Alpha-tocopheryl succinate induces ER stress, disregulates lipid metabolism and leads to apoptosis in normal and tumorous cell lines of epidermal origin

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Vitamin E succinate (VES, α-tocopheryl succinate), is a potential antitumor agent known to selectively affect the mitochondria of tumor cells. However, the data on the proapoptotic mechanism of action of VES are unclear, and the effect of VES on normal, non-tumorigenic cells has not been fully investigated. Previously, we showed that VES induces apoptosis via the mitochondrial pathway in A431 human epidermoid carcinoma cells. The goal of this work is to investigate the effect of VES on non-tumorigenic cells and to reveal commonalities and differences in pathways activated in normal and tumorous cells. To achieve this, we studied how VES affects such organelles as the ER and the Golgi apparatus, analyzed the expression of ER stress-associated genes, and also assessed the ROS content and the accumulation of lipid droplets in A431 human epidermoid carcinoma cells and HaCaT immortalized human keratinocytes. We show that in both cell lines there are signs of ER stress, the amount of ROS and lipid droplets increases, as does the number of apoptotic cells. At the same time, the key difference in the mechanisms apoptotic cell death induction in A431 and HaCaT cells treated with VES lies in the reaction of mitochondria: in A431 cells, apoptotic cell death is triggered via the mitochondrial pathway, while HaCaT cells initiate apoptosis without involving mitochondria. Thus, the targets of VES in normal and tumor cells may differ and can possibly complement each other during apoptosis induction.

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M. Savitskaya

Lomonosov Moscow State University

Email: nakomis@mail.ru

Department of Cell Biology and Histology

俄罗斯联邦, Москва, 119234

I. Zakharov

Lomonosov Moscow State University

Email: nakomis@mail.ru

Department of Cell Biology and Histology

俄罗斯联邦, Moscow, 119234

А. Saidova

Lomonosov Moscow State University

Email: nakomis@mail.ru

Department of Cell Biology and Histology

俄罗斯联邦, Moscow, 119234

Е. Smirnova

Lomonosov Moscow State University

Email: nakomis@mail.ru

Department of Cell Biology and Histology

俄罗斯联邦, Moscow, 119234

G. Onishchenko

Lomonosov Moscow State University

编辑信件的主要联系方式.
Email: nakomis@mail.ru

Department of Cell Biology and Histology

俄罗斯联邦, Moscow, 119234

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2. Fig. 1. Response of cultured cells of the A431 and HaCaT lines to the effect of vitamin E succinate (VES): a – cell viability after cultivation in the presence of VES for 48 hours; b – the value of the apoptotic index of HaCaT cells at different concentrations and time of exposure to SVE. Data are presented as mean and standard deviation; Mann–Whitney test with Benjamini–Hochberg correction for multiple comparisons; differences with the control with the addition of alcohol are significant at (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 or (****) P < 0.0001; n = 3–4 (a) and n = 3 (b).

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3. Fig. 2. Apoptosis in HaCaT cells under the influence of 60 µM SBE: a – hematoxylin and eosin staining; b, c – phase contrast and immunocytochemical detection of caspase 3 in the same cell, respectively. Shown is a representative result from three independent experiments.

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4. Fig. 3. Mitochondria in HaCaT cells after exposure to SVE for 48 hours: a–d – mitochondria in HaCaT cells stained with MitoTracker Orange in the control (a) and after exposure to SVE at a concentration of 40 (b), 60 (c) and 100 ( d) µM; d–i – immunocytochemical detection of cytochrome c in HaCaT cells in the control without alcohol (e) and with the addition of it (f), as well as after exposure to SVE at a concentration of 40 (g), 60 (h) and 100 (i) µM. Shown is a representative result from three independent experiments.

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5. Fig. 4. Ultrastructure of mitochondria in HaCaT cells in control (a, b) and after exposure to SVE for 48 hours at a concentration of 40 (c, d), 60 (e–g) and 100 (h–i) μM; nj – lipid inclusions (neutral fat).

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6. Fig. 5. Intravital detection of hydrogen peroxide in HaCaT cells in control (a, c) and after exposure to 40 μM SBE for 48 hours (b, d); phase contrast (a, b) and staining using the DCFH-DA probe (c, d); e – proportion of ROS-positive cells after cultivation in the presence of SVE. Data are presented as mean and standard deviation; Mann–Whitney test, difference from the control with the addition of alcohol, significant at (*) P < 0.05 (n = 3).

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7. Fig. 6. Detection of neutral fat in A431 (a, b) and HaCaT (c, d) cells after cultivation in control (a, c) and in the presence of 40 μM SBE (b, d).

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8. Fig. 7. Ultrastructure of granular ER in A431 (a–f) and HaCaT (g–m) cells in control and after 48-hour exposure to SVE. A431 cells: a – control; b, c – control with the addition of alcohol; d–f – 40 µM SBE. NaCaT cells: g – control, h – 40 µM CBE, i, j – 60 µM CBE, l, l – 100 µM CBE. Designations: ER – endoplasmic reticulum, MTX – mitochondria, NL – lipid droplets (neutral fat).

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9. Fig. 8. Immunocytochemical detection of the p58k protein as part of the Golgi apparatus in A431 (a, b) and HaCaT (c–f) cells in the control and after 48-hour exposure to SVE. A431 cells: a – control, b – 40 µM SBE. HaCaT cells: c – control, d – 40 µM CBE, d – 60 µM CBE, f – 100 µM CBE.

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10. Fig. 9. Ultrastructure of the Golgi apparatus in A431 (a–e) and HaCaT (f–o) cells in control and after 48-hour exposure to SVE. A431 cells: a, c – control; b, d, e – 40 µM SBE. HaCaT cells: f, h, i, j – control; g, n – 60 µM SBE; l, m – 40 µM SBE; o – 100 µM SBE. Designations: AG – Golgi apparatus, MTX – mitochondria.

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11. Fig. 10. Relative expression of ER stress marker genes: GRP78, ATF4 and CHOP in A431 and HaCaT cells exposed to 40 µM SBE. Real-time PCR. Data are presented as mean and standard deviation; Mann–Whitney test; differences with the control are significant at (*) P < 0.05 or (***) P < 0.001 (n = 3).

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