Melanoma cells adhesive properties activation in 3d spheroids

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Abstract

In this study, the viability and adhesive features of BRO and SK-MEL-2 cell lines were evaluated using a melanoma model. It was revealed that in BRO cells, the development of apoptosis after exposure to dacarbazine was combined with the transition of the proportion of cells to the G0 phase of the cell cycle, that corresponded to previously obtained results. The absence of apoptosis in 3D spheroids and the absence of exit from the cell cycle were observed in SK-MEL-2 melanoma cells. It was also revealed that in the control spheroids (cells without exposure) of the BRO and SK-MEL-2 melanoma lines, adhesion to fibronectin was higher compared with the cells of the control monolayer, which is explained by the three-dimensional structure requiring cell communication with the extracellular matrix. In spheroids formed by SK-MEL-2 cells, dacarbazine induced a decrease in adhesion to fibronectin, which may be associated with the development of drug resistance. An increase in the expression of integrins AV and β8 in BRO and SK-MEL-2, as well as integrin β5 in SK-MEL-2, was determined in cells after exposure to dacarbazine, which may indicate the involvement of aforementioned molecules in the exit from proliferative stage of tumor cells.

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About the authors

D. V. Chernykh

Voino-Yasenetsky Krasnoyarsk State Medical University

Author for correspondence.
Email: tatyana_ruksha@mail.ru
Russian Federation, Krasnoyarsk

I. S. Zinchenko

Voino-Yasenetsky Krasnoyarsk State Medical University

Email: tatyana_ruksha@mail.ru
Russian Federation, Krasnoyarsk

T. G. Ruksha

Voino-Yasenetsky Krasnoyarsk State Medical University

Email: tatyana_ruksha@mail.ru
Russian Federation, Krasnoyarsk

References

Supplementary files

Supplementary Files
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2. Fig. 1. Structure of a spheroid (after: Zanoni et al., 2020).

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3. Fig. 2. Spheroids consisting of BRO (a, b) and SK-MEL-2 (c, d) cell lines. Cell nuclei are stained with DAPI (blue), green (secondary antibodies) – Ki67 expression, immunocytochemistry, fluorescence microscopy.

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4. Fig. 3. Distribution of apoptotic cells in spheroids of BRO (a, b) and SK-MEL-2 (c, d) lines. Green color (annexin) – apoptotic cells.

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5. Fig. 4. The proportion of Ki-67-negative cells in the monolayer (Mon) and spheroids (Sph) of BRO (a) and SK-MEL-2 (b) cells in the control (C, without treatment) and after the action of DMSO and dacarbazine (Dac). The mean values ​​(n = 3) and their standard deviations are shown; differences are significant at: *P = 0.0092, **P < 0.05 and ***P = 0.0045 (Mann–Whitney test).

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6. Fig. 5. The proportion of apoptotic cells in the monolayer and spheroids of BRO (a) and SK-MEL-2 (b) cells in the control (C) and after the action of DMSO and dacarbazine. The mean values ​​(n = 3) and their standard deviations are shown; differences are significant at: *P = 0.0213, **P = 0.0006 and ***P = 0.0055 (Mann–Whitney test). The designations are the same as in Fig. 4.

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7. Fig. 6. Color intensity at different degrees of adhesion to the fibronectin substrate (a, the brighter the color, the higher the degree of adhesion) and the degree of adhesion to the fibronectin substrate of BRO (b) and SK-MEL-2 (c) cells. Data are shown for the monolayer and spheroids in the control and after the action of DMSO and dacarbazine. Designations are the same as in Fig. 4. Average values ​​(n = 3) and their standard deviations are given; differences are significant at: *P = 0.0495 (Mann–Whitney test).

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8. Fig. 7. Relative expression of the integrin genes ITGВ 5 (a), ITGB8 (b), and ITGAV (c) in BRO and SK-MEL-2 cell line spheroids and after exposure to DMSO and dacarbazine (DAC). Integrin gene expression in cells after exposure to dacarbazine was assessed relative to cells exposed to DMSO. Average values ​​(n = 3) and their standard deviations are given; (*) – differences are significant at P = 0.0495 (Mann–Whitney test).

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