Synthesis of New Chromene Derivatives Targeting Triple-Negative Breast Cancer Cells

Abstract

Breast cancer continues to be the leading cause of cancer-related deaths among women worldwide. The most aggressive type of breast cancer is triple-negative breast cancer (TNBC). Indeed, not only does TNBC not respond well to several chemotherapeutic agents, but it also frequently develops resistance to various anti-cancer drugs, including taxane mitotic inhibitors. This necessitates the search for newer, more efficacious drugs. In this study, we synthesized two novel chromene derivatives (C1 and C2) and tested their efficacy against a battery of luminal type A and TNBC cell lines. Our results show that C1 and C2 significantly and specifically inhibited TNBC cell viability but had no effect on the luminal A cell type. In addition, these novel compounds induced mitotic arrest, cell multinucleation leading to senescence, and apoptotic cell death through the activation of the extrinsic pathway. We also showed that the underlying mechanisms for these actions of C1 and C2 involved inhibition of microtubule polymerization and disruption of the F-actin cytoskeleton. Furthermore, both compounds significantly attenuated migration of TNBC cells and inhibited angiogenesis in vitro. Finally, we performed an in silico analysis, which revealed that these novel variants bind to the colchicine binding site in β-tubulin. Taken together, our data highlight the potential chemotherapeutic properties of two novel chromene compounds against TNBC.

Keywords: apoptosis; chromenes; microtubules; mitotic slippage; multinucleation; senescence.

Figure 1

Chromene motifs in natural products.

Figure 2

Chromenes C1 and C2 inhibit the cell viability of triple-negative breast cancer cells. The T47D, MCF-7, MDA-MB-231, and Hs578T cell lines after treatment with vehicle (0.1% DMSO) and the indicated concentrations of chromene C1 (A) or C2 (B) for 24 and 48 h. Cell viability was measured using the MTT assay as described in the text. All experiments were performed in three technical replicates and repeated at least four times. Columns and bars represent means and SEM, respectively. (*), (**), and (***) indicate that a condition was significantly different from the vehicle control at p < 0.05, p < 0.005, and p < 0.001, respectively.

Figure 3

Chromenes C1 and C2 induce cell death and inhibit the anchorage-dependent, colony-forming ability of MDA-MB-231 triple-negative breast cancer cells. (A,B) Determination of cell viability via cell counting. MDA-MB-231 cells were treated with vehicle (0.1% DMSO) and the indicated concentrations of chromene C1 (A) or C2 (B) for 24 h. Cell viability was assessed using a Muse cell analyzer as described in the Materials and Methods. Data represent the mean  ±  SEM of three independent experiments. (C,D) Inhibition of MDA-MB-231 colony growth. MDA-MB-231 cells were cultured for 3 days before adding freshly prepared growth medium with or without various concentrations of chromene C1 or C2.

Figure 4

Chromenes C1 and C2 activate the extrinsic apoptotic pathway in MDA-MB-231 cells. (A,B) Western blotting analysis of caspase-8 and PARP cleavage in MDA-MB-231 cells treated with the indicated concentrations of C1 (A) or C2 (B). (C) The induction of caspase 3/7 in MDA-MB-231 cells after exposure to C1 or C2 for 24 h. Caspase 3/7 activity was normalized to the number of viable cells per well and was expressed as a fold difference in the degree of activation compared to control cells. Data represent the mean  ±  SEM of three independent experiments conducted in triplicate. (*) and (**) indicate statistically significantly different mean values at p < 0.05 and p < 0.005, respectively.

Figure 5

Chromenes C1 and C2 induce multinucleation in MDA-MB-231 cells. (A) Morphological changes observed in MDA-MB-231 cells treated for 24 h with or without 0.25 μM C1 or C2. Cells were observed under an EVOS XL Core Cell Imaging System (Life Technologies, Carlsbad, CA, USA) at a magnification of 400×. (B) Treated MDA-MB-231 cells produced under the same conditions as those in section (A) were subjected to hematoxylin and eosin staining. Cells were photographed under an Olympus light microscope at 200× equipped with DP74. (C,D) Quantification of multinucleated cells treated with C1 (C) or C2 (D). (E) Upregulation of markers of double-strand DNA breaks (γH2AX) in MDA-MB-231 treated with C1 or C2. MDA-MB-231 cells were then treated with or without the indicated concentrations of C1 (A) or C2 (B) for 24 h. Next, Western blotting was performed to assess the level of DNA damage by determining the level of γH2AX accumulation using anti-phospho-H2AX (ser 139) antibody. (*), (**) and (***) indicate statistically significantly different mean values at p < 0.05, p < 0.005 and p < 0.001, respectively.

Figure 6

C1 and C2 induce a mitotic arrest in MDA-MB-231 cells. (A–C) Cell cycle distribution analysis showing MDA-MB-231 cells treated with and without C1 or C2 for 24 h. Values represent mean ± SEM of three independent experiments conducted in duplicate (* p < 0.05, ** p < 0.005). (D) Western blotting analysis of H3pSer10, a marker of the M phase, in MDA-MB-231 cells treated with or without C1 or C2.

Figure 7

Senescence and upregulation of the cell cycle inhibitors p16 and p21 in C1- and C2-treated MDA-MB-231 cells. (A–D) Detection of senescence in C1- and C2-treated cells. MDA-MB-231 cells were incubated with or without chromene C1 or C2 for 48 h and were then stained for SA-β-galactosidase activity to detect senescence. Data are represented as mean ± SEM of three independent experiments (* p  <  0.05, *** p  <  0.001). (E,F) Upregulation of p16 and p21 protein levels. Cells were treated with the indicated concentrations of C1 (E) or C2 (F) for 24 h, after which the protein levels of p16 and p21 were determined through Western blotting analyses.

Figure 8

Chromenes C1 and C2 affect the integrity of cytoskeletal microtubules. (A) Fluorescence microscopy analysis of MDA-MB-231 cells after 24 h treatment with 1 μM of C1, C2, or a negative control. Cells were stained with anti-α-tubulin, followed by an appropriate rhodamine-conjugated secondary antibody. The centrosome is indicated by a white arrowhead. (B,C) Chromene C1 and C2 inhibit microtubule polymerization in MDA-MB-231 cells. Cells were treated with or without C1 or C2 at concentrations of 0.5 and 1.0 μM for 24 h and lysed at 37 °C. Tubulin in polymers was separated from soluble tubulin by centrifugation as described in the Materials and Methods section. Equal amounts of each fraction of the supernatant (S) and the pellet (P) were analyzed by Western blotting for α-tubulin. (D–F) Docked pose of C1 and C2 in the colchicine binding site. (D,E) β-tubulin is presented in gray and α-tubulin in green. Docked C1 is presented in violet, C2 in yellow, and crystallized colchicine in blue. (F) A zoomed-in view of the boxed region in (A), with the key residues that C1 and C2 interact with.

Figure 8

Chromenes C1 and C2 affect the integrity of cytoskeletal microtubules. (A) Fluorescence microscopy analysis of MDA-MB-231 cells after 24 h treatment with 1 μM of C1, C2, or a negative control. Cells were stained with anti-α-tubulin, followed by an appropriate rhodamine-conjugated secondary antibody. The centrosome is indicated by a white arrowhead. (B,C) Chromene C1 and C2 inhibit microtubule polymerization in MDA-MB-231 cells. Cells were treated with or without C1 or C2 at concentrations of 0.5 and 1.0 μM for 24 h and lysed at 37 °C. Tubulin in polymers was separated from soluble tubulin by centrifugation as described in the Materials and Methods section. Equal amounts of each fraction of the supernatant (S) and the pellet (P) were analyzed by Western blotting for α-tubulin. (D–F) Docked pose of C1 and C2 in the colchicine binding site. (D,E) β-tubulin is presented in gray and α-tubulin in green. Docked C1 is presented in violet, C2 in yellow, and crystallized colchicine in blue. (F) A zoomed-in view of the boxed region in (A), with the key residues that C1 and C2 interact with.

Figure 9

Defective polymerization of cytoskeletal actin filaments in C1- and C2-treated MDA-MB-231 cells. Fluorescence microscopy analysis of MDA-MB-231 cells treated with 1 μM of C1 or C2 for 24 h and stained with phalloidin.

Figure 10

Chromenes C1 and C2 impair MDA-MB-231 cell motility. (A,B) Wounds were introduced in confluent monolayers of MDA-MB-231 cells before treating the cells with 0.25 μM of C1 (A) or C2 (B) or a negative control. The size of the wound was measured under a microscope (40× magnification) and was photographed using an EVOS XL Core Cell Imaging System (Life Technologies). (C,D) Quantification analysis of the wound-healing assay. Data are represented as mean ± SEM of the distance (in arbitrary units) migrated by cells after 4 and 10 h of treatment. Data are representative of three independent experiments. (* p  <  0.05, *** p  <  0.001). (E,F) Cell viability of MDA-MB-231 cells treated with C1 (E) or C2 (F) measured after 10 h treatment.

Figure 11

Inhibition of capillary-like structure formation by HUVECs in vitro. (A) Patterns of angiogenesis induced by human umbilical vein endothelial cells (HUVECs) cultured on matrigel matrix in 96-well plates with or without chromene C1 or C2. (B) Quantification of tubule formation by control and chromene-treated HUVECs. Tube formation was evaluated by the length of tube-like structures containing connected cells. (C,D) Effect of C1 or C2 on the cell viability of HUVECs. The viability of HUVECs was measured 8 and 24 h post-treatment. Data represent mean  ±  SEM of three independent experiments each conducted in triplicate (* p  <  0.05, ** p  <  0.005, *** p  <  0.001).

Figure 12

A hypothetical model for the anti-TNBC effect of chromenes C1 and C2.