39±2,91	0,81±0,07	0,13±0,0121	0,10±0,0091
Females
Intact mice	1979,1±181	0,01±0,001	0,45±0,03	0,49±0,03	0,3± 0,029	0,16± 0,014
Melanoma	2420,67±202	0,01±0,001	0,13±0,0111	0,95±0,061	0,13±0,0111	0,1±0,011

Note: 1statistically significant differences compared with intact mice (p<0.05) 50 | Cardiometry | Issue 27. May 2023

of E2, E1 and T were reduced by 1.6 times, 1.8 times and 2 times, respectively, compared with those in the intact animals, but the contents of GnRH, LH and FSH were increased by 4.5 times, 1.3 times (p<0.05) and 2.8 times, respectively, without significant differences in the P4 level. In the tumor samples taken from the males after 2 weeks of the melanoma growth, the level of E2 and T was 1.6 and 1.7 times lower than that recorded in the unaffected skin in the intact animals, and the concentrations of E1, GnRH and LH were 2.1 times, 5.6 times and 2.5 times higher, respectively.

After 2 weeks of the experiment in the females, in samples of their unaffected skin, the level of GnRH was 1.6 times and LH 1.9 times lower compared with the intact animals, and the content of E1 was recorded to be 1.3 times higher (p<0.05). In samples of the un- affected skin of the females, no significant differences were found in the concentrations of E2, T, P4, and FSH compared with those revealed in the intact animals. At the same time, in tumor samples, the level of E1, P4, GnRH, LH and FSH exceeded the values in the skin of the intact animals by 2 times, 1.4 times, 1.7 times (p<0.05), and by 3 times and 3,3 times, respectively.

The growth of B16/F10 melanoma affected the content of steroid hormone receptors in the skin samples, and especially that in the tumor samples (see Table 6 herein).

In the males, in their unaffected skin, only the level of the progesterone receptor increased by 1.25 times (p<0.05), while in the tumor samples, the content of all studied receptors significantly increased compared with the skin of the intact animals and the unaffected

Table 5

The level of hormones in the tumor and surrounding tissues in mice with transplanted B16/F10 melanoma

Groups	Е2 nM/g tissue	T ng/g tissue	Р4 ng/g tissue	GnRH ng/g tissue	LH IU/g tissue	FSH IU/g tissue	Е1 pM/g tissue
MALES with В16/F10
Intact skin	0,47±0,02	53,7±1,4	1,02±0,07	0,30±0,03	7,7±0,65	5,5±0,51	202,8±15,4
Skin	0,3±0,011	27,3±1,31	0,94±0,06	1,36±0,111	9,9±0,951	15,4±1,01	114,4±9,81
Tumor	0,29±0,011	31,9±1,71	0,95±0,06	1,69±0,151	19,5±1,81	6±0,54	417,98±20,51
FEMALES with В16/F10
Intact skin	0,57±0,04	12,8±1,0	1,01±0,07	0,26±0,02	13,4±0,9	4,7±0,41	262,4± 14,4
Skin	0,68±0,05	14,4±1,3	0,96±0,063	0,16±0,0141,2,3	7,2±0,61,2	3,8±0,332	353± 251,3
Tumor	0,63±0,03	13,1±1,1	1,4±0,061	0,44±0,031	40,8±3,51	15,3±1,51	519,3± 22,51

Notes:1statistically significant differences compared with intact mice; 2statistically significant differences compared with those in the tumor (p<0.05)

Table 6

The content of steroid hormone receptors during the growth of B16/F10 melanoma

Groups	ERɑ (ng/g tissue)	ERβ (ng/g tissue)	RP4 (pg/g tissue)	RA (ng/g tissue)
Intact skin Males	2,075±0,20	2,23±0,21	0,32±0,03	0,28±0,02
Skin Males with В16/F10	1,7±0,152	1,85±0,172	0,4±0,031,2	0,29±0,028
Tumor Males with В16/F10	14,46±1,21	12,8±1,01	0,55±0,051	0,35±0,031
Intact skin Females	2,2±0,2	2,02±0,19	0,21±0,02	0,22±0,02
Skin Females with В16/F10	0,86±0,081,2	1,8±0,152	0,12±0,0111,2	0,23±0,022
Tumor Females with В16/F10	8,6±0,631	4,7±0,381	0,44±0,031	0,3±0,0251

Notes: 1statistically significant in comparison with indicators in intact animals; 2statistically significant differences compared with the values in the tumor (p<0.05)

skin of the animals with the tumor: ER ɑ experienced an increase by 6.9-8.5 times; ERβ by 5.7 – 6.9 times; RP4 by 1.7-1.4 times; RA by an average of 1.3 times (p<0.05), respectively.

In the females with melanoma, in their samples of the unaffected skin, compared with those in the skin of the intact animals, the levels of ER ɑ and RP4 decreased by 2.6 times and 1.8 times, respectively, without significant differences in other indicators. In the tumor samples taken from the females with melanoma, the content of all receptors was recorded to be increased in comparison with the indices in the intact animals and with the samples of the unaffected skin from the females with the tumor as follows: ER ɑ by 3.9-10 times; ERβ by 2.3-2.6 times; RP4 by 2.1-3.7 times; RA by averaged 1.4 times (p<0.05), respectively.

Discussion

It is known that in humans, the severity of the disease and the mortality from melanoma in men is greater than in women [10], that is, melanoma is a hormone-sensitive tumor, so the study of the role of the hypothalamic-pituitary-gonadal axis and the skin as its local analogue is the relevant topic. It turned out that the male mice were characterized by a more severe course of melanoma, characterized by a shorter latent period before the appearance of the primary tumor nodule, by larger tumor volumes and shorter life spans, compared with the females.

There is also evidence that the use of certain types of hormonal fertility drugs (clomiphene, gonadotropins, human chorionic gonadotropin, GnRH) may be associated with an increased risk of malignant melanoma [11]. Our studies have shown that changes in the content of GnRH in the hypothalamus, as well as LH and FSH in the pituitary gland, in response to the growth of transplanted melanoma, have demonstrated their gender specifics. In the males, the central regulatory connection of the hypothalamic-pituitary tract was disrupted, while in the females it remained preserved. That is, depending on the gender, on day 14 after the tumor inoculation, it was possible to establish gender differences in the degree of damage to the central regulatory structures. It is possible that in the males, the identified disorders could cause a shorter latent period before the appearance of transplanted melanoma. In addition, it turned out that the changes in the production of the regulatory hormones by the pituitary gland and the sex hormones by the gonads were not always reflected in blood counts.

Modulation of the work of the HPG axis was reflected in the local skin analogue and in the growing malignant tumor. So, in the males, both in the skin unaffected by the tumor process and in the melanoma itself, the level of regulatory peptides GnRH, LH and FSH sharply increased, while in the females in their unaffected skin, in response to the growth of the neoplasm, the level of regulatory hormones was either reduced or did not differ from that recorded in the intact animals, and the concentrations of GnRH, LH and FSH increased only in the melanoma samples. That is, in the females, their unaffected skin was not yet such a biologically active tumor field as it was the case with the males.

Estrogens are known to regulate the growth and differentiation of normal and some neoplastic tissues (such as breast, ovarian, and endometrial tumors). In particular, estrogens exert their action through specific nuclear receptors α (ER-α), β (ER-β) and the G protein-coupled estrogen receptor (GPER) on the cell membrane [2]. At the same time, it was shown that in vitro incubation with 17-β-estradiol stopped the growth of the human metastatic melanoma cells with a subsequent decrease in the interleukin-8 mRNA concentration. The authors thereof have concluded that estrogen acts as a suppressor by inhibiting the IL-8 expression, and that estrogen mediates the inhibitory effect on melanoma via ER and IL8, since this effect is observed not in the ER (-) cells, but exclusively in the ER (+) cell population [12]. In our study, in the animals with transplanted melanoma, in the skin and tumor samples, either the level of E2 decreased compared with the values in the skin of the intact animals (in the males), or its concentration did not change in the females. However, we assume that the main active estrogen for melanoma is estrone, the level of which has sharply increased directly in the tumor. In addition, it should be noted that only the male mice with melanoma have shown a decrease in the testosterone levels both in the unaffected skin and the tumor samples.

Despite the fact that the role of hormonal receptors in the pathogenesis of melanocytic lesions remains poorly understood, there are a number of facts confirming their involvement therein. A study by De Giorgi et al, who evaluated the expression of sex steroid receptors in human melanoma tissues and in adjacent healthy tissues, shows that ERβ is the main estrogen receptor in melanoma as well as in other melanocytic lesions, both benign and malignant. Moreover, ER-β mRNA and ER-β protein have been found in some thicker, more invasive tumors [13]. We have revealed an increase in the content of the steroid hormone receptors with varying degrees of severity in tumor samples both in the males and the females, and a decrease in the level of ERα and RP4 in the unaffected skin in the females only. The data obtained indicated the formation of the autonomous autocrine/paracrine regulation of proliferative processes by the melanoma tissue, which ensured the growth and development of the tumor with the gradual capture of ever larger regions.

Thus, our study showed a significant role of the hypothalamic-pituitary-gonadal regulatory axis in the logarithmic growth phase of B16/F10 grafted melanoma, which had sex-related specifics and was characterized by the disorders in the direct and reverse feedback loops. Melanoma contained an increased amount of all the regulatory peptides, hormones and receptors of the HPG axis, which allowed it to provide the autonomous regulation of its own growth.