New Insights into Curcumin- and Resveratrol-Mediated Anti-Cancer Effects

Abstract

Curcumin and resveratrol are bioactive natural compounds displaying anti-inflammatory, anti-oxidant and anti-cancer properties. In this study, we compared the cytotoxic effects of these molecules and the molecular mechanisms involved against Her-2/neu-positive breast and salivary cancer cell lines. We found that both curcumin and resveratrol were efficient in reducing cancer cell survival and that they differently affected autophagy, ROS and activation of the PI3K/AKT/mTOR pathway. Moreover, we found that resveratrol and curcumin in combination exerted a stronger cytotoxic effect in correlation with the induction of a stronger ER stress and the upregulation of pro-death UPR molecule CHOP. This effect also correlated with the induction of pro-survival autophagy by curcumin and its inhibition by resveratrol. In conclusion, this study unveils new molecular mechanisms underlying the anti-cancer effects of resveratrol, curcumin and their combination, which can help to design new therapeutic strategies based on the use of these polyphenols.

Keywords: ER stress; Her-2/neu cancers; PI3K/AKT/mTOR; autophagy; curcumin; resveratrol.

Figure 1

Curcumin (CUR), resveratrol [9] and their combination (CUR/RES) induce a cytotoxic effect in TUBO and SALTO cell lines. TUBO and SALTO cells were untreated (CT) or treated with curcumin (15 μM), resveratrol (15 μM) or combination of both for 48 h, and (A) cell survival and (B) cell proliferation were evaluated by trypan blue and MTT assays, respectively. Histograms represent the mean plus standard deviation (S.D.) of three independent experiments. (C) Western blot analysis showing cleavage PARP (clPARP) in TUBO and SALTO cells treated with curcumin, resveratrol or combination of both, as above reported. Histograms represent the mean plus S.D. of densitometric analysis of three independent experiments. Actin was used as loading control (ACT). One representative experiment is shown. * p value < 0.05.

Figure 2

Curcumin promotes autophagy while resveratrol reduces this process also in combination with curcumin. (A) p62 expression level in TUBO in SALTO cells untreated or treated with curcumin, resveratrol or combination of both, at the above reported concentrations, for 48 h, as assessed by Western blot analysis. (B) LC3I/II expression was evaluated in TUBO cells treated by curcumin, resveratrol or combination of both in the absence or in the presence of chloroquine (10 µM) added for the last 10 h. One representative experiment is shown. Histograms represent the mean plus standard deviation of densitometric analysis of three independent experiments. Actin was used as a loading control. (C) Cell survival as evaluated by trypan blue assay in cells treated by curcumin, resveratrol or combination of both in the absence or in the presence of chloroquine. Histograms represent the mean plus standard deviation of three independent experiments. * p value < 0.05.

Figure 3

Curcumin and resveratrol affect PI3K/AKT/mTOR activation. (A) 4EBP1 and P70S6K mTOR targets and (B) AKT phosphorylation as evaluated by Western blot analysis in curcumin-, resveratrol- and curcumin/resveratrol-treated TUBO and SALTO cell lines, after 48 h of treatment. One representative experiment is shown. Histograms represent the mean plus standard deviation of densitometric analysis of three independent experiments. Actin was used as a loading control. * p value < 0.05.

Figure 4

ROS modulation in curcumin-, resveratrol- and curcumin/resveratrol-treated cells. (A) Intracellular ROS levels after 48 h of curcumin, resveratrol and curcumin/resveratrol treatment, as evaluated by FACS analysis after DCFDA staining. Histograms represent the mean plus standard deviation of three independent experiments. (B) Dose-dependent effect of NAC (10 and 20 mM) on AKT phosphorylation in TUBO cells treated for 48 h with curcumin, resveratrol and curcumin/resveratrol. One representative experiment is shown. Actin was used as a loading control. Histograms represent the mean plus standard deviation of densitometric analysis of three independent experiments. (C) Cell survival as evaluated in TUBO cells treated by NAC (20 mM) for 48 h. Histograms represent the mean plus standard deviation of three independent experiments. * p value < 0.05.

Figure 5

Curcumin and resveratrol combination activates the PERK/eIF2alpha/CHOP axis of UPR. (A) CHOP, BIP and (B) eIF2alpha activation was evaluated by Western blot in curcumin-, resveratrol- and curcumin/resveratrol-treated cells, as above reported for 48 h. One representative experiment is shown. Actin was used as a loading control. Histograms represent the mean plus standard deviation of densitometric analysis of three independent experiments. * p value < 0.05.