Resveratrol inhibits cancer cell proliferation by impairing oxidative phosphorylation and inducing oxidative stress

Highlights

• RSV preferentially blocks metastatic cancer cell growth.

• RSV deters cancer cell growth by impairing cellular respiration and glycolysis.

• RSV also promotes ROS over-production and mitophagy activation.

Abstract

The resveratrol (RSV) efficacy to affect the proliferation of several cancer cell lines was initially examined. RSV showed higher potency to decrease growth of metastatic HeLa and MDA-MB-231 (IC₅₀ = 200–250 μM) cells than of low metastatic MCF-7, SiHa and A549 (IC₅₀ = 400–500 μM) and non-cancer HUVEC and 3T3 (IC₅₀≥600 μM) cells after 48 h exposure. In order to elucidate the biochemical mechanisms underlying RSV anti-cancer effects, the energy metabolic pathways and the oxidative stress metabolism were analyzed in HeLa cells as metastatic-type cell model. RSV (200 μM/48 h) significantly decreased both glycolysis and oxidative phosphorylation (OxPhos) protein contents (30–90%) and fluxes (40–70%) vs. non-treated cells. RSV (100 μM/1–5 min) also decreased at a greater extent OxPhos flux (net ADP-stimulated respiration) of isolated tumor mitochondria (> 50%) than of non-tumor mitochondria (< 50%), particularly with succinate as oxidizable substrate. In addition, RSV promoted an excessive cellular ROS (2–3 times) production corresponding with a significant decrement in the SOD activity (but not in its content) and GSH levels; whereas the catalase, glutahione reductase, glutathione peroxidase and glutathione-S-transferase activities (but not their contents) remained unchanged. RSV (200 μM/48 h) also induced cellular death although not by apoptosis but rather by promoting a strong mitophagy activation (65%). In conclusion, RSV impaired OxPhos by inducing mitophagy and ROS over-production, which in turn halted metastatic HeLa cancer cell growth.

Introduction

The beneficial role of several phytochemicals for multiple illnesses (cardiovascular events, obesity, diabetes and cancer) has been widely documented (Szkudelska and Szkudelski, 2010; Jiang et al., 2017). Particularly, resveratrol (trans-3,5,4′-tryhydroxystilbene, RSV) a natural polyphenol found in large quantities in grapes, berries and peanuts has shown multiple positive effects on normal cells (Mukherjee et al., 2010; Baarine et al., 2011). At 0.1–50 μM doses, RSV (a) activates signaling and transcription factors involved in cell-cycle regulation, apoptosis, angiogenesis, antioxidant mechanism; and down-regulates cyclooxygenase 2 and NF-kB in immune cells (Gao et al., 2001; Švajger and Jeras, 2012); (b) shows ROS scavenger capacity (increasing NOS expression and SIRT1 activation) improving mitochondrial function in heart (Turan et al., 2012) whereas in neurons, RSV shows a protective role against Alzheimer's and Parkinson's diseases in aging models (Richard et al., 2011); and (c) inhibits initiation, promotion and progression of cancer development with apparent low toxicity for normal cells (Kueck et al., 2007; Gwak et al., 2015).

In cancer cells, RSV (15–50 μM for 24–72 h) arrests the cellular cycle, induces apoptosis and promotes a massive reactive oxygen species (ROS) production (García-Zepeda et al., 2013). RSV also decreases the mRNA content (30–85%) of several glycolytic transporters (GLUT1) and enzymes (HKII, PFK-1 and PGM) and glycolytic flux (20–60%) in human hepatocellular carcinomas (HCC-LM3 and Bel-7402), diffuse large B-cell lymphoma (OCI-Ly1 and OCI-Ly18), breast cancer (MCF-7) and brain carcinomas (H460, HCC827 and H165) (Faber et al., 2006; Gomez et al., 2013; Jung et al., 2013; Dai et al., 2015). Although the mechanisms associated with the effect of RSV on glycolysis have not been clearly elucidated, it has been demonstrated that the activation of PI-3K/Akt/mTOR signaling pathway decreases the activities of the glucose transporter and several glycolytic enzymes in cancer cells (Faber et al., 2006; Kueck et al., 2007; Iqbal and Bamezai, 2012; Gwak et al., 2015). In addition, the inhibition of the PI-3K pathway by RSV also suppresses the accumulation of the key transcriptional regulator HIF-1α and of its glycolytic targets, leading to a lower glycolytic flux (Jung et al., 2013).

On the other hand, RSV (10–50 μM) increases the total oxygen consumption (30–80%) after 24–48 h as well as the contents of respiratory chain and mitochondrial biogenesis proteins in colon cancer cells (SW620, CaCo2, HCT116, HCEC, ICT, RPA) and mouse Lewis lung carcinoma (Blanquer-Rosselló et al., 2017; Saunier et al., 2017). As the mitochondrial membrane potential only increased by 10%, the data suggested that the RSV-induced oxygen uptake increase was not coupled to ATP synthesis. However, since the oxidative phosphorylation (OxPhos) flux was not assessed in these cancer cell lines, it is difficult to ascertain whether there are in fact RSV effects on mitochondrial function. This apparent mitochondrial function improvement at low RSV doses (20 μM, 24 h) has not been observed in non-cancer (epithelial) cells (Sheu et al., 2013). In addition, the effect of RSV on OxPhos, at doses in which cancer cell death occurs, has not been systematically assessed.

Therefore, the present study analyzed whether RSV only affects glycolysis or whether it also affects OxPhos flux, the predominant energy supplier in cancer cells under normoxia (Rodríguez-Enríquez et al., 2010; Hernández-Reséndiz et al., 2015; Pacheco-Velázquez et al., 2018). To this end, an integral analysis of the glycolytic and OxPhos enzymes contents, activities and fluxes was carried out in HeLa cells. ROS levels and antioxidant metabolites and enzymes as well as mitophagy processes were also examined. Our results clearly indicated that RSV, at doses near the growth IC₅₀ values, acts as pro-oxidant drug inhibiting both OxPhos and glycolysis leading to ROS accumulation which in turn activated cellular death through mitophagy.