Биологические науки/9. Биохимия и биофизика

PhD Shakhristova E.V., MD, PhD, professor Stepovaya E.A., PhD Nosareva O.L., MD, PhD, professor Ryazantseva N.V., MD, PhD, professor Novitsky V.V.

Siberian state medical university, Tomsk, Russia

The impact of induced oxidative stress on cell cycle phase distribution of breast cancer cells

Nowadays researchers pay special attention to molecular mechanisms of cell system dysfunction in pathologies which are associated with oxidative stress, accompanied by redox status change, proliferation dysregulation and apoptosis [1,2]. Breast tumors are number one cancer type among women all over the world, and Russia is not an exception. The objective of the present research is to study the intensity of intracellular reactive oxygen species production, the degree of protein oxidative modification as well as cell cycle phase distribution of MCF-7 breast cancer cells under N-ethylmaleimide-induced oxidative stress.

The research was carried out on the MCF-7 cell line (human breast adenocarcinoma), obtained from the Russian culture collection at the Cytology Institute of the Russian Academy of Science (Saint-Petersburg). MCF-7 cells were cultured in complete growth medium composed of 90% EMEM (“PanEco”, Russia) with 10% (v/v) fetal calf serum (“Invitrogen”, USA), 1% nonessential amino acids (“PanEco”, Russia), 10 mcg/ml bovine insulin (“PanEco”, Russia), 0.3 mg/ml L-glutamine (“PanEco”, Russia) and 100 mcg/ml gentamycin (“INS”, USA).

In breast cancer cells oxidative stress was induced by adding 5mM N-ethylmaleimide (NEM, “Sigma Aldrich”, USA) to the medium [3] with further culturing for 18 hours at 37°C and 5% CO2. N-ethylmaleimide irreversibly binds protein and peptide SH groups, which results in the intracellular oxidant/antioxidant ratio imbalance and oxidative stress.

The intensity of free radical oxidation in MCF-7 cells was judged by the concentration of carbonyl protein derivatives, determined by spectrophotometry (the method is based on the reaction of oxidized amino acid residues with 2,4-dinitrophenylhydrazine [4]), and the concentration of reactive oxygen species (ROS), determined by flow cytofluorometry with 2,7-dischlorfluorescein-diacetate (DCFH-DA) fluorescence probe (DCFH-DA, 5mcM, “Sigma Aldrich”, USA) [5]. Phase distribution of MCF-7 cells was evaluated by flow cytofluorometry using Cycle Test Plus kit (“Becton Dickinson”, USA). The results were processed by the nonparametric Mann-Whitney test.

In the course of the research it was established that NEM induces intracellular ROS production in breast cancer cells, which was indicated by the rise in the fluorescence of DCFH-DA-loaded cells (Table).

Table

The concentration of reactive oxygen species and protein carbonyl derivatives in MCF-7 breast cancer cells under the effect of N-ethylmaleimide (5mM), Ме (Q₁–Q₃)

Studied parameters			MCF-7 cancer cell line	MCF-7 cancer cell line + N-ethylmaleimide
Reactive oxygen species, conventional units			0,81 (0,80-0,81)	2,35^* (2,25-2,50)
Carbonyl derivatives of proteins, conventional units /mg protein	Spontaneous oxidative modification of proteins	λ=274 нм	4,52 (3,26-7,34)	20,21^* (13,76-20,61)
		λ=363 нм	5,48 (5,01-6,28)	26,91^* (26,22-28,36)
	Metal-catalyzed oxidative modification of proteins	λ=363 нм	16,34 (15,27-19,38)	29,88^* (29,21-32,16)
		λ=274 нм	20,22 (20,09-20,84)	35,41^* (32,72-38,98)

Note: ^* – р<0,01 statistical significance calculated with respect to MCF-7 tumor cells. MCF-7 cell culturing in the presence of NEM resulted in the increase in protein oxidative modification. At the wavelength of 274 nm aldehyde phenylhydrazones were observed, which are early markers of protein oxidative modification; at 363 nm ketone dinitrophenylhydrazones were registered, which are markers of late protein destruction. Against the backdrop of NEM addition to the cancer cell medium, a rise (р<0,01) in the concentration of carbonyl derivatives was detected at 274 nm and 363 nm under the conditions of spontaneous and metal-catalyzed protein oxidation, as opposed to the degree of protein oxidative modification in the intact cells (Table). The increase in spontaneous and metal-catalyzed protein oxidation is a marker of oxidative damage to MCF cells, during which proteins act as efficient traps for generated ROS [6].

It was found out that the blocking agent of protein and peptide SH groups altered phase distribution of MCF-7 cells. It was identified that the number of tumor cells in the S-phase jumped (р<0,01) under the effect of NEM due to a fall in their amount in the G₀/G₁ phase, as opposed to the intact MCF-7 cells (Fig.).

Figure. Cell cycle phase distribution of MCF-7 tumor cells under the effect of N-ethylmaleimide (^* – р<0,01 statistical significance calculated with respect to intact MCF-7 cells).

Therefore, NEM enhanced tumor cell transition from G₀/G₁ phase to S-phase, however it did not significantly change the amount of cells in the G₂/М phase. Stopping of breast cancer cell cycle in the S-phase under the conditions of NEM-induced free radical oxidation indicates violation of DNA replication, which may be associated with alterations in the functions of redox-sensitive proteins, in particular, transcription factors, cyclins and cyclin-dependent kinases.

The study was supported by the Russian Foundation for Humanities as part of the research project No. 15-36-01289.

References:

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4. Arutyunyan A.V., Dubinina E.E., Zybina N.N. Metody ocenki svobodnoradikal'nogo okisleniya i antioksidantnoj zashchity organizma. – Saint-Petersburg: IKF «Foliant» 2000.

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