Features of changes in the content of hormones of the hypothalamic-pituitary tract depending on the independent or combined variant of B16/F10 melanoma growth in mice of both sexes

Oleg I. Kit, Elena M. Frantsiyants, Valeriya A. Bandovkina*, Irina V. Kaplieva, Alla I. Shikhlyarova, Ekaterina I. Surikova, Irina V. Neskubina, Natalia D. Cheryarina, Yulia A. Pogorelova, Lidia K. Trepitaki, Inga M. Kotieva, Larisa N. Vaschenko, Yulia Y. Kozel, Anna S. Egorova. Features of changes in the content of hormones of the hypothalamic-pituitary tract depending on the independent or combined variant of B16/F10 melanoma growth in mice of both sexes. Cardiometry; Issue No. 27; May 2023; p. 33-39; DOI: 10.18137/cardiometry.2023.27.3339; Available from:

Melanoma is the most aggressive type of skin cancer. Reference literature data suggest that the incidence rate thereof tends to be higher in women than in men, aged under 50, and thereafter higher incidence rates are found to be dominant in men. In addition, some specific differences in the incidence of melanoma in certain anatomical regions have been identified (for example, a higher incidence of melanoma of the lower extremities at an early age in women and a higher incidence of melanoma of the head and neck in men at their later age) [1, 2]. The offered explanation for the observed differences remains intangible, and it is not clear to what extent they are due to innate biological differences (e.g., those of cellular and hormonal type) and to what extent to some gender-specific behavior.

In the search for factors responsible for discrepancies in the incidence rates of skin melanoma and its localization, the decisive role of sex hormones was

Issue 27. May 2023 | Cardiometry | 33

revealed, although the participation of some other mechanisms cannot be excluded [3]. In particular, it is known that innate and adaptive immune responses in women develop more strongly than those in men that, on the one hand, is an advantage against infectious diseases and cancer, and, on the other hand, makes women more susceptible to autoimmune diseases [4–6].

The hypothalamus is one of the oldest brain structure, which controls complex physiological responses to maintain internal homeostasis, containing several types of neurons, which secrete different hormones: thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone (CRH), and somatostatin (STH-R) [7]. The hypothalamus functions together with the pituitary gland through the hypothalamic-pituitary axis, and releasing hormones are released from the hypothalamus into the bloodstream and enter the anterior pituitary gland [8]. Thus, TRH stimulates the release of thyroid-stimulating hormone (TSH) and prolactin from the anterior pituitary gland; GnRH controls the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH); CRH stimulates the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland; somatostatin STH-R inhibits the release of both growth hormone and TSH, as well as various intestinal and gastric hormones [8].

Tropic hormones of the pituitary gland, the release of which is controlled by inhibitory or stimulating signals by the hypothalamus, are involved in the regulation of the functions of other endocrine glands [9]. TSH stimulates thyroid follicular cells to produce thyroxine [10]; ACTH acts on the adrenal cortex by activating the adrenal axis, and LH and FSH play a key role in the regulation of the reproductive axis [11].

The hypothalamic-pituitary axis is known to be affected by hypoxia, hypercoagulability, endothelial dysfunction and autoimmune changes caused by various infectious and non-infectious diseases [12]. In general, the hormonal regulation of the hypothalamic-pituitary function is extremely complex and integrates several regulatory levels, ranging from the subcellular to extracellular processes [13].

The regulatory axes exhibit a surprising variety of dynamic signaling processes that communicate information between the brain, the endocrine glands and the peripheral target tissues. These dynamic processes include fluctuations, responses to perturbations, and 34 | Cardiometry | Issue 27. May 2023

long-term plastic changes observed from the cellular to systemic levels. The neuroendocrine axes are involved in complex mechanisms mediated by the release of some key regulatory molecules, hypothalamic releasing hormones and pituitary tropic hormones to maintain body homeostasis, modulating various physiological and pathological processes in humans and animals, including the antitumor resistance of the body [14]. Comorbid diseases affect both the central and peripheral links of the regulatory axes, and malignant tumors themselves, when simultaneously transplanted, can increase the biological aggressiveness of each other, or one of them can suppress the growth of the other [15, 16]. Therefore, it is of interest to study the involvement of each component of the central regulatory tract of the hypothalamus-pituitary gland in the development of B16/F10 melanoma in its independent and combined variants with Lewis carcinoma, taking into account the gender of the animals.

The aim of the present research work was to study changes in the level of hormones of the hypothalamus and the pituitary gland in mice of both sexes in different variants of B16/F10 melanoma growth.

Materials and methods

The studies were carried out in 84 BALB/c Nude mice of both sexes, aged 8–9 weeks, weighing 21–22 g, supplied by the Andreevka Research Center for Biomedical Technologies, FMBA (Moscow Region). The animals were kept under the same conditions in standard plastic boxes, 5 animals in each box, under the natural light conditions, at an ambient air temperature 22–26°C, with free access to water and food. Mice received daily standard food and drinking water. The animals were constantly monitored with regular examination and body weighing.

All studies were conducted in accordance with the requirements and conditions specified by 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 animals was carried out in accordance with the rules of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Directive 86/609/EEC), Reference: Record No. 7/111 dated 31.03.2021 prepared by our Ethics Committee.

We used murine B16/F10 melanoma metastasizing to the lungs and Lewis carcinoma delivered by the Russian Cancer Research Center named after N.N. Blokhin at RAMS (Moscow). Tumor strains were inoculated into females and males with 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 BALB/c Nude line (n=14), mice of both sexes, with standard subcutaneous inoculation of B16/F10 melanoma (n=14); mice with reproduction of the model of multiple primary malignant tumor (MPMT) growth (n=14). To reproduce MPMT, B16/F10 melanoma and Lewis carcinoma were inoculated into mice sequentially subcutaneously on two sides: one on the left side and the other on the right side.

At the terminal stage of the tumor growth, with ELISA using standard kits in 1% hypothalamic tissue homogenates we determined releasing hormones as follows: corticotropic hormone (CRH), thyrotropic hormone (TRH), gonadotropic hormone (GnRH), somatotropic hormone (STH-R); in the pituitary gland with RIA method we identified the levels of TSH, LH , FSH and ACTH (Immunotech, Czech Republic).

Statistical processing of the obtained results was completed using the parametric Student’s test with a personal computer employing 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 were found in correspondence to the law, the others were considered not to be in correspondence thereto. For those indicators that corresponded to a normal distribution, we used parametric statistics, and for the outliers we utilized non-parametric statistics. Differences between the two samples were considered statistically significant at p<0.05.

Results and discussion

Some features of the growth of B16/F10 melanoma with the independent and combined transplantation of the tumors are presented in Table 1 given herein.

We selected the same time spans for the growth of melanoma: 15 days in its independent and combined variants. As can be seen from the results of the Table given herein, in the mice with combined inoculation (MPMT model), on day 15, the tumors had different volumes, which differed from the volume of the tumors with the independent single inoculation: in the females, the volume of B16/F10 melanoma with the combined inoculation was 2.8 times greater than that with the independent single inoculation, while in the males, on the contrary, it was 7.2 times smaller. At the same time, on day 15 of the independent tumor growth, the volume of melanoma in the males was 2.7 times greater than that measured in the females. The life span of animals of both sexes with the independent growth variant was longer than in the mice with MPMT.

Next, it was necessary to identify some sex differences in the hormonal function of the hypothalamic-pituitary tract in the independent variant of B16/ F10 melanoma growth and that combined with Lewis carcinoma.

We have revealed the sex differences in the content of the releasing hormones in the hypothalamus in the intact BALB/c Nude mice. Thus, in the females, the TRH concentration was 7.5 times higher than those recorded in the males, while the contents of Gn-RH and STH-R, on the contrary, were 2.8 times and 7 times lower. Only the level of CRH in the hypothalamus of animals of both sexes did not have significant differences depending on the sex.

Table 1

Peculiarities of tumor growth with independent B16 melanoma transplantation and that combined with Lewis carcinoma in mice of both sexes

	Melanoma В16 in its independent variant		Melanoma В16 combined with Lewis carcinoma growth
	Females	Males	Females	Males
Tumor volume on day 15, cm3	0,82±0,22	2,24±0,072	2,31±0,541	0,31±0,0951,2
Life span, days	26,00±0,58	29,00±1,672	22,67±0,881	22,30±0,671

Notes: 1significance of differences compared with independent growth of B16/F10 melanoma, 2significance of differences compared with independent growth of B16/F10 melanoma in females.

Table 2

The content of releasing hormones in the hypothalamus tissue in C57BL/6 mice with B16/F10 melanoma in its independent growth variant and with MPMT (ng/g tissue)

Indicators		Intact mice	Melanoma В16/F10	MPMT
TRH	Females	20,2±2,5 р1=0,0000	5,4±0,11 р1=0,0000 р2=0,0000	3,7±0,1 р1=0,0000 р2=0,0000 р3=0,0000
	Males	2,7±0,24	0,6±0,09 р2=0,0000	0,3±0,05 р2=0,0000 р3=0,0095
GnRH	Females	0,2±0,02 р1=0,0000	0,7±0,02 р1=0,0000 р2=0,0000	0,79±0,05 р1=0,0000 р2=0,0000
	Males	0,56±0,03	0,17±0,008 р2=0,0000	0,21±0,03 р2=0,0000
CRH	Females	0,4±0,02	0,76±0,03 р1=0,0000 р2=0,0000	0,9±0,06 р1=0,0000 р2=0,0000
	Males	0,5±0,05	0,35±0,02 р2=0,0185	0,3±0,02 р2=0,0036
STH-R	Females	0,01±0,001 р1=0,0000	0,03±0,002 р1=0,0000 р2=0,0000	0,06±0,003 р1=0,0000 р2=0,0000 р3=0,0000
	Males	0,07±0,004	0,01±0,0008 р2=0,0000	0,03±0,002 р2=0,0000 р3=0,0000

Notes: Statistically significant difference compared with the following: p1 with the indicator in males; р2 with intact mice; р3 with isolated B16/F10 melanoma growth (р<0.05)

It turned out that in the females and the males with MPMT and the independent growth of B16/F10 melanoma, in the hypothalamus, the level of TRH decreased unidirectionally, but with a different intensity: under the independent growth of the tumor we recorded a decrease by 3.7 times in the females and by 4.5 times in the males, respectively, and in case with MPMT we noted a decrease by 5.5 times in the females and by 9 times on the males, correspondingly. As for the rest of the studied hypothalamic releasing hormones, in response to the tumor growth, both in its independent and combined variants, multidirectional changes in their concentrations depending on sex were established.

In the females, compared with the indices in the intact animals, the growth of B16 / F10 resulted in an increase in the level of GnRH by 3.5 times, CRH by 1.9 times and STH-R by 3 times, while with MPMT the concentrations of the above mentioned hormones increased by 3, 9 times, 2.3 times and 6 times, respec- 36 | Cardiometry | Issue 27. May 2023

tively. At the same time, in case of MPMT, in the females, a significantly 2-fold higher content of STH-R was found in the hypothalamus, compared with independently developing melanoma.

In the males, compared with the intact animals, the level of releasing hormones of the hypothalamus, on the contrary, was reduced both with the independent and with combined tumor growth variants: GnRH was reduced by 3.3 times and 2.7 times; CRH by 1.4 times and 1.7 times, STH-R by 7 times and 2.3 times, respectively. Significant differences in case with MPMT, compared with the independent growth of B16 / F10 in the males, were established only in relation to STH-R content: it was 3 times higher with the combined tumor growth.

Our analysis of the concentrations of the pituitary tropic hormones in the intact BALB/c Nude mice showed that in the females, as against the males, the levels of TSH and FSH were 1.6 times and 23 times lower, respectively, while the contents of LH and

ACTH in the pituitary gland had no significant differences therein considering the gender aspect.

In response to the growth of B16/F10 melanoma in its independent growth and combined variants with Lewis carcinoma, we found in the mice of both sexes, in comparison with the intact animals, unidirectional changes in FSH, but however those of varying intensity, the level of which decreased in the females with B16/F10 by 1.5 times and in the males by 7.8 times, respectively, while the concentration of that hormone under MPMT was 1.7 times lower in the females and 13,6 times lower in the males, accordingly. At the same time, only in the males with MPMT, the FSH content was recorded to be 1.8 times lower compared with the independent growth of melanoma, while no differences therein were observed in the females.

Considering the LH data, it turned out that with the independent growth of B16 / F10 melanoma, its concentration increased by 1.6 times in the females and 1.4 times in the males, while with MPMT an increase was noted only in the males by 1.3 times, and in the females its level did not differ from that recorded in the intact animals and was found 1.5 times lower than that reported in case of the independent growth of melanoma (р<0.05).

The concentration of ACTH in the pituitary gland in the males increased in response to the independent growth of B16/F10 by 2.5 times and by 4 times in case of MPMT, while in the females, on the contrary, it decreased by 1.4 times and 1.8 times, respectively. In the males with MPMT, the level of ACTH was 1.6 times higher than that recorded under the independent growth of melanoma, while in the females there were no significant differences depending on the type of the melanoma growth.

The level of TSH in the pituitary gland in the females, as against the intact animals, increased by 2.8 times only win case with MPMT, and, as a result, the content of TSH under MPMT exceeded that recorded under the independent melanoma tumor growth by 2.6 times. In the males, there were no significant changes in the concentration of TSH in the pituitary gland in any of the groups.

Table 3

Tropic hormones in the pituitary tissue in C57BL/6 mice with melanoma B16/F10 in its independent growth variant and with MPMT

Indicators		Intact mice	Melanoma В16/F10	MPMT
TSH mIU/g tissue	Females	0,12±0,008 р1=0,0000	0,13±0,01 р1=0,0075	0,34±0,01 р1=0,0000 р2=0,0000 р3=0,0000
	Males	0,19±0,01	0,17±0,008	0,19±0,01
LH mIU/g tissue	Females	605,3±21,4 р1=0,0285	960±42,4 р2=0,0000	635,4±24,2 Р1=0,0000 р3=0,0000
	Males	671,3±16,6	925,9±24,5 р2=0,0000	873,7±35,5 р2=0,0001
FSH mIU/g tissue	Females	19±0,94 р1=0,0000	12,8±0,6 р1=0,0000 р2=0,0000	11,3±0,77 р1=0,0000 р2=0,0000
	Males	437,5±21,9	56±2,4 р2=0,0000	32,1±2,1 р2=0,0036 р3=0,0000
ACTH pg/g tissue	Females	67,2±3,5 р1=0,0391	48,1±3,3 р1=0,0000 р2=0,0014	36,8±2,8 р1=0,0000 р2=0,0000 р3=0,0199
	Males	57,2±2,6	145,2±3,8 р2=0,0000	230,8±24,0 р2=0,0000 р3=0,0033

Notes: Statistically significant difference compared with the following: p1 with the relevant indicator in males; р2 with the intact mice; р3 with isolated B16/F10 melanoma growth (р<0.05)

Discussion

The growth of B16/F10 melanoma in combination with Lewis carcinoma (MPMT) has demonstrated its own gender characteristics, consisting in multidirectional changes in the average tumor volumes, but however the average life spans both in the males and the females decreased. From this, we have deduced that the simultaneous growth of two tumors, even despite the decrease in the average volume of B16/F10 melanoma in the males, has resulted in a premature death of the animals that is an indication of greater biological aggressiveness of the malignant process. It has been revealed that any variant of the growth of the malignant tumor, both alone and in combination, in the experimental animals has initiated a disorder in the central regulation, consisting in a change in the levels of hypothalamic and pituitary regulatory hormones.

It is known that the hypothalamus together with the pituitary gland are responsible for the central regulation of the main axes of the body, namely, the gonadal, the thyroid and the adrenal axes, and disorders in the hypothalamic-pituitary interactions can manifest themselves in various diseases [10]. The hypothalamus is the central command center for the hormonal regulation, and its effects are mainly produced by hormones released by the hypothalamus and delivered to the target organ, the pituitary gland [17].

As it follows from our results, low levels of TRH in the hypothalamus of the animals both with the independent growth of B16/F10 melanoma and with MPMT did not cause adequate release of TSH by the pituitary gland that indicated a disorder in the hypothalamic-pituitary relationship. It turned out to be interesting that only the interaction of the hypothalamus-pituitary gland at the level of TRH and TSH turned out to be unidirectional for the animals of both sexes in the studied malignant processes, and the other releasing peptides and tropic hormones demonstrated changes in their concentrations in the females and the males in different directions in response to the growth of the malignant tumors.

According to the evidence data obtained by us, the interaction between GnRH of the hypothalamus and LH of the pituitary gland was supported only in the females, and that between GnRH and FSH was kept only in the males. That was reflected in the fact that an increase in the level of GnRH in the females was accompanied by a regular increase in the level of LH in the pituitary against the background of a decrease 38 | Cardiometry | Issue 27. May 2023

in FSH, while in the males, on the contrary, a decrease in the level of GnRH in the hypothalamus was accompanied by a decrease in the content of FSH in the pituitary against the background of an increased amount of LH. Moreover, this pattern is preserved both under the independent variant of the melanoma growth and with its growth in case of MPMT. This fact can be considered as a pathognomonic feature specific to a certain sex of animals.

It is known that the production of GnRH and pituitary tropic hormones LH and FSH by the hypothalamus is characterized by non-linear pulsatile dynamics, as well as interpulse secretion. The GnRH pulse generator is a key target for the negative feedback regulation of sex steroids in both sexes [18]. As the levels of estradiol (E2) and androgens decrease, the GnRH pulsation increases rapidly, enhancing the synthesis of sex steroids in the gonads due to the pulsatile release of LH from the pituitary gland. However, the operation of the pulse generator at even higher frequencies can lead to desensitization of the signaling cascade and the collapse of pulsatile hormone secretion [19].

We found that there was a sex-specific response by pituitary hormones to changes in the level of the hypothalamus hormones. Thus, an increase in the content of the releasing hormone CRH of the hypothalamus, both with the independent variant of the melanoma growth and as a part of MPMT, was accompanied in the females by a reduced level of ACTH in the pituitary gland. In contrast thereto, a decrease in the content of the releasing hormone CRH of the hypothalamus, both in the case of the independent variant of the melanoma growth and as a part of MPMT, was accompanied in the males by an elevated level of ACTH in the pituitary gland. As it is the case with the GnRH-LH-FSH axis, this fact can be considered as a pathognomonic sign inherent in a certain sex of animals.

It should be noted that the aggravation of the dysfunction of the hypothalamic-pituitary tract in a malignant process, especially in case of MPMT, can also have a detrimental effect on the body due to the development of concomitant metabolic disorders. It is known that damage to the hypothalamic-pituitary axis is associated with metabolic diseases, including abdominal obesity, elevated cholesterol/triglyceride levels, and insulin resistance that additionally lead to a higher risk of a cardiovascular disease and poor longterm prognosis [7].

Conclusion

Obviously, in the development of B16/F10 melanoma, both in the independent growth variant and in case of MPMT, there was reported a sex-dependent dysfunction of the hypothalamic-pituitary tract. Changes in the concentrations of GnRH, CRH, and STH-R in the hypothalamus in different directions recorded in the males and the females indicate different mechanisms of a hormonal imbalance in response to the growth of the malignant tumor. The fact that in the animals of the same sex, changes in the concentrations of hormones of the hypothalamic-pituitary tract in case of MPMT differed from those recorded under the independent growth of B16 / F10 melanoma may indicate both a decrease in antitumor resistance, which depends on the central regulatory links of the body, under the simultaneous growth of several malignant tumors, and the occurrence of concomitant shifts in the body’s homeostasis, which require further identification and studies.