Units of fibrinolytic system in mice with urokinase gene knockout in presence of growing B16/F10 melanoma Текст научной статьи по специальности «Клиническая медицина»

South Russian Journal of Cancer. 2024. Vol. 5, No. 2. P. 14-24

4.0

https://doi.org/10.37748/2686-9039-2024-5-2-2

https://elibrary.ru/incomr

South Russian

Journal of Cancer

ORIGINAL ARTICLE

Южно-Российский

онкологический журнал

Vol. 5

Units of fibrinolytic system in mice with urokinase gene knockout No. 2, 2024

in presence of growing B16/F10 melanoma

E. M. Frantsiyants1, V. A. Bandovkina1, E. I. Surikova1, I. V. Kaplieva1, Yu. A. Pogorelova1, I. V. Neskubina1, L. K. Trepitaki1, N. D. Cheryarina1, N. D. Ushakova1, O. G. Ishonina1,2, M. A. Gusareva1, I. A. Udalenkova1

1 National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation 2 Rostov State Medical University, Rostov-on-Don, Russian Federation valerryana@yandex.ru

ABSTRACT

Purpose of the study. Was to reveal the effect of urokinase gene knockout in male and female mice with transplanted B16/F10 melanoma on the functions of the fibrinolytic system units.

Materials and methods. Male and female mice were used: main group with genetically modified mice C57BL/6-Plautm1.

1Bug – ThisPlauGFDhu/GFDhu (uPA-/-); control group with С57Bl/6 (uPA+/+) mice. B16/F10 melanoma was transplanted by the standard methods to the animals, and levels of plasminogen (PG), plasmin (PAP), urokinase receptor uPAR, content (AG) and activity (act) of uPA, t- PA and PAI-I were measured with ELISA (Cussabio, China) in 10 % tumor homogenates and peritumoral area after 3 weeks of tumor growth.

Results. The activity and levels of urokinase in intact uPA-/- animals were significantly (by 100–860 times) inhibited, compared to uPA+/+, but uPAR levels were unchanged in females and were 1.9 times lower in males. PAP levels in uPA-/- mice were 2.1–4.2 times higher than in uPA+/+ animals. The growth of B16/F10 melanoma in uPA-/- mice was slower and metastasizing was suppressed, but their survival was not improved. The dynamics of changes in components of the fibrinolytic system in presence of melanoma growth differed in uPA-/- mice, compared to uPA+/+ animals: PAP levels in tumor samples decreased by over 2 times, uPA levels and activity were not increased, PAI was practically unchanged, but activity of t- PA elevated by 3.8–8.2 times, as well as in uPA+/+ mice.

Conclusion. Despite the suppression of the growth and metastasis of the primary tumor nodes in uPA-/- mice, their average survival was not improved, which indicates that the mechanisms of tumor are complex and there are alternative biological pathways supporting melanoma to survive in conditions of the urokinase gene knockout.

Keywords: urokinase gene knockout, mice, melanoma B16/F10, fibrinolytic system For citation: Frantsiyants E. M., Bandovkina V. A., Surikova E. I., Kaplieva I. V., Pogorelova Yu. A., Neskubina I. V., Trepitaki L. K., Cheryarina N. D., Ushakova N. D., Ishonina O. G., Gusareva M. A., Udalenkova I. A. Units of fibrinolytic system in mice with urokinase gene knockout in presence of growing B16/F10 melanoma. South Russian Journal of Cancer. 2024; 5(2):14-24. https://doi.org/10.37748/2686-9039-2024-5-2-2, https://elibrary.ru/incomr

For correspondence: Valeriya A. Bandovkina – Dr. Sci. (Biol.), senior researcher at the laboratory for the study of pathogenesis of malignant tumors, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation Address: 63 14 line str., Rostov-on-Don 344037, Russian Federation E-mail: valerryana@yandex.ru

ORCID: https://orcid.org/0000-0002-2302-8271

SPIN: 8806-2641, AuthorID: 696989

ResearcherID: AAG-8708-2019

Scopus Author ID: 57194276288

Compliance with ethical standards: the work with animals was carried out 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) and the Helsinki Declaration, as well as in compliance with the International Guiding Principles for Biomedical Research Involving Animals, and Order No. 267 of the Ministry of Health of the Russian Federation dated 06/19/2003 "On approval of the rules for laboratory practice". The Bioethics Commission of the National Medical Research Center of Oncology dated 12/24/2019, approved the research protocol (Protocol of the Ethical Committee No. 15/75) on working with Balb/c Nude mice

Funding: this work was not funded

Conflict of interest: the authors declare that there are no obvious and potential conflicts of interest associated with the publication of this article The article was submitted 13.10.2023; approved after reviewing 01.04.2024; accepted for publication 09.05.2024

© Frantsiyants E. M., Bandovkina V. A., Surikova E. I., Kaplieva I. V., Pogorelova Yu. A., Neskubina I. V., Trepitaki L. K., Cheryarina N. D., Ushakova N. D., Ishonina O. G., Gusareva M. A., Udalenkova I. A., 2024

14

Южно-Российский онкологический журнал. 2024. Т. 5, № 2. С. 14-24

https://doi.org/10.37748/2686-9039-2024-5-2-2

https://elibrary.ru/incomr

3.1.6. Онкология, лучевая терапия

ОРИГИНАЛЬНАЯ СТАТЬЯ

Звенья фибринолитической системы у мышей с нокаутом по гену урокиназы

на фоне роста меланомы В16/F10

Е. М. Франциянц1, В. А. Бандовкина1, Е. И. Сурикова1, И. В. Каплиева1, Ю. А. Погорелова1, И. В. Нескубина1, Л. К. Трепитаки1, Н. Д. Черярина1, Н. Д. Ушакова1, О. Г. Ишонина1,2, М. А. Гусарева1, И. А. Удаленкова1

1 ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация

2 ФГБОУ ВО «Ростовский государственный медицинский университет» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация

valerryana@yandex.ru

РЕЗЮМЕ

Цель исследования. Изучение влияния нокаута по гену урокиназы у мышей обоего пола с перевитой меланомой

В16/F10 на функционирование звеньев фибринолитической системы.

Материалы и методы. Были использованы мыши обоего пола: основная группа генмодифицированная линия C57BL/6-Plautm1.1Bug – ThisPlauGFDhu/GFDhu (uPA-/-); группа контроля – линия С57Bl/6 (uPA+/+). Животным по стандартной

методике перевивали меланому В16/F10 и через 3 недели роста в 10 % гомогенатах опухоли и ее перифокальной

зоне ИФА методом определяли уровень: плазминогена (ПГ), плазмина (РАР), рецептора урокиназы uPAR, содержание

(АГ) и активность (акт) uPA, t- PA и PAI-I (Cussabio, Китай).

Результаты. У интактных животных uPA-/- в коже оказалась существенно подавлена, по сравнению с uPA+/+ активность и содержание урокиназы (в 100–860 раз), однако у самок не изменился уровень uPAR, тогда как у самцов

снизился в 1,9 раза. Уровень плазмина у uPA-/- мышей был выше в 2,1–4,2 раза, по сравнению с uPA+/+ животными.

Рост меланомы В16/F10 у uPA-/- мышей был замедлен, тормозилось метастазирование, однако не увеличивалась

продолжительность жизни. Динамика изменений компонентов фибринолитической системы при росте меланомы

у uPA-/- мышей отличалась от uPA+/+: в образцах опухоли снижался уровень РАР более чем в 2 раза, не повышал-ся уровень и активность uPA, практически не реагировала PAI, однако, как и у uPA+/+ возрастала активность t- PA в 3,8–8,2 раза.

Заключение. Несмотря на подавление роста первичного узла опухоли и процессов метастазирования у мышей

uPA-/-, средняя продолжительность жизни не увеличивалась, что свидетельствует о сложных механизмах опухоле-вой болезни и наличии альтернативных биологических путей, позволяющих меланоме прогрессировать в условиях

нокаута гена урокиназы.

Ключевые слова: нокаут по гену урокиназы, мыши, меланома В16/F10, фибринолитическая система

Для цитирования: Франциянц Е. М., Бандовкина В. А., Сурикова Е. И., Каплиева И. В., Погорелова Ю. А., Нескубина И. В., Трепитаки Л. К., Черярина Н. Д., Ушакова Н. Д., Ишонина О. Г., Гусарева М. А., Удаленкова И. А. Звенья фибринолитической системы у мышей с нокаутом

по гену урокиназы на фоне роста меланомы В16/F10. Южно- Российский онкологический журнал. 2024; 5(2):14-24.

https://doi.org/10.37748/2686-9039-2024-5-2-2, https://elibrary.ru/incomr Для корреспонденции: Бандовкина Валерия Ахтямовна – д.б.н., старший научный сотрудник лаборатории изучения патогенеза

злокачественных опухолей ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения

Российской Федерации,

г. Ростов-на-Дону, Российская Федерация

Адрес: 344037, Российская Федерация, г. Ростов-на-Дону, ул. 14 линия, д. 63

E-mail: valerryana@yandex.ru

ORCID: https://orcid.org/0000-0002-2302-8271

SPIN: 8806-2641, AuthorID: 696989

ResearcherID: AAG-8708-2019

Scopus Author ID: 57194276288

Соблюдение этических стандартов: работа с животными проводилась в соответствии с правилами «Европейской конвенции о

защите животных, используемых в экспериментах» (Директива 86/609/ЕЕС) и Хельсинкской декларации, а также в соответствии

с «Международными рекомендациями по проведению медико-биологических исследований с использованием животных» и

приказом Минздрава России от 19.06.2003 г. № 267 «Об утверждении правил лабораторной практики». Комиссией по биоэтике ФГБУ

«Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации от

24.12.2019 г., был одобрен протокол исследования (протокол этического комитета № 15/75) по работе с мышами линии Balb/c Nude Финансирование: финансирование данной работы не проводилось

Конфликт интересов: все авторы заявляют об отсутствии явных и потенциальных конфликтов интересов, связанных с публикацией

настоящей статьи

Статья поступила в редакцию 13.10.2023; одобрена после рецензирования 01.04.2024; принята к публикации 09.05.2024

15

South Russian Journal of Cancer 2024. Vol. 5, No. 2. P. 14-24

Frantsiyants E. M., Bandovkina V. A., Surikova E. I., Kaplieva I. V., Pogorelova Yu. A., Neskubina I. V., Trepitaki L. K., Cheryarina N. D., Ushakova N. D., Ishonina O. G., Gusareva M. A., Udalenkova I. A. Units of fibrinolytic system in mice with urokinase gene knockout in presence of growing B16/F10 melanoma INTRODUCTION

date, transgenic models have become traditional and

are successfully used in carcinogenesis studies [12].

The fibrinolytic system is considered one of the

For us, the mice with uPA gene knockout were of

leading mechanisms of carcinogenesis, due to the

the greatest interest, obtained using a molecular ge-

destruction of cell membranes, proliferation, migra-

netic method during which changes are made to the

tion and invasion of cells [1].

nucleotide sequence of the uPA gene, as a result of

Urokinase-type (uPA) and tissue-type (t- PA) plas-

which urokinase is not bound by the urokinase-type

minogen activators are serine proteases that convert

plasminogen activator receptor (uPAR). These mu-

plasminogen into plasmin after binding to the uPA

tant animals can be used in the study of inflamma-

receptor (uPAR) [2]. uPA is found on the surface of

tion, oncogenesis, and fibrinolysis mechanisms in

tumor cells, and its overexpression at the final stage

tumors and surrounding tissues.

of transformation of malignant cells contributes to

The aim of the study was to study the effect of

the processes of metastasis [3]. The activation of

knockout by the urokinase gene in mice of both

the fibrinolytic system and the formation of plasmin

sexes with B16/F10 transplanted melanoma on the

stimulates metalloproteinases, vascular growth fac-

functioning of the fibrinolytic system links.

tors, this in turn consequently destroys the physical

barrier to the migration of tumor cells and stimulates

MATERIALS AND METHODS

tumor growth [4].

Several researchers believe that understanding

The study used genetically modified female and

the molecular mechanisms of the biological action

male mice of the C57BL/6-Plautm1.1Bug – This-

of the plasmin/plasminogen system and inhibition

PlauGFDhu/GFDhu (uPA-/-) line with an initial weight

of angiogenesis by blocking serine proteases may

of females – 24–26 g, 31–33g for males. The rodents

allow improving therapeutic strategies for regulating

were btained from the nursery of laboratory animals

the growth of malignant tumors and disorders asso-

"Pushchino" Branch of the Institute of Bioorganic

ciated with neovascularization [5, 6].

Chemistry named after Academicians M. M. Shem-

It has been previously shown that changes in the

yakin and Yu. A. Ovchinnikov (Pushchino, Moscow

links of the fibrinolytic system of the skin occur in the

region). Animals with urokinase knockout gene

growth dynamics of B16/F10 transfused melanoma

(uPA-/-) can be used in studies of chronic tissue in-

in C57Bl/6 mice with wild type genes, characterized

flammation, mechanisms of fibrinolysis, oncogenesis

by increased activity of all components of the plas-

and vascular growth in the tumor and surrounding

minogen activation system, subsequently leading to

tissue. Mice of both sexes of the C57BL/6 (uPA+/+)

an increased content of plasmin in it. The comorbid

line with an initial weight of 21–23 g obtained from

disease, i. e. chronic neurogenic pain, has a modify-

the Andreevka Scientific Center for Biomedical

ing effect on the studied indicators [7–9].

Technologies (FMBA) (Moscow Region) were used

Experimental models of tumors make it possible

as controls. The animals were kept under natural

to find out the causes, study the pathogenesis of the

lighting conditions with free access to water and

tumor process, develop methods for its prevention

food. The study was conducted in accordance with

and treatment, while the use of various animal lines,

the "International Recommendations for conducting

including those with genetically determined char-

biomedical research using animals" and the Order of

acteristics, is justified [10]. Models of genetically

the Ministry of Health of the Russian Federation No.

engineered mice have been successfully used for

267 dated 06/19/2003 "On approval of the rules of

decades in modeling the tumor process [11]. There

laboratory practice".

are certain types of transgenic mice used in studies

The study was performed on 64 male and 64 fe-

of the malignant process in which oncogenes can

male mice. The animals were divided into groups of

be constitutively or conditionally expressed. In such

10 individuals each: intact females and males of the

animal models, tumor suppressor genes can be sup-

C57BL/6 line (uPA+/+); intact females and males of

pressed using traditional methods such as retroviral

the C57BL/6 line -Plautm1.1Bug – ThisPlauGFDhu/

infection, microinjection of DNA constructs and the

GFDhu (uPA-/-); control group females and males

so-called "gene-directed" transgenic approach. To

of the C57BL/6 line (uPA+/+) 3 weeks after trans-

16

Южно-Российский онкологический журнал 2024. Т. 5, № 2. С. 14-24

Франциянц Е. М., Бандовкина В. А., Сурикова Е. И., Каплиева И. В., Погорелова Ю. А., Нескубина И. В., Трепитаки Л. К., Черярина Н. Д., Ушакова Н. Д., Ишонина О. Г., Гусарева М. А., Удаленкова И. А. Звенья фибринолитической системы у мышей с нокаутом по гену урокиназы на фоне роста меланомы В16/F10

plantation of melanoma B16/F10; the main group of

the law of normal distribution (Shapiro- Wilk crite-

females and males of the C57BL/6-Plautm1.1Bug –

rion (for small samples)). When the sample cor-

ThisPlauGFDhu/GFDhu (uPA-/-) line 3 weeks after

responded to the normal distribution, parametric

transplantation of melanoma B16/F10. The study

statistics were used (Student's criterion), and when

period – 3 weeks after the transplantation of mela-

there was a discrepancy, nonparametric statistics

noma B16/F10 was chosen because it was the stage

were used (Wilcoxon- Mann- Whitney criteria). The

of mass death for male mice and the beginning of

differences were considered statistically significant

death of females, in addition, after 3 weeks, maxi-

at p < 0.05.

mum differences in average tumor volumes in ani-

mals of uPA-/- and uPA+/+ lines were noted. Groups

STUDY RESULTS

of uPA-/- (12 individuals of each sex) and uPA+/+

(25 individuals of each sex) animals were separately

It was found that the process of carcinogene-

identified for the study of average life expectancy.

sis in genetically modified female mice (uPA-/-)

This work used a cell line of mouse melanoma

compared with the control (uPA+/+) had features

B16/F10 metastasizing to the lungs, obtained from

consisting in a reduction in the preclinical period of

the N. N. Blokhin Russian Research Center of the

melanoma development and a decrease in the aver-

Russian Academy of Medical Sciences (Moscow).

age volume of tumor nodes at all stages of obser-

Melanoma B16/F10 was transferred by subcutane-

vation (from 1 to 4 weeks). Single lung metastases

ous injection of 0.5 ml of tumor tissue suspension

were diagnosed in the females of the experimental

in saline solution (1:10) into the right hind leg of

group, whereas metastatic lung and liver damage

a mouse according to the standard procedure [13].

was observed in the control group. In males, tumors

With standard grafting, the tumor appears in 100 %

were characterized by a fairly active, "spasmod-

of cases, grows quite quickly and on the 12th-16th

ic" growth, and their average volume at 4 weeks

day of growth metastasizes mainly hematogenously

after transplantation did not differ from those in

to the lungs (60–90 %), less often to the liver and

mice with a normal genome. In uPA-/-males, no

spleen [14]. For the experiment, the second passage

visible metastases to internal organs were detected

of melanoma B16/F10 transplantation in C57BL mice

at all stages of the growth of B16/F10 transfused

was used/6.

melanoma, but hemorrhages to the lungs were de-

Tumor growth was assessed by daily measuring

tected. The average life expectancy in uPA-/- and

its diameters in three mutually perpendicular areas,

uPA+/+ mice had no significant differences and

followed by calculating the volume of the tumor as

was 34.67±0.67 versus 30.25 ± 1.67 in females,

the product of its three measurements.

and 23.33 ± 3.18 versus 22.1 ± 0.82 in males, re-

Intact animals, as well as mice of the control and

spectively [15].

main groups, were decapitated 3 weeks after the

Based on the previously obtained results of differ-

transplantation of melanoma, and the following were

ences in the growth of malignant tumors in animals

isolated in the cold: tumor, perifocal zone, skin. The

with a knockout of the urokinase gene and a wild

samples were mechanically homogenized, 10 % ho-

type of gene, it was interesting to find out what differ-

mogenates were obtained from the tissues, prepared

ences in the content and activity of the main links of

on a 0.1M potassium phosphate buffer pH 7.4 con-

the fibrinolytic system of the skin are characteristic

taining 0.1 % Twin-20 and 1 % BSA In tissue homoge-

of animals with a knockout of the urokinase gene

nates, the level of plasminogen (PG), plasmin (PAP),

(Tables 1, 2).

urokinase receptor uPAR, content (AG) and activity

Compared with animals of the control group, only

were determined using enzyme immunoassay meth-

traces of uPA were recorded in the skin of intact uPA-

ods (act) uPA, t- PA and PAI-I (Cussabio, China).

/- mice of both sexes: a decrease in uPA levels and

Statistical processing of the obtained results

activity was noted by 100–860 times (Tables 1, 2).

was carried out using the Statistica 10.0 program.

In intact uPA-/- females, the high content of PPB

For all quantitative data, the group arithmetic mean

attracts attention, exceeding 4.2 times the same indi-

(M) and standard error (m) were calculated. All the

cator in female uPA+/+ mice with a 1.3-fold ( p 0.05)

results obtained were checked for compliance with

reduced PG content.

17

South Russian Journal of Cancer 2024. Vol. 5, No. 2. P. 14-24

Frantsiyants E. M., Bandovkina V. A., Surikova E. I., Kaplieva I. V., Pogorelova Yu. A., Neskubina I. V., Trepitaki L. K., Cheryarina N. D., Ushakova N. D., Ishonina O. G., Gusareva M. A., Udalenkova I. A. Units of fibrinolytic system in mice with urokinase gene knockout in presence of growing B16/F10 melanoma In intact uPA-/-males, concentrations of both

The amount of the uPAR receptor in uPA-/- female

PAP and PG were increased 2.1-fold and 1.8-fold in

mice was at the same level as in uPA+/+ mice, where-

the skin, compared with those in the skin of intact

as in males it was reduced by 1.9 times. Significant

uPA+/+ mice (Table 2).

differences with the norm were also observed for

In conditions of uPA deficiency with a high con-

PAI-1: in females, uPA-/- PAI-I activity and its content

tent of PAP, an increase in the level of the second

were 15.0 and 3.0 times lower than normal, respective-

activator of PG – tPA was expected, However, its

ly, in males by 4.9 times and 9.8 times, respectively.

content and activity were reduced only in uPA-/-

Since the average tumor size in female uPA-/-

males by 4.3 times and 1.7 times, respectively. In

mice was smaller after 3 weeks of the experiment

uPA-/- females, a decrease was revealed, relative

than in uPA+/+ females [15], a comparative analysis

to the data in uPA+/+ mice, only tPA activity by

of the links of the fibrinolytic system in samples of

2.5 times, despite an increase in its content by

melanoma of animals with knockout and mice with

1.7 times.

wild genome type was further performed.

Table 1. The content and activity of fibrinolytic system components in the skin, tumor and perifocal zone of female uPA -/-

mice with melanoma B16/F10 3 weeks after transplantation (M ± m) Indicators

Intact mice skin

(normal)

Skin

Tumor volume cm3

Perifocal zone

uPA -/- female mice ( n = 10)

uPA-act (u/g t)

0.010 ± 0.0013

0.009 ± 0.00092,3

0.025 ± 0.0023

0.01 ± 0.0013

uPA-AG (ng/g t)

0.220 ± 0.023

0.24 ± 0.0182,3

0.14 ± 0.0111,3

0.73 ± 0.061,2,3

uPAR (pg/g t)

58.20 ± 4.3

56.2 ± 4.7

66.2 ± 5.33

59.5 ± 5.33

РАР (ng/g t)

45.0 ± 3.43

18.75 ± 1.61

19.7 ± 1.41,3

24.4 ± 2.21,3

PG (ng/g t)

7.70 ± 0.63

10 ± 0.971,3

8 ± 0.773

20 ± 1.71,2,3

tPA-act (u/g t)

0.240 ± 0.023

0.16 ± 0.0141,2,3

1.95 ± 0.161

0.155 ± 0.0141,2,3

tPA-AG (ng/g t)

0.670 ± 0.053

0.34 ± 0.0281,2,3

0.69 ± 0.053

1.55 ± 0.131,2,3

PAI-I-act (u/g t)

1.60 ± 0.13

1.85 ± 0.152,3

3.05 ± 0.291,3

5.25 ± 4.41,2,3

PAI-I-АG (ng/g t)

3.30 ± 0.33

2.2 ± 0.171.3

2.7 ± 0.243

2.8 ± 0.223

uPA +/+ female mice ( n = 10)

uPA-act (u/g t)

1.6 ± 0.12

2.5 ± 0.21

2.8 ± 0.191

2.6 ± 0.171

uPA-AG (ng/g t)

31.7 ± 2.1

187.5 ± 131,2

335.5 ± 231

186.5 ± 12.51,2

uPAR (pg/g t)

56.06 ± 4.5

65.36 ± 5.62

141.8 ± 11.71

112.0 ± 9.61

РАР (ng/g t)

10.7 ± 0.7

19.9 ± 1.11.2

36.4 ± 1.51

18.9 ± 1.21,3

PG (ng/g t)

10.25 ± 0.9

13.5 ± 1.11

16.7 ± 1.41.

11.03 ± 0.9

tPA-act (u/g t)

0.6 ± 0.04

0.7 ± 0.032

2.2 ± 0.191

0.7 ± 0.063

tPA-AG (ng/g t)

0.4 ± 0.02

2.0 ± 0.151,2

12.3 ± 0.91

2.5 ± 0.131,3

PAI-I-act (u/g t)

24.0 ± 0.16

24.0 ± 1.42

71.1 ± 4.21

81.0 ± 5.81

PAI-I-AG (ng/g t)

9.9 ± 0.4

24.5 ± 1.81,2

79.5 ± 6.31

71.6 ± 5.21

Note: 1 – the differences are statistically significant relative to the norm in animals; 2 – compared with a tumor; 3 – compared with similar samples in uPA+/+ animals ( р < 0.05)

18

Южно-Российский онкологический журнал 2024. Т. 5, № 2. С. 14-24

Франциянц Е. М., Бандовкина В. А., Сурикова Е. И., Каплиева И. В., Погорелова Ю. А., Нескубина И. В., Трепитаки Л. К., Черярина Н. Д., Ушакова Н. Д., Ишонина О. Г., Гусарева М. А., Удаленкова И. А. Звенья фибринолитической системы у мышей с нокаутом по гену урокиназы на фоне роста меланомы В16/F10

In the tumor samples of uPA-/- females, compared

skin, an increase in all studied parameters of the

with the tumor samples of uPA+/+ females, the indi-

fibrinolytic system was noted, whereas in uPA-/- fe-

cators of the determined factors were significantly

males in melanoma samples such stimulation was

lower: the activity and content of uPA by 112 times

not detected, except for an increase in activity, but

and by 2396 times, the level of uPAR by 2.1 times,

not the content tPA.

PP and PG by 1.8 times and 2.1 times, the content of

There were also differences in the studied param-

tPA by 17.8 times, the activity and content of PAI-1

eters in the perifocal zone and the skin unaffected by

by 23.3 times and 29 times, respectively. Only the

tumor growth. Thus, in the perifocal zone of uPA -/-

activity of tPA did not have significant differences

females after 3 weeks of melanoma growth, uPA ac-

in tumor samples depending on the urokinase gene.

tivity and level were lower than in the perifocal zone

That said, 3 weeks after the transplantation of

of uPA+/+ mice by 260 and 255 times, respectively,

melanoma B16/F10 in uPA+/+ females in tumor

and the concentration of uPAR was also 1.9 times

samples, compared with the corresponding intact

lower. The level of tPA, as well as its activity, were

Table 2. Content and activity of fibrinolytic system components in the skin, tumor and perifocal zone in male uPA -/- mice with melanoma B16/F10 3 weeks after transplantation (M ± m) Indicators

Intact mice skin

Skin

(normal)

Tumor volume cm3

Perifocal zone

uPA -/- male mice

uPA-act (u/g t)

0.010 ± 0.0013

0.009 ± 0.00072,3

0.013 ± 0.00111,3

0.015 ± 0.0011,3

uPA-AG (ng/g t)

0.250 ± 0.023

0.25 ± 0.0182,3

0.10 ± 0.0091,3

0.39 ± 0.031,3

uPAR (pg/g t)

56.90 ± 4.33

67.4 ± 5.9

90.8 ± 7.61

67.55 ± 5.52

РАР (ng/g t)

30.0 ± 2.53

18.13 ± 1.41,3

14.4 ± 0.91,3

18.13 ± 1.71

PG (ng/g t)

12.50 ± 0.93

9.1 ± 0.771,3

10 ± 0.073

12.2 ± 0.8

tPA-act (u/g t)

0.320 ± 0.023

0.17 ± 0.0151,2,3

1.22 ± 0.781.3

0.17 ± 0.0141,2,3

tPA-AG (ng/g t)

0.70 ± 0.053

0.66 ± 0.062,3

0.46 ± 0.041.3

0.88 ± 0.0712,3

PAI-I-act (u/g t)

2.60 ± 0.23

1.77 ± 0.131,2,3

2.87 ± 0.213

2.37 ± 0.183

PAI-I-AG (ng/g t)

4.10 ± 0.33

2.43 ± 0.211,2,3

3.9 ± 0.333

4.8 ± 0.433

uPA +/+ male mice

uPA-act (u/g t)

1.561±0.10

1.65 ± 0.143

2.7 ± 0.21

1.9 ± 0.17

uPA-AG (ng/g t)

215.3 ± 16.8

181.6 ± 17.13

300.4 ± 24

210.3 ± 19

uPAR (pg/g t)

110.3 ± 6.5

65.13 ± 5.71

73.48 ± 5.31

85.96 ± 7.21

РАР (ng/g t)

14.52 ± 0.9

30.7 ± 2.9

48.8 ± 4.1

19.8 ± 1.7

PG (ng/g t)

6.851 ± 0.5

15 ± 1.2

21.6 ± 1.9

13 ± 1.1

tPA-act (u/g t)

0.551 ± 0.04

0.86 ± 0.07

2.4 ± 0.19

0.8 ± 0.06

tPA-AG (ng/g t)

2.981 ± 0.2

4.8 ± 0.381

11.6 ± 0.9

5.5 ± 0.42

PAI-I-act (u/g t)

12.61 ± 1.02

52.5 ± 4.7

41.3 ± 3.8

59.4 ± 5.6

PAI-I-AG (ng/g t)

40.0 ± 3.5

28 ± 2.5

39 ± 3.4

54.8 ± 4.5

Note: 1 – the differences are statistically significant relative to the norm in animals; 2 – compared with a tumor; 3 – compared with similar samples in uPA+/+ animals ( р < 0.05)

19

South Russian Journal of Cancer 2024. Vol. 5, No. 2. P. 14-24

Frantsiyants E. M., Bandovkina V. A., Surikova E. I., Kaplieva I. V., Pogorelova Yu. A., Neskubina I. V., Trepitaki L. K., Cheryarina N. D., Ushakova N. D., Ishonina O. G., Gusareva M. A., Udalenkova I. A. Units of fibrinolytic system in mice with urokinase gene knockout in presence of growing B16/F10 melanoma 1.6 times and 4.5 times lower than in animals without

uPA+/+ in tumor samples, compared with intact skin

knockout, respectively. The activity of PAI-I and its

(normal), almost all the studied parameters of the

content were reduced by 15.4 times and 25.6 times.

fibrinolytic system have increased, with the exception

Despite this, the level of PAP and PG in the perifocal

of the receptor level, in melanoma in males uPA-/-

zone of uPA-/- females turned out to be 1.3 times and

on the other hand only an increase in tPA and uPAR

1.8 times higher than in uPA+/+ females, respectively.

activity was noted.

That said, in the perifocal zone in female uPA+/+

In the perifocal zone of uPA-/- males, compared

mice, after 3 weeks of melanoma growth, almost all

with the perifocal zone of uPA+/+ males, the activity

links of the fibrinolytic system (with the exception of

and level of uPA were 127 times and 538 times lower,

PG and tPA activity) exceeded the indicators in the

and tPA was 25 times and 11.4 times, and PAI-I was

skin of intact animals, whereas in uPA-/- mice in the

25 times and 8.8 times, respectively. At the same

perifocal zone, compared with the skin of intact an-

time, the level of uPAR, plasmin and PG in the perifo-

imals, only an increase in the content was detected

cal zone did not carry any significant differences de-

PG, uPA and tPA, without increasing their activity, as

pending on the state of the urokinase gene. It turned

well as increased activity of PAI-I.

out that with tumor growth in uPA+/+ males in the

In the skin of uPA-/- females unaffected by tu-

perifocal zone, the content of PAP, PG, as well as the

mor growth, almost all indicators of the fibrinolyt-

activity and content of tPA and PAI-I increased com-

ic system were reduced, except for the absence of

pared with the skin of the corresponding intact ani-

differences in PAP and uPAR, compared with skin

mals, whereas in males uPA-/- either did not change

samples in uPA+/+ females. Thus, in skin samples

compared with intact skin, or decreased.

with tumor growth in females, uPA-/- activity and uPA

In the samples of unaffected skin in uPA-/- males

content were lower by 277.8 times and 781.3 times;

with melanoma B16/F10, compared with the indica-

tPA activity and content by 4.4 times and 5.9 times;

tors in unaffected skin in uPA+/+ males, the concen-

PAI-I activity and content by 13 times and 11.4 times,

trations of PAP and PG were on average 1.7 times

respectively. After 3 weeks of tumor growth, the

lower, the activity and content of uPA 183 times and

dynamics of changes in the studied parameters in

726 times, the activity and content of tPA 5.1 times

unaffected skin in uPA+/+ females, compared with

and by 7.3 times, the activity and concentration of

intact mice, generally corresponded to the orienta-

PAI-I by 29.7 times and 11.5 times. Only the uPAR lev-

tion in the tumor and perifocal zone – activation of

el had no significant differences. At the same time,

the fibrinolytic system was observed, whereas in

it should be noted that in uPA+/+ males and in mice

uPA-/- females, on the contrary, either no changes

with urokinase gene knockout, only the absence of

or a decrease in the level of RAR, activity and the

changes in the activity and content of iRA, as well as

content of tPA and the content of PAI-I, compared

a decrease in PAI-I levels, turned out to be the same

with intact mice of the same line.

in unaffected skin with melanoma growth, as well

At the stage 3 weeks after transplantation, the

as a decrease in the level of PAI-I, compared with

volumes of primary tumors in uPA-/- males were

the indicators of healthy skin of the corresponding

smaller than in animals with wild type genes [15].

intact animals. The rest of the studied parameters

In the tumor samples of uPA-/- males, compared

changed in different directions – in uPA-/- males

with similar samples in uPA+/+ males, the level of plas-

either decreased (PAP, PG, tPA activity) or did not

min was reduced by 3.4 times and plasminogen by

change (PAI-I activity, tPA content, uPAR), whereas

2.2 times (Table 2). The activity and content of uPA in

activation was detected in uPA+/+ (with the excep-

males with urokinase gene knockout were 208 times

tion of uPAR).

and 3004 times lower than in wild-type animals, re-

spectively, and the content and activity of tPA were

DISCUSSION

25.2 times and 2 times lower, respectively. In addition,

a decrease by 14.4 times and 10 times in the activity

Currently, it is known that urokinase (uPA) is

and concentration of PAI-I was revealed as well.

secreted in many malignant cells, including pan-

Only the uPAR content did not differ depending

creatic, breast, and colorectal cancers, and its ex-

on the state of the urokinase gene. So if in males

pression often correlates with the prognosis of the

20

Южно-Российский онкологический журнал 2024. Т. 5, № 2. С. 14-24

Франциянц Е. М., Бандовкина В. А., Сурикова Е. И., Каплиева И. В., Погорелова Ю. А., Нескубина И. В., Трепитаки Л. К., Черярина Н. Д., Ушакова Н. Д., Ишонина О. Г., Гусарева М. А., Удаленкова И. А. Звенья фибринолитической системы у мышей с нокаутом по гену урокиназы на фоне роста меланомы В16/F10

disease [4, 16]. The biological role of this protease

cells by assembling them in complex regulatory units

is to bind to the uPAR receptor to stimulate the pro-

with transmembrane receptors [21].

teolytic cascade and convert inactive proteases such

Our study showed that on the background of

as plasmin and matrix metallopeptidase 9 (MMP-9)

significant suppression of urokinase, the level of

into active forms, thereby endowing tumor cells with

the uPAR receptor in intact skin in females did not

the ability to destroy the components of the extra-

change, and in males. Although an almost twofold

cellular matrix, activate the growth and metastasis

decrease in its concentration was detected. However,

of tumor cells [17–19]. Therefore, the role of uPA in

its content in the tumor and surrounding tissues on

migration, invasion and metastasis of tumor cells is

the background of the growth of melanoma B16/F10

undeniable [18].

has no significant differences from those in animals

Previously, we received confirmation of the effect

with wild the type of genome. It is known that uPAR

of urokinase gene knockout on the tumor process,

competes with uPA for participation in many non-pro-

namely, significant suppression of tumor volume

teolytic biological processes, such as migration, ad-

growth and metastasis in animals of both sexes [15].

hesion, cell proliferation and angiogenesis [22]. Thus,

We found that in intact uPA-deficient mice of the

uPA-/- uPA functions could be performed by uPAR

C57BL/6-PlautmI.IBug- ThisPlau6FDhu/GFDhu line,

in mice. In our study, the uPA-/- urokinase receptor

almost the entire cascade of PG regulators was

level in the studied samples did not change in rela-

suppressed in the skin (with the exception of the

tion to the parameters in intact animals, whereas in

urokinase receptor uPAR and tPA content only in

males the tumor samples increased, which was ac-

females). We expected to detect an increase in the

companied by large volumes of melanoma in males,

activity of a number of enzymes, but in intact uPA-

compared with females.

/- mice, an increased content of plasmin alone was

Series of studies confirm that a decrease in uPAR

recorded. With uPA deficiency in C57BL/6-PlautmI.

expression on the cell surface mitigates the develop-

IBug- ThisPlau6FDhu/GFDhu mice, plasmin activity

ment of characteristic cancer signs caused by PIK-

could have found other targets in our experiment.

3CA and KRas mutations in colorectal cancer [23],

We believe that an increase in the content of PAP in

and by interacting with uPA and IGF1R, uPAR is able

knockout mice is a kind of compensation, contrib-

to enhance the malignant potential of triple negative

uting to a sharp decrease in urokinase, cleavage of

breast cancer [24]. Clinical observations are con-

its receptor.

firmed by experimental studies in which knockout of

Despite the significant suppression of the fibri-

the uPAR gene in mice leads to G2/M arrest, thereby

nolytic system in mice, uPA-/- transfused melano-

suppressing cell proliferation [25]. There are studies

ma grew, and although it had significantly smaller

on the possibility of using uPA inhibitors to slow tu-

volumes (especially in females) and rarely metas-

mor growth and metastasis [26].

tasized (males had no visible metastases), the life

It is believed that overexpression of uPAR in hu-

expectancy of animals of the two lines did not have

man melanoma cells controls the invasive and gly-

significant differences. In addition, the level of plas-

colytic phenotype. uPAR-mediated pathways have

min in the skin of intact uPA-/- mice exceeded the

already been established, including the integrin-

values in animals of the C57BL/6 line. These points

dependent association of uPAR with at least four

prove the presence of alternative biological pathways

IL- TKR systems: EGFR, IGFR, PDGFR and MET [27].

that melanoma "uses" for its survival in conditions

The results of our studies showed a significant

of knockout of the urokinase gene.

increase in the level of uPAR in tumor samples

One of the alternative pathways in urokinase gene

and its perifocal zone in uPA+/+ mice, whereas in

knockout conditions may be uPAR, known as CD-87,

uPA-/- females such a pattern was not observed

which is highly expressed in various tumor cells, and

and only in uPA-/- males the concentration of the

various signals regulated by uPAR play an important

urokinase receptor increased in tumor samples.

role in neoplasm proliferation and metastasis, tumor-

The complexity of various molecular pathways al-

related glycolysis, as well as tumor microenviron-

lows malignant cells to continue to proliferate and

ment and angiogenesis [20]. There is evidence that

migrate even in conditions of urokinase deficiency,

it is uPAR that regulates the migration of melanoma

using uPA-independent pathways of proteolytic

21

South Russian Journal of Cancer 2024. Vol. 5, No. 2. P. 14-24

Frantsiyants E. M., Bandovkina V. A., Surikova E. I., Kaplieva I. V., Pogorelova Yu. A., Neskubina I. V., Trepitaki L. K., Cheryarina N. D., Ushakova N. D., Ishonina O. G., Gusareva M. A., Udalenkova I. A. Units of fibrinolytic system in mice with urokinase gene knockout in presence of growing B16/F10 melanoma activation of angiogenesis factors. This was con-CONCLUSIONS

firmed by our previous studies of angiogenesis

factors in animals with urokinase gene knockout,

The fact that in uPA-/- mice, despite the extremely

demonstrating an increased content of VEGF-A

small volumes of the primary tumor and rare metas-

and especially VEGF-C in unaffected skin in female

tasis, tumor disease caused the death of animals

uPA-/- mice [28].

at the same time as in uPA+/+ animals, indicates

At the same time, it should be considered that

a significant effect of tumor disease on all regula-

knockout by the urokinase gene is a kind of artificial-

tory systems of the body, regardless of the size of

ly induced genetic comorbid disease, as a result of

the neoplasm. Our study confirmed the claim that

which the fibrinolytic system is suppressed not only

the use of drugs that inhibit the urokinase pathway

in the skin, but also in other organs and systems.

may be promising in the treatment of the disease

The involvement of the fibrinolytic system in various

by slowing the growth of neoplasm volume and its

physiological processes, wound healing, as well as

metastasis, but is not a panacea, since the effect of

in the preservation of brain neurons after various

a malignant tumor on the body is much more com-

ischemic injuries indicates a possible insufficiency

plex, therefore further studies of the pathogenesis

of these processes in uPA-/- animals.

of malignant growth are required.

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Information about authors:

Elena M. Frantsiyants – Dr. Sci. (Biol.), professor, deputy CEO for science, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0003-3618-6890, SPIN: 9427-9928, AuthorID: 462868, ResearcherID: Y-1491-2018, Scopus Author ID: 55890047700

Valeriya A. Bandovkina – Dr. Sci. (Biol.), senior researcher at the Laboratory for the Study of Pathogenesis of Malignant Tumors, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0002-2302-8271, SPIN: 8806-2641, AuthorID: 696989, ResearcherID: AAG-8708-2019, Scopus Author ID: 57194276288

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South Russian Journal of Cancer 2024. Vol. 5, No. 2. P. 14-24

Frantsiyants E. M., Bandovkina V. A., Surikova E. I., Kaplieva I. V., Pogorelova Yu. A., Neskubina I. V., Trepitaki L. K., Cheryarina N. D., Ushakova N. D., Ishonina O. G., Gusareva M. A., Udalenkova I. A. Units of fibrinolytic system in mice with urokinase gene knockout in presence of growing B16/F10 melanoma Ekaterina I. Surikova – Cand. Sci. (Biol.), senior researcher at the Laboratory for the Study of Pathogenesis of Malignant Tumors, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0002-4318-7587, SPIN: 2401-4115, AuthorID: 301537, ResearcherID: AAG-8748-2019, Scopus Author ID: 6507092816

Irina V. Kaplieva – Dr. Sci. (Med.), head of the Laboratory for the Study of the Pathogenesis of Malignant Tumors, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-3972-2452, SPIN: 5047-1541, AuthorID: 734116, ResearcherID: ААЕ-3540-2019, Scopus Author ID: 23994000800

Yuliya A. Pogorelova – Cand. Sci. (Biol.), senior researcher at the Laboratory of Malignant Tumor Pathogenesis Study, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-2674-9832, SPIN: 2168-8737, AuthorID: 558241, ResearcherID: AAE-4168-2022, Scopus Author ID: 37026863400

Irina V. Neskubina – Cand. Sci. (Biol.), senior researcher at the Laboratory for the Study of the Pathogenesis of Malignant Tumors, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0002-7395-3086, SPIN: 3581-8531, AuthorID: 794688, ResearcherID: AAG-8731-2019, Scopus Author ID: 6507509066

Lidiya K. Trepitaki – Cand. Sci. (Biol.), researcher at the Laboratory for the Study of Pathogenesis of Malignant Tumors, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-9749-2747, SPIN: 2052-1248, AuthorID: 734359, ResearcherID: AAG-9218-2019, Scopus Author ID: 55357624700

Natalya D. Cheryarina – MD, laboratory assistant at the Laboratory for the Study of the Pathogenesis of Malignant Tumors, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0002-3711-8155, SPIN: 2189-3404, AuthorID: 558243, Scopus Author ID: 56204439400

Natalia D. Ushakova – Dr. Sci. (Med.), professor, MD, anesthesiologist-resuscitator of the Department of Anesthesiology and Resuscitation, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ORCID: https://orcid.org/0000-0002-0068-0881, SPIN: 9715-2250, AuthorID: 571594, Scopus Author ID: 8210961900

Oksana G. Ishonina – Cand. Sci. (Biol.), head at the Department of Training and Retraining of Specialists, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation; associate professor of the Department of Medical Biology and Genetics, Rostov State Medical University, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-5300-1213, SPIN: 4051-5165, AuthorID: 612417, Scopus Author ID: 37115461900

Marina A. Gusareva – Cand. Sci. (Med.), head of the Radiotherapy Department, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0002-9426-9662, SPIN: 9040-5476, AuthorID: 705242

Irina A. Udalenkova – Cand. Sci. (Med.), MD, oncologist, Department of Tumor Medical Therapy, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation

ORCID: https://orcid.org/0000-0003-0075-6935, SPIN: 2175-4570, AuthorID: 974753

Contribution of the authors:

Franzyants E. M. Bandovkina V. A., Kaplieva I. V. – development of the concept and design of the experiment, writing the source text, analysis and interpretation of data, final approval of the manuscript for publication; Surikova E. I., Cheryarina N. D. – statistical processing of the results, technical editing and preparation of the manuscript for publication; Pogorelova Yu. A., Neskubina I. V. Trepitaki L. K. – conducting an experiment, performing an ELISA analysis; Ushakova N. D., Ishonina O. G. – scientific editing, revision of the text, selection of literature, bibliography design; Gusareva M. A., Udalenkova I. A. – scientific editing, revision of the text.

All authors have contributed equally to the preparation of the publication.

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