International Journal of Molecular Sciences Review Modulation of Nrf2 and NF-κB Signaling Pathways by Naturally Occurring Compounds in Relation to Cancer Prevention and Therapy. Are Combinations Better Than Single Compounds? Violetta Krajka-Ku´zniak and Wanda Baer-Dubowska * Department of Pharmaceutical Biochemistry, Poznan University of Medical Science, 4, Swi˛ecickiStreet,´ 60-781 Pozna´n,Poland; [email protected] * Correspondence: [email protected] Abstract: Nrf2 (nuclear factor erythroid 2-related factor 2) and NF-κB (nuclear factor–kappa B) signaling pathways play a central role in suppressing or inducing inflammation and angiogenesis processes. Therefore, they are involved in many steps of carcinogenesis through cooperation with multiple signaling molecules and pathways. Targeting both transcription factors simultaneously may be considered an equally important strategy for cancer chemoprevention and therapy. Several hundreds of phytochemicals, mainly edible plant and vegetable components, were shown to activate Nrf2 and mediate antioxidant response. A similar number of phytochemicals was revealed to affect NF-κB. While activation of Nrf2 and inhibition of NF-κB may protect normal cells against cancer initiation and promotion, enhanced expression and activation in cancer cells may lead to resistance Citation: Krajka-Ku´zniak,V.; to conventional chemo- or radiotherapy. Most phytochemicals, through different mechanisms, Baer-Dubowska, W. Modulation of activate Nrf2, but others, such as luteolin, can act as inhibitors of both Nrf2 and NF-κB. Despite many Nrf2 and NF-κB Signaling Pathways experimental data confirming the above mechanisms currently, limited evidence exists demonstrating by Naturally Occurring Compounds such activity in humans. Combinations of phytochemicals resembling that in a natural food matrix in Relation to Cancer Prevention and but allowing higher concentrations may improve their modulating effect on Nrf2 and NF-κB and Therapy. Are Combinations Better ultimately cancer prevention and therapy. This review presents the current knowledge on the effect Than Single Compounds? Int. J. Mol. of selected phytochemicals and their combinations on Nrf2 and NF-κB activities in the above context. Sci. 2021, 22, 8223. https://doi.org/ 10.3390/ijms22158223 Keywords: Nrf2; NF-κB; inflammation; naturally occurring compounds; cancer chemoprevention; Academic Editor: See-Hyoung Park cancer therapy; polyphenols; phytochemical combinations Received: 28 June 2021 Accepted: 26 July 2021 Published: 30 July 2021 1. Introduction Dysfunction of the tumor microenvironment is related to at least 30% of cancers. Publisher’s Note: MDPI stays neutral Inflammatory cells, macrophages, neutrophils, or lymphocytes, the elements of the mi- with regard to jurisdictional claims in croenvironment, possess the ability to generate highly reactive species, which directly published maps and institutional affil- damage DNA and lead to the initiation of carcinogenesis and subsequently promote the iations. clonal growth of the initiated cells [1]. Inflammatory cells also contribute to the process of angiogenesis. Therefore, targeting chronic inflammation and angiogenesis is considered an important strategy of both cancer chemoprevention and therapy. Nrf2 (nuclear factor erythroid 2-related factor 2) and NF-κB (nuclear factor–kappa B) signaling pathways, acti- Copyright: © 2021 by the authors. vated by various stimuli, play a central role in suppressing or inducing inflammation and Licensee MDPI, Basel, Switzerland. angiogenesis processes. This article is an open access article Moreover, although Nrf2 and NF-κB transcription factors are directly involved in distributed under the terms and many steps of carcinogenesis, cooperation with multiple other signaling molecules and conditions of the Creative Commons pathways may ultimately affect cell differentiation and proliferation [2]. Since interference Attribution (CC BY) license (https:// exists between these two pathways, their concomitant modulation, i.e., induction of Nrf2 creativecommons.org/licenses/by/ and inhibition of NF-κB in normal cells and inhibition of both in cancer cells, may be 4.0/). Int. J. Mol. Sci. 2021, 22, 8223. https://doi.org/10.3390/ijms22158223 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 8223 2 of 19 considered the best strategy of cancer chemoprevention and therapy, respectively. Modu- lation of multiple signaling pathways is an element of the therapeutic approach named anakoinosis [3]. The concept of anakoinosis is an alternative to conventional chemotherapy, which is usually based on a single target or focused on a single area of the tumor and can equally be applied in chemoprevention. Several hundreds of phytochemicals, mainly edible vegetables and fruits components, were shown to activate Nrf2 and mediate antioxidant response. One of the first in this group was naturally occurring and subsequently chemically modified triterpenoids [4]. Similar numbers of phytochemicals were shown to modulate the NF-κB pathway. In this group, curcumin (diferuloylmethane) was one of the first widely described as an inhibitor of NF-κB in cancer cells [5]. Among them, only a relatively small amount affected both pathways, and several of their modulators reached clinical trials level [6]. The activity of these phytochemicals was assessed mainly as single compounds but also in the natural food matrix. Recently, attempts were made to select the best combination of naturally occurring compounds for chemoprevention or therapeutic purposes, including modulation of Nrf2 and NF-κB pathways. This review focuses on presenting the comparison of the modulating effect of selected phytochemicals and their combinations on Nrf2 and NF-κB activities in relation to their possible chemopreventive or therapeutic applications. 2. Overview of Nrf2 and NF-κB Signaling Pathways and Their Interconnections The Nrf2 pathway is responsible for cytoprotection. Its activation leads to the expres- sion of several cytoprotective enzymes and proteins involved in cellular defense against reactive oxygen species (ROS) and electrophilic species [7]. Therefore, it represents the first line of defense against cancer initiating and promoting agents. Under normal conditions, Nrf2 is sequestered in the cytoplasm bound to its repressor Keap1 (Kelch-like ECH associated protein 1). Keap1 is rich in cysteine residues protein and is divided into five domains [8]. Polyubiquitination by Keap1/Cul3 ubiquitin ligase leads to rapid Nrf2 degradation by proteasomes [9]. Nrf2 activators, including phytochemicals, such as naturally occurring isothiocyanates, bind to SH groups of the Keap1 protein, leading to a conformational change precluding Nrf2 degradation and allowing its translocation into the nucleus. In the nucleus, Nrf2 heterodimerizes with small Maf proteins (MafF, MafG, and MafK) through its bZip domains and binds to the ARE 50-GTCACAGTGACTCAGCAGAATCTG-30 target sequence in DNA [10]. Besides the canonical pathway of activation, Nrf2 can also be regulated by phospho- rylation. Post-translational modification of Nrf2 by various protein kinases can affect the release of Nrf2 from the complex with Keap1, its nuclear translocation, and stability [9]. In one of the mechanisms of the non-canonical pathway of Nrf2 activation, the p62 (SQSTM1) protein is involved. This protein is an autophagy receptor for protein and mitochondria degradation. Similar to the interaction of Keap1 with Nrf2, p62 protein is able to interact with Keap1 by the KIR domain in p62. The interaction of Keap1 with p62 induces a depen- dent autophagy degradation of Keap1 and subsequent Nrf2 stabilization and activation in MEF and HEK293 cells [11]. The Nrf2 activation dependent on p62 increases the expression of NAD(P)H:quinone oxidoreductase (NQO1), glutathione -S-transferases (GSTs) and anti-apoptotic proteins such as Bcl-2 (B-cell lymphoma 2) and Bcl-xL (B-cell lymphoma- extra-large), decreasing ROS levels and protecting the cell against oxidative stress [10]. However, sustained Nrf2 activation by impairment of autophagy and an increase in p62 phosphorylation promotes cancer cell proliferation. Mutations in the KIR domain in p62, which prevents Keap1-p62 interaction, is associated with a ROS increase [10,12]. Epigenetic mechanisms, such as Nrf2 or Keap1 promoter methylation and microRNA have also been implicated in the complex regulation of Nrf2 pathway activity [9]. Int. J. Mol. Sci. 2021, 22, 8223 3 of 19 Although Nrf2 pathway plays an essential role in maintaining cellular redox and electrophilic homeostasis, it was also demonstrated that Nrf2 is overexpressed in cancer cells and may contribute to increased proliferation, invasion, and chemoresistance [13]. Several mechanisms are involved in the prooncogenic activation of the Nrf2 pathway in cancer cells and include both genetic alterations and epigenetic changes [14]. Evidence exists that this pathway is associated with the proliferation of cancer cells through metabolic reprogramming [15]. Thus, induction of Nrf2 activation by phytochemicals in normal cells or at the early stages of carcinogenesis is an important strategy of chemoprevention. In cancer cells, the naturally occurring inhibitors of this pathway are desired. While Nrf2 protects against reactive species and, in general, inflammatory insult, NF-κB plays a crucial role in inflammatory response and production of pro-inflammatory molecules, including cytokines and enzymes such as cyclooxygenase-2 (COX-2) and in- ducible nitric oxide synthetase (iNOS).