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. 2021 Jul 31;22(15):8264.
doi: 10.3390/ijms22158264.

Identification of Spiro-Fused [3-azabicyclo[3.1.0]hexane]oxindoles as Potential Antitumor Agents: Initial In Vitro Evaluation of Anti-Proliferative Effect and Actin Cytoskeleton Transformation in 3T3 and 3T3-SV40 Fibroblast

Nickolay A Knyazev  1   2 , Stanislav V Shmakov  3 , Sofya A Pechkovskaya  2 , Alexander S Filatov  4 , Alexander V Stepakov  4 , Vitali M Boitsov  3   5 , Natalia A Filatova  2
Affiliations

Identification of Spiro-Fused [3-azabicyclo[3.1.0]hexane]oxindoles as Potential Antitumor Agents: Initial In Vitro Evaluation of Anti-Proliferative Effect and Actin Cytoskeleton Transformation in 3T3 and 3T3-SV40 Fibroblast

Nickolay A Knyazev et al. Int J Mol Sci. .

Abstract

Novel heterocyclic compounds containing 3-spiro[3-azabicyclo[3.1.0]hexane]oxindole framework (4a, 4b and 4c) have been studied as potential antitumor agents. The in silico ADMET (adsorption, distribution, metabolism, excretion and toxicity) analysis was performed on 4a-c compounds with promising antiproliferative activity, previously synthetized and screened against human erythroleukemic cell line K562 tumor cell line. Cytotoxicity of 4a-c against murine fibroblast 3T3 and SV-40 transformed murine fibroblast 3T3-SV40 cell lines were evaluated. The 4a and 4c compounds were cytotoxic against 3T3-SV40 cells in comparison with those of 3T3. In agreement with the DNA cytometry studies, the tested compounds have achieved significant cell-cycle perturbation with higher accumulation of cells in G0/G1 phase. Using confocal microscopy, we found that with 4a and 4c treatment of 3T3 cells, actin filaments disappeared, and granular actin was distributed diffusely in the cytoplasm in 82-97% of cells. The number of 3T3-SV40 cells with stress fibers increased to 7-30% against 2% in control. We discovered that transformed 3T3-SV40 cells after treatment with compounds 4a and 4c significantly reduced the number of cells with filopodium-like membrane protrusions (from 86 % in control cells to 6-18% after treatment), which indirectly suggests a decrease in cell motility. We can conclude that the studied compounds 4a and 4c have a cytostatic effect, which can lead to a decrease in the number of filopodium-like membrane protrusions.

Keywords: 3-spiro[azabicyclo[3.1.0]hexane]oxindoles; 3T3; 3T3-SV40; ADMET analysis; actin; cell cycle; cytoskeleton; metastasis; tumor cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of spiro-fused [3-azabicyclo[3.1.0]hexane]exindoles derivatives 4a–c and cisplatin on growth 3T3 and 3T3 SV40 cells. The graph demonstrates the effect of the compounds on cell growth in relation to the control. Results are averages of 3 replicates in each experiment, given as the mean ± SD values. * p ≤ 0.05. ** intergroup significance levels p ≤ 0.05.
Figure 2
Figure 2
Effect of spiro-fused [3-azabicyclo[3.1.0]hexane]exindoles derivatives 4a–c and cisplatin on the distribution of 3T3 and 3T3 SV40 cells in the cell cycle. The most typical histograms (A) of distribution of cells in the cell cycle are presented (3T3A and 3T3 SV40A). The graphs 3T3B and 3T3 SV40B (B) demonstrate the average distribution of the cells in the cell cycle based on 3 replicates in each experiment, given as the mean ± SD values. * p ≤ 0.05.
Figure 3
Figure 3
State of actin cytoskeleton of 3T3 and 3T3-SV40 cells after treatment with spiro-fused [3-azabicyclo[3.1.0]hexane]exindole derivatives 4a–c and cisplatin. I: Images demonstrate the different stages of cell actin cytoskeleton (I-3T3 and I-3T3-SV40). II: Pie charts demonstrate percentage of cells with normal stress fibers (A) and disassembled stress fibers (B) (II-3T3 and II-3T3-SV40). III: Pie charts demonstrate percentage of cells with filopodia-like deformations (C), and without filopodia-like deformations (D). Inserts: 1—stress fibers; 2—disassembled stress fibers; 3—filopodia-like deformations; 4—without filopodia-like deformations.
Figure 4
Figure 4
Synthesis of spiro-fused [3-azabicyclo[3.1.0]hexane]oxindoles 4a, 4b, and 4c. Synthesized compounds containing spiro-fused 3-azabicyclo[3.1.0]hexane and oxindole moieties, by means of 1,3-dipolar cycloaddition reaction of appropriately substituted cyclopropene 1 to azomethine ylides generated in-situ from isatin 2 and corresponding α-amino acids 3a–c.
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