GLUTATHIONE AND GLUTAREDOXIN IN ROSCOVITINE-MEDIATED INHIBITION OF BREAST CANCER CELL PROLIFERATION

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Abstract


Background: Breast tumors are number one cause of cancer morbidity and mortality among women around the world, and Russia is not an exception. Many proteins that control proliferation of immortalized cells are redox-regulated, which is essential for modulating cellular proliferative activity, especially during tumor growth. Studying the role of glutaredoxin and glutathione in cell cycle phase distribution will allow not only to identify the molecular targets regulating cell proliferation, but also to develop methods of diagnosis and targeted therapy of socially sensitive diseases, including breast cancer, in the future.

Aims: To evaluate the role of glutathione and glutaredoxin in the molecular mechanisms regulating MCF-7 breast cancer cell proliferation under the effects of roscovitine, a cyclin-dependent protein kinase inhibitor.

Materials and methods: The MCF-7 cell line (human breast adenocarcinoma) was used in the study. The cell culture was incubated in the presence and absence of roscovitine in the final concentration of 20 µmol for 18 h. The production of reactive oxygen species, the distribution of cells between cell cycle phases and the amount of Annexin V positive cells were determined using flow cytometry. The concentrations of total, reduced and oxidized glutathione, protein SH groups and protein-bound glutathione were measured by spectrophotometry. The levels of glutaredoxin, cyclin E and cyclin-dependent protein kinases were estimated by Western blotting with monoclonal antibodies.

Results: The effects of roscovitine in the MCF-7 cells resulted in cell cycle arrest in G2/М phases with the decreased levels of cyclin E and cyclin-dependent protein kinase 2. It was accompanied by activation of programmed cell death. In tumor cells incubated in the presence of roscovitine, oxidative stress was triggered, which was accompanied by the elevated generation of reactive oxygen species, the decrease in the concentration of reduced glutathione, and the rise in the level of glutaredoxin. It contributed to the increase in protein glutathionylation against the backdrop of the decreased SH group concentration.

Conclusions: Breast cancer cell proliferation under the effects of roscovitine is reduced following not only the decrease in the cyclin level and cyclin-dependent protein kinase activity, but also the shift in the intracellular oxidant/antioxidant ratio. Roscovitine-induced oxidative stress in the MCF-7 cells contributed to protein glutathionylation with the changes in the protein structure and functions. It results in impaired cell cycle progression, indicating a possibility to regulate cellular proliferation through modulating functional properties of redox-dependent proteins using the glutathione/glutaredoxin system.


E. V. Shakhristova

Siberian state medical university

Email: shaxristova@yandex.ru
ORCID iD: 0000-0003-2938-1137

Russian Federation

Кандидат медицинских наук, доцент кафедры биохимии и молекулярной биологии с курсом клинической лабораторной диагностики.

634050, Томск, Московский тракт, д. 2, тел.: +7 (3822) 90-11-01 доб. 1853.

SPIN-код: 8125-6414

E. A. Stepovaya

Siberian state medical university

Author for correspondence.
Email: muir@mail.ru
ORCID iD: 0000-0001-9339-6304

Russian Federation

Доктор медицинских наук, профессор, профессор кафедры биохимии и молекулярной биологии с курсом клинической лабораторной диагностики.

634050, Томск, Московский тракт, д. 2, тел.: +7 (3822) 90-11-01 доб. 1853.

SPIN-код: 5562-4522

O. L. Nosareva

Siberian state medical university

Email: olnosareva@yandex.ru
ORCID iD: 0000-0002-7441-5554

Russian Federation

Кандидат медицинских наук, доцент кафедры биохимии и молекулярной биологии с курсом клинической лабораторной диагностики.

634050, Томск, Московский тракт, д. 2, тел.: +7 (3822) 90-11-01 доб. 1853.

SPIN-код: 5688-7566

E. V. Rudikov

Siberian state medical university

Email: korvin_w@mail.ru
ORCID iD: 0000-0003-3283-3616

Russian Federation

Интерн кафедры биохимии и молекулярной биологии с курсом клинической лабораторной диагностики.

634050, Томск, Московский тракт, д. 2, тел.: +7 (3822) 90-11-01 доб. 1853.

SPIN-код: 5559-4313

V. V. Novitsky

Siberian state medical university

Email: patfizssmu@yandex.ru
ORCID iD: 0000-0002-9577-8370

Russian Federation

Доктор медицинских наук, профессор, академик РАН, заведующий кафедрой патофизиологии.

634050, Томск, Московский тракт, д. 2, тел.: +7 (3822) 90-11-01 доб. 1740.

SPIN-код: 7160-6881

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