Study of Cytotoxicity of Spiro-Fused [3-Azabicyclo[3.1.0]hexane]oxindoles and Cyclopropa[a]pyrrolizidine-oxindoles Against Tumor Cell Lines

Abstract

Background: A series of spiro-fused heterocyclic compounds containing cyclopropa[a]pyrrolizidine-2,3'-oxindole and 3-spiro[3-azabicyclo[3.1.0]-hexane]oxindole frameworks were synthesized and studied for their in vitro antiproliferative activity against human erythroleukemia (K562), cervical carcinoma (HeLa), acute T cell leukemia (Jurkat), melanoma (Sk-mel-2) and breast cancer (MCF-7) as well as mouse colon carcinoma (CT26) cell lines. Methods: Cell proliferation was evaluated in vitro by MTS assay. Confocal microscopy was used to study actin cytoskeleton structure and cell motility. Cell cycle analysis was evaluated by flow cytometry. Results: It was found that compounds 4, 8, 18 and 24 showed antiproliferative activity against the Jurkat, K-562, HeLa and Sk-mel-2 cell lines with IC₅₀ ranging from 2 to 10 μM (72 h). Evaluation of the impact on cell cycle progression showed that the tested compounds achieved significant cell-cycle perturbation with a higher accumulation of cells in the SubG1 and G0/G1 phases of the cell cycle, in comparison to the negative control. I Incubation with tested compounds led to the disappearance of stress fibers (granular actin was distributed diffusely in the cytoplasm in up to 38% of treated HeLa cells) and changes in the number of filopodia-like deformations (reduced from 93% in control cells to 64% after treatment). The impact on the Sk-mel-2 cell actin cytoskeleton structure was even greater: granular actin was distributed diffusely in the cytoplasm in up to 90% of treated cells while the number of filopodia-like deformations was reduced by up to 23%. A scratch test performed on the human melanoma cell line showed that these cells did not fill the scratched strip and lose their ability to move under treatment. Conclusions: The obtained results support the antitumor effect of the tested spiro-compounds and encourage the extension of this study in order to improve their anticancer activity as well as reduce their toxicological risks.

Keywords: 1,3-dipolar cycloaddition; 3-spiro[3-azabicyclo[3.1.0]-hexane]oxindole; antiproliferative activity; azomethine ylides; cell cycle; cell motility; cyclopropa[a]pyrrolizidine-2,3′-oxindole; cyclopropenes; morphological changes (cytoskeleton); tumor cell lines.

Figure 1

Selected examples of biologically active 1-azabicyclo[3.3.0]octanes (pyrrolizines or pyrrolizidines), 3-azabicyclo[3.1.0]hexanes and spiro[pyrrolidine-3,3′-oxindoles], based on the studies by Fohlen et al. (2021) [10], Appadurai et al. (2014) [12], AbdelSamad et al. (2023) [13], Sugie et al. (2001) [14], Tanaka et al. (2017) [19], Chang et al. (2012) [21], McHardy et al. (2011) [23], Moffat et al. (2010) [28], Yu et al. (2015) [36] and Zhao et al. (2013) [37].

Figure 2

Cytotoxicity of racemic spiro-adducts against the K562 cell line for 24 h (A) and 72 h (B).

Figure 3

Cytotoxicity of racemic spiro-adducts against the HeLa cell line for 24 h (A) and 72 h (B).

Figure 4

Cytotoxicity of selected racemic spiro-adducts against the Jurkat cell line for 24 h (A) and 72 h (B).

Figure 5

Cytotoxicity of racemic spiro-adducts against the Sk-mel-2 cell line for 24 h (A) and 72 h (B).

Figure 6

Cytotoxicity of selected racemic spiro-adducts against the MCF-7 cell line for 24 h (A) and 72 h (B).

Figure 7

Cytotoxicity of selected racemic spiro-adducts against the CT26 cell line for 24 h (A) and 72 h (B).

Figure 8

State of actin cytoskeleton of HeLa cells after treatment with compounds 2, 4, 8, 17, 18, 22 and 24 (10 μg/mL). (A) Images demonstrating the different stages of the cell actin cytoskeleton. (B) Histograms demonstrating the percentage of cells with filopodia-like deformations. (C) Histograms demonstrating the percentage of cells with normal stress fibers.

Figure 8

State of actin cytoskeleton of HeLa cells after treatment with compounds 2, 4, 8, 17, 18, 22 and 24 (10 μg/mL). (A) Images demonstrating the different stages of the cell actin cytoskeleton. (B) Histograms demonstrating the percentage of cells with filopodia-like deformations. (C) Histograms demonstrating the percentage of cells with normal stress fibers.

Figure 9

State of actin cytoskeleton of Sk-mel-2 cells after treatment with compounds 2, 4, 8, 11, 17 and 18 (10 μg/mL). (A) Images demonstrating the different stages of the cell actin cytoskeleton. (B) Histograms demonstrating the percentage of cells with filopodia-like deformations. (C) Histograms demonstrating the percentage of cells with normal stress fibers.

Figure 10

Microscopic images of the Sk-mel-2 cell wound area in the scratch assay and wound area (%) in the scratch assay after 36 h incubation with spiro-fused [3-azabicyclo[3.1.0]hexane]oxindoles 4, 11, 12, 18, 24 and cyclopropa[a]pyrrolizidine-2,3′-oxindole 2 (10 μg/mL). * p value < 0.05.

Figure 11

The effect of cycloadducts 2, 4, 8, 17, 18 at a concentration of 10 μg/mL on the distribution of K562 cells in the cell cycle.