Resveratrol and acetyl-resveratrol modulate activity of VEGF and IL-8 in ovarian cancer cell aggregates via attenuation of the NF-κB protein

Abstract

Background: Key features of advanced ovarian cancer include metastasis via cell clusters in the abdominal cavity and increased chemoresistance. Resveratrol and derivatives of resveratrol have been shown to have antitumour properties. The purpose of this study was to investigate the effect of resveratrol and acetyl-resveratrol on 3D cell aggregates of ovarian cancer, and establish if NF-κB signalling may be a potential target.

Methods: Poly-HEMA coated wells were used to produce 3D aggregates of two ovarian cancer cell lines, SKOV-3 and OVCAR-5. The aggregates were exposed to 10, 20 or 30 μM resveratrol or acetyl-resveratrol for 2, 4 or 6 days. Cell growth and metabolism were measured then ELISA, western blot and immunofluorescence were utilised to evaluate VEGF, IL-8 and NF-κB levels.

Results: Resveratrol and acetyl-resveratrol reduced cell growth and metabolism of SKOV-3 aggregates in a dose- and time-dependent manner. After 6 days all three doses of both compounds inhibited cell growth. This growth inhibition correlated with the attenuated secretion of VEGF and a decrease of NF-κB protein levels. Conversely, the secretion of IL-8 increased with treatment. The effects of the compounds were limited in OVCAR-5 cell clusters.

Conclusions: The results suggest that resveratrol and its derivative acetyl-resveratrol may inhibit in vitro 3D cell growth of certain subtypes of ovarian cancer, and growth restriction may be associated with the secretion of VEGF under the control of the NF-κB protein.

Keywords: Acetyl-resveratrol; Cell clusters; IL-8; NF-κB; Ovarian cancer; Resveratrol; VEGF.

Fig. 1

Cellular metabolism relative to control of SKOV-3 aggregates after treatment with resveratrol (a) or acetyl-resveratrol (c) for 2, 4 or 6 days. Cellular metabolism relative to control of OVCAR-5 clusters after treatment with resveratrol (b) or acetyl-resveratrol (d) for 2, 4 or 6 days. * indicates statistical significance p < 0.05 compared to the control. n = 4 independent experiments in triplicate

Fig. 2

Cell growth relative to control of SKOV-3 aggregates after treatment with resveratrol (a) or acetyl-resveratrol (c) for 2, 4 or 6 days. Cell growth relative to control of OVCAR-5 clusters after treatment with resveratrol (b) or acetyl-resveratrol (d) for 2, 4 or 6 days. * indicates statistical significance p < 0.05 compared to the control. n = 4 independent experiments in triplicate

Fig. 3

The secretion of vascular endothelial growth factor (VEGF) of SKOV-3 aggregates after treatment with resveratrol (a) or acetyl-resveratrol (c) for 2, 4 or 6 days. The secretion of VEGF of OVCAR-5 clusters after treatment with resveratrol (b) or acetyl-resveratrol (d) for 2, 4 or 6 days. * indicates statistical significance p < 0.05 compared to the control. n = 4 independent experiments in triplicate

Fig. 4

The secretion of interleukin-8 (IL-8) of SKOV-3 aggregates after treatment with resveratrol (a) or acetyl-resveratrol (c) for 2, 4 or 6 days. The secretion of IL-8 of OVCAR-5 clusters after treatment with resveratrol (b) or acetyl-resveratrol (d) for 2, 4 or 6 days. * indicates statistical significance p < 0.05 compared to the control. n = 4 independent experiments in triplicate

Fig. 5

Immunofluorescent images of NF-κB and pNF-κB in frozen cut sections of SKOV-3 control aggregates (a and b) treated with resveratrol (c and d) or acetyl-resveratrol (e and f) for 6 days. NF-κB and pNF-κB in OVCAR-5 control clusters (g and h) treated with resveratrol (i and j) or acetyl-resveratrol (k and l) for 6 days. NF-κB and pNF-κB were stained red and nuclei were stained blue

Fig. 6

Western blot analysis of SKOV-3 aggregates (a) and OVCAR-5 clusters (b) treated with resveratrol for 6 days. Densitometry ratios relative to GAPDH of pNF-κB, NF-κB, pIκBα and PCNA of SKOV-3 aggregates (c, d, e and f) and of OVCAR-5 clusters (g, h, i and j). * indicates statistical significance p < 0.05 compared to the control. n = 4 independent experiments in triplicate. GAPDH expression is a house keeping protein (k)

Fig. 7

Western blot analysis of SKOV-3 aggregates (a) treated with acetyl-resveratrol for 6 days. Densitometry ratios relative to GAPDH of pNF-κB (b), NF-κB (c), pIκBα (d) and PCNA (e). * indicates statistical significance p < 0.05 compared to the control. n = 4 independent experiments in triplicate

Fig. 8

Treatment of SKOV-3 aggregates and OVCAR-5 clusters with Bay 11-7085. Cellular metabolism (a) and cell growth (b) are relative to control after 6 days treatment. The secretion of VEGF (c) and IL-8 (d) measured after 6 days of treatment. * indicates statistical significance p < 0.05 compared to the control. n = 4 independent experiments in triplicate