Stilbene Treatment Reduces Stemness Features in Human Lung Adenocarcinoma Model

Abstract

Lung cancer is among the most clinically challenging tumors because of its aggressive proliferation, metastasis, and the presence of cancer stem cells (CSCs). Natural bioactive substances have been used for cancer prevention, and, in particular, resveratrol (RSV), a stilbene-based compound with wide biological properties, has been proposed for chemoprevention. Its lesser-known analogue 4,4'-dihydroxy-trans-stilbene (DHS) has demonstrated superior activity both in cell-based assays and in mouse and zebrafish in vivo models. The present study analyzed the effects of DHS and RSV on A549 lung cancer cells, with a particular focus on stemness features and CSCs, isolated by sorting of the side population (SP). The results show that both stilbenes, especially DHS, strongly inhibited cell cycle progression. A reduction in the S phase was induced by DHS, whereas an increase in this phase was obtained with RSV. In addition, 50% reductions in the clonogenicity and soft agar colony formation were observed with the DHS treatment only. Finally, both stilbenes, especially DHS, reduced stemness marker expression in A549 cells and their sorted SP fraction. Spheroid formation, higher in SP cells than in the main population (MP), was significantly reduced after pretreatment with DHS, which was found to decrease SOX2 levels more than RSV. These findings indicate that stilbenes, and particularly DHS, affect stemness features of A549 cells and the SP fraction, suggesting their potential utility as anticancer agents, either alone or combined with chemotherapeutic drugs.

Keywords: 4,4’-dihydroxy-trans-stilbene; A549 cells; resveratrol; stemness markers.

Figure 1

Cell viability and cell cycle profile analysis after treatment with DHS and RSV. (a) Cell viability was determined by MTT assay after 24 h of treatment with different concentrations of DHS or RSV (range: 1–100 μM). (b) Cell cycle phase distribution was evaluated by flow cytometry after staining with PI. Twenty thousand cells were analyzed from each sample, and (c) statistical analysis of the mean fluorescence intensity was performed with Attune NxT software v February 4, 1627.1. The results shown are the means ± SD and are representative of three independent experiments. (* p < 0.05 and ** p < 0.01 compared with control cells; # p < 0.05 and ## p < 0.01 compared with DMSO-treated cells).

Figure 2

Anchorage-dependent and -independent growth of A549 cells treated with DHS or RSV. (a) Representative images of clonogenic efficiency of A549 cells treated with 1, 15, and 30 μM DHS or RSV for 24 h and (b) relative quantitative analysis. (c) Soft agar assay was performed treating the A549 cells with the highest concentrations of both stilbenes, and (d) the relative quantitative analysis. Soft agar images were obtained by stereomicroscopy at magnitude 4×, scale bar: 250 μm. The results shown are the means ± SD and are representative of three independent experiments. (* p < 0.05 and ** p < 0.01 compared with control cells; # p < 0.05 and ## p < 0.01 compared with DMSO-treated cells).

Figure 3

Gene and protein levels of CD44 and SOX2 in 2D A549 cells treated with 1, 15, and 30 μM DHS or RSV for 24 h. (a) Analysis of CD44 and (b) SOX2 transcripts by quantitative real-time PCR in A549 cells treated with stilbenes. (c) Representative images of CD44 and SOX2 Western blots and (d,e) the relative quantification of proteins by densitometric analysis of the Western blots and normalization to the internal loading control, β-actin. Data are the means ± SD from at least three independent experiments; values are expressed as arbitrary units (a.u.). (* p < 0.05 and ** p < 0.01 compared with control cells; # p < 0.05 and ## p < 0.01 compared with DMSO-treated cells).

Figure 4

3D spheroid culture of A549 cells and their treatment with stilbenes. (a) Representative images of spheroids obtained in suspension and treatment with 30 μM DHS or RSV in pre- and postformation conditions of A549 cells. Images obtained by stereomicroscopy at a magnitude of 1.8×, scale bar: 250 μm. (b) Numbers of spheroids obtained per well under the control and treated conditions. Analysis of (c) CD44 and (d) SOX2 transcripts by quantitative real-time PCR in A549 spheroids treated with stilbenes. (e) Representative images of CD44 and SOX2 Western blots in A549 spheroids and (f,g) the relative quantification of proteins by densitometric analysis of the Western blots and normalization to the internal loading control, β-actin. Data are shown as the means ± SD from at least three independent experiments; values are expressed as arbitrary units (a.u.). (* p < 0.05 and ** p < 0.01 compared with control spheroids; # p < 0.05 and ## p < 0.01 compared with DMSO-treated spheroids).

Figure 5

Characterization of SP fraction from A549 cells by Western blot analyses of stemness markers CD44, SOX2, and OCT4. (a) Representative images of CD44, SOX2, and OCT4 Western blots in A549 sorted cells (SP and MP) and non-sorted cells (NS). (b) Relative quantification of the proteins by densitometric analysis of the Western blots and normalization to the internal loading control, β-actin. Data are shown as the means ± SD from at least three independent experiments; values are expressed as arbitrary units (a.u.). (* p < 0.05 and ** p < 0.01 compared with NS cells).

Figure 6

Protein levels of CD44 and SOX2 in SP and MP cells after 7.5, 15, and 30 μM RSV or DHS for 24 h of treatment. (a) Representative images of CD44 and SOX2 Western blots in SP cells and (b,c) the relative quantification of proteins by densitometric analysis of the Western blots and normalization to the internal loading control, β-actin. (d) Representative images of CD44 and SOX2 Western blots in MP cells and relative quantification of proteins by densitometric analysis of the Western blots and normalization to the internal loading control, β-actin (e,f). Data are shown as the means ± SD from at least three independent experiments; values are expressed as arbitrary units (a.u.). (* p < 0.05 and ** p < 0.01 compared with control cells).

Figure 7

3D culture of sorted A549 cells (SP and MP) and their treatment with stilbenes. (a) Representative images of spheroids obtained in suspension and treatment with 30 μM DHS or RSV in pre- and postformation conditions of SP cells. (b) Numbers of spheroids obtained per well in the control and treated conditions. (c) Representative images of spheroids obtained in suspension and treatment with 30 μM DHS or RSV in pre- and postformation conditions of MP cells. (d) Numbers of spheroids obtained in each well in control and treated conditions. (e) Analysis of CD44, (f) SOX2 transcripts by quantitative real-time PCR in SP spheroids treated with stilbenes. (g) Analysis of CD44, (h) SOX2 transcripts by quantitative real-time PCR in MP spheroids treated with stilbenes. Images were obtained by stereomicroscopy at a magnitude of 1.8×, scale bar: 250 μm. Data are shown as the means ± SD from at least three independent experiments; values are expressed as arbitrary units (a.u.). (* p < 0.05 and ** p < 0.01 compared with control spheroids; # p < 0.05 and ## p < 0.01 compared with DMSO-treated spheroids).

Figure 8

Protein levels of CD44 and SOX2 in SP and MP spheroids after 30 μM DHS or RSV in pre- and postformation conditions. (a) Representative images of CD44 and SOX2 Western blots in SP spheroids and (b,c) the relative quantification of proteins by densitometric analysis of the Western blots and normalization to the internal loading control, β-actin. (d) Representative images of CD44 and SOX2 Western blots in MP spheroids and (e,f) the relative quantification of proteins by densitometric analysis of the Western blots and normalization to the internal loading control, β-actin. Data are shown as the means ± SD from at least three independent experiments; values are expressed as arbitrary units (a.u.). (* p < 0.05 compared with control spheroids; # p < 0.05 and ## p < 0.01 compared with DMSO-treated spheroids).