Inhibition of NF-κB nuclear translocation via HO-1 activation underlies α-tocopheryl succinate toxicity

Abstract

α-Tocopheryl succinate (α-TOS) inhibits oxidative phosphorylation at the level of mitochondrial complex I and II, thus promoting cancer cell death through mitochondrial reactive oxygen species (ROS) generation. Redox imbalance activates NF-E2 p45-related factor 2 (Nrf2), a transcription factor involved in cell protection and detoxification responses. Here we examined the involvement of heme oxygenase-1 (HO-1) in the regulation of nuclear factor κB (NF-κB) signaling by short exposure to α-TOS in prostate cancer cells. A short-term (4 h) exposure to α-TOS causes a significant reduction in cell viability (76%±9%) and a moderate rise in ROS production (113%±8%). α-TOS alters glutathione (GSH) homeostasis by inducing a biphasic effect, i.e., an early (1 h) decrease in intracellular GSH content (56%±20%) followed by a threefold rise at 4 h. α-TOS increases nuclear translocation and electrophile-responsive/antioxidant-responsive elements binding activity of Nrf2, resulting in up-regulation of downstream genes cystine-glutamic acid exchange transporter and HO-1, while decreasing NF-κB nuclear translocation. This effect is suppressed by the pharmacological inhibition of HO-1 and mimicked by the end-products of HO activity, i.e., bilirubin and carbon monoxide. Results suggest a little understood mechanism for α-TOS-induced inhibition of NF-κB nuclear translocation due to HO-1 up-regulation.

Introduction

α-Tocopheryl succinate (α-TOS) is one of the most potent anticancer derivatives of vitamin E [1], [2], [3] with in vitro efficacy on prostate cancer cells [4]. This derivative is thought to promote cancer cell death through mitochondrial effects that include mitochondrial complex I and II inhibition and reactive oxygen species (ROS) generation [5], [6], [7], [8]. Intracellular oxidative stress is sensed by NF-E2 p45-related factor 2 (Nrf2), a transcription factor which, binding to the antioxidant/electrophile response element (EpRE/ARE), induces the expression of phase II/antioxidant genes. Nrf2 protein is regulated by kelch-like ECH-associated protein 1 (Keap1), which binds to and sequesters Nrf2 in the cytoplasm [9]. ROS signaling induces thiol modifications in Keap1 leading to Nrf2 release and nuclear translocation. Nrf2 regulates the expression of several antioxidant enzymes, such as NAD(P)H:quinone oxidoreductase, heme oxygenase-1 (HO-1), thioredoxin reductase 1, l-cystine and l-glutamic acid exchange transporter (XCt), glutamate-cysteine ligase modifier subunit (GCLM) and glutamate-cysteine ligase catalytic subunit (GCLC) [10], [11].

HO-1 is a microsomal enzyme catalyzing the first, rate-limiting step in the degradation of heme and playing an important role in iron recycling. By cleaving heme α-meso carbon bridge, HO-1 yields equimolar quantities of carbon monoxide (CO), iron ions (Fe²⁺) and biliverdin. The enzymatic activity of HO-1 results in decreased oxidative stress [12]. HO-1 also controls cell growth and proliferation and mitigates inflammation. Hence, induction of HO-1 is a key event in cellular responses to pro-oxidative and proinflammatory insults [13]. To date, HO-1 expression and nuclear localization define a new subgroup of prostate cancer primary tumors, suggesting that HO-1 may represent a new approach to prostate cancer therapy [14].

Nuclear factor-κB (NF-κB) is an inducible, widely expressed, pleiotropic transcription factor implicated in several physiological and pathological processes, such as infection, inflammation and cancer [15]. The term NF-κB commonly refers to a p50-p65 heterodimer, which is the major Rel/NF-κB complex in most cells. In basal conditions, NF-κB is sequestered in the cytoplasm by inhibitor proteins, usually IκBα. Several NF-κB inducers phosphorylate IκBα that becomes a substrate for ubiquitination and subsequent degradation by the 26S proteasome. The released NF-κB dimer can then translocate to the nucleus and activate target genes by binding with high affinity to κB elements in their promoters [16].

Since anti-inflammatory and anticarcinogenetic agents suppress NF-κB signaling while activating Nrf2-EpRE/ARE pathway [17], [18], [19], we decided to investigate the involvement of α-TOS-induced ROS generation in the up-regulation of Nrf2-driven genes and the contribution of these genes to NF-κB inhibition in prostate cancer cell lines.