Schiff Base Moiety Could be a Possible Inhibitor of Mutated Nrf2/KEAP1 Associated Cancer

Abstract

In recent studies, the cap’n’collar (CNC) bZIP transcription factor Nrf2 plays a pivotal role in cancer drug development. The Nrf2/KEAP1 pathway is the most important signalling cascade involved in the resistance of oxidative damage induced by external chemicals. The Nrf2 maintains cellular homeostasis, anti-inflammatory, and anticancer properties by activating downstream signalling pathways and their cell survival. But, a recent literature survey suggested that mutated KEAP1/Nrf2 is responsible for cancer formation by suppressing apoptosis and metabolic reprogramming. More importantly, Nrf2 is proven to contribute to the chemoresistance and radioresistance of cancer cells as well as inflammation-induced carcinogenesis. A number of Nrf2 inhibitors, particularly Schiff base molecules discovered for cancer treatment, were reviewed in this chapter. Schiff bases or azomethines are compounds formed by a condensation reaction between primary amines and aldehydes, and have various biological, medicinal, clinical, pharmacological and analytical applications. These provide a new strategy that targeting Nrf2 could be a promising therapeutic approach against cancer. This review emphasises the role of Schiff base to summarise the effects of Nrf2 in cancer, revealing its function both in cancer prevention and inhibition, to further synthesise the novel Schiff base-related anticancer treatment.

1. Introduction

Cancer is a multistage progressive disease in which a group of cells start replicating uncontrolled after a significant transformation occurs in hereditary modifications of normal cells [1, 2, 3, 4, 5]. Genes regulating reproduction, cellular growth and cell cycle get mutated and convert normal cells into cancerous cells [6, 7, 8]. Such cells escape cell cycle checkpoints, evade apoptosis, produce bigger populations, increase their mass, and start invasion into nearby tissues [9, 10]. Cancer affects almost all body systems, organs and tissues, except dead cells like hair and nails, and is continuously detected in individuals of all ages. After affecting a specific body part or system, the disease also progresses to other areas of the body. Cancer can be managed and treated effectively if diagnosed early, but it can be fatal if it remains undiagnosed and the disease progresses to an end-stage [11, 12, 13, 14]. There are more than 270 types of cancer that have a great tendency to resist chemotherapeutics, leading to a relapse after an initial cure. Therefore, the discovery of novel molecules and strategies to combat this challenge is necessary [15]. Apoptosis is a programmed cell death which prevents abnormal cells from further replication. In cancer cells, apoptosis is blocked by activating anti-apoptotic proteins that lead to the over-activation of cancer cells to grow further. Hence, activation of apoptosis in cancer cells is a significant way to develop anticancer drugs that trigger the production of pro-apoptotic enzymes and inhibit downstream signalling pathways. Thus, Schiff bases could be one of the promising drug targets for many cancers in humans [16]. Schiff bases or azomethines are compounds formed by a condensation reaction between primary amines and aldehydes and have various biological, medicinal, clinical, pharmacological and analytical applications [17, 18, 19, 20]. Aromatic primary amines can also contain additional donor functional groups like –Cl, –OH, –CH3, etc., which help enhance and regulate biological activities [21, 22, 23]. Schiff bases have been shown to exhibit various biological activities that include anticancer and anti-inflammatory [24, 25, 26].

The formation of tumours and its malignancy is tightly controlled by the external exposure of cells to harmful or protective environments. The possible link between these mechanisms is seen at the molecular level via modulation of the signalling cascade for which the initiation or inhibition can either sustain or impair cell proliferation of normal cells and increase the progression of cancer cells. The Nrf2 is involved in both normal and cancer pathways. In cancer, the Nrf2 signalling pathway is the most important for cell protection from oxidative stress and electrophilic effects [27]. The NFE2L2 quality encodes the Nrf2 protein, which is essential for the “Cap ‘n’ Collar” (CNC) group of transcriptional factors (TFs).

2. Canonical and non-canonical pathway of Nrf2

The reactive oxygen species level is the key element in the Nrf2 and KEAP1 pathways. In most of the KEAP1/Nrf2 pathway, KEAP1 undergoes point mutation, and KEAP1 helps Nrf2 release from the inhibitor and translocates into the nucleus. KEAP1 protein family belongs to the Kelch family of proteins (KLHL); KEAP1 interacts with the Nrf2-ECH homology (Neh2) domain of Nrf2 in a specific amino acid sequence containing 69–84 and is flanked by the ETGE motif [28, 29, 30]. The cullin-3 is a ubiquitination ligase, and it has a direct link with the Nrf2 proteins. The KEAP1 is responsible for the continuous degradation of lysine in the Heh2 domain and its proteasomal degradation; this evidence clearly indicates that there is a possible link between Nrf2 and its cullin-3. The epigenetic regulation in the KEAP1 leads to the over-activation of Nrf2, and the adaptor proteins called p62 are involved in the over-activation of Nrf2. The p62 is the key mediator and the target for the Nrf2 transcriptional factor and thus increases the overstimulation of Nrf2 for cell proliferation. The P21 also plays a pivotal role in the activation of Nrf2 because it has a positive regulatory effect on the Nrf2 protein. Some of the cytokines like IL-1β and TNF-α also triggered due to the production of adiponectin in adipocyte-associated macrophages, and it triggers the production of p62, a negative regulator of pro-inflammatory cytokines, p62 interacts with KEAP1 and mediates its degradation (Figure 1) [31].

Figure 1.

The role of adaptor protein p62 in Nrf2 signalling pathway – The phosphorylation of P62 by mTOR, and TAK1 leads to the stronger binding affinity towards KEAP1 due to reassemble of ETGE in Nrf2. As a result, Nrf2 translocates into the nucleus and binds on the ARE region along with MAF and finally triggers transcription.

The primary purposes for the over articulation level of Nrf2 in malignant growth are DNA mutation, epigenetic modifications, and alteration in the protein structure [32]. In some disease types, one or both KEAP1 and Nrf2 are changed, which does not permit legitimate chemical modifications between both Nrf2 as well as KEAP1. In most cancers, the regular methylation pattern of the DNA is altered, and the promoter region of KEAP1 becomes hyper methylated, resulting in the low level of transcription of the KEAP1 protein and the release of Nrf2 in specific cells (Figure 2).

Figure 2.

Mechanistic approach of KEAP1/Nrf2 in cancer – Due to the modification of KEAP1 cysteine leads to the release of Nrf2, ETGE and DLG. The bunch of Nrf2, ETGE, and DLG enters into the nucleus and interacts with ARE promoter region on the DNA along with sMAF that leads to the over activations of transcription in mutated Nrf2 cells.

3. Liver cancer and Nrf2

In hepatocellular carcinoma, the role of Nrf2 and MMP9 was well studied, and there is a direct correlation between Nrf2 and MMP9 levels in the activation of transcription factors in many human cancers. MMP9 and its role in angiogenesis are crucial in invasion and metastasis via degradation of the basal membrane and Bcl family protein. In most cancers, Nrf2 contributes to decreased BAX and over-simulations of Bcl2 [33]. The degradation of collagen IV by the help of MMP9 is responsible for the cancer cell progression, invasion, metastasis, angiogenesis and their proliferation. Apart from liver cancer, MMP9 is highly expressed in mammary carcinoma cell lines. The Gelatinase B plays a pivotal role in the progression of cancer cells via angiogenesis to stromal remodelling and metastasis. In Glioma cells, levels of MMP9 and Nrf2 are highly expressed. Once the ARE1 promoter regions are more specific to the mutated Nrf2 and MMP9 (due to KEAP1 mutation), the bunch of transcriptional factors along with Nrf2 translocates into the nucleus and activates transcription [34].

4. Cervical cancer and Nrf2

Cervical cancer is the main global health issue and it is the second most diagnosed cancer in the world, with 5 lakhs new cases diagnosed each year [35]. The main role of Nrf2 is to control the redox potential by controlling the physiological balance in the body. The antioxidant response element (ARE) is the promoter where Nrf2 initiates its first mode of action along with other transcriptional factors [36]. The antioxidant response element (ARE) contains the Leucine zipper structure, and it belongs to Cap n collar family, and its transactivation induces the gene expressions to tolerate oxidative damage and electrophilic effect. In a normal physiological process, the Nrf2 is expressed at a moderate level because the KEAP1 regulates Nrf2 in a controlled manner [37]. Due to oxidative stress or a xenobiotic stress, KEAP1 is degraded and cysteine residue gets modified in KEAP1. Due to its modification of cysteine residue in the KEAP1, Nrf2 forms heterodimer with sMAF family protein and finally interacts with the ARE region on the DNA site and activates the transcription. Hence, KEAP1 acts as a tumour suppressor role; loss of Mutation of KEAP1 regulates over expression of Nrf2 in the cancer cells [38]. The hyper methylation pattern of KEAP1 in cervical cancer is poorly understood. Breast cancer gene (BRCA) is another tumour suppressor gene that can interact with Nrf2 to increase its stability. When BRCA is absent, oestrogen restores Nrf2 activation and diminishes the expression of ROS in many mammary carcinomas. Hence, our future goal is more focused towards epigenetic regulation of KEAP1 in the cervical cancer and their inhibiting drugs, particularly Schiff bases, are more preferable [39].

5. Lung cancer and Nrf2

The hinge and latch model explains the direct indication of Nrf2/KEAP1 regulation in ETGE and DLG [40]. During this interaction, the Nrf2/KEAP1 protein adopts conformational changes with the KEAP1 molecule. In cancer cells, the redox potential is completely imbalanced, and it exhibits high ROS production due to oncogenic production by KEAP1 modification. In this contest, Nrf2 plays a significant role in both tumour suppressor as well as oncogenic roles. Both canonical and non-canonical pathways are key pathways to study about Nrf2 [41]. In the canonical pathway, the Nrf2 are low expressive and have a low affinity towards cul-3/RBX1 E3 ubiquitin-dependent degradation [42]. During its conformational change in Nrf2/KEAP1, newly formed Nrf2 interacts with ETGE to alternative subunits of KEAP1 and finally interacts with DLG for its closed conformation and its final degradation from 26 s proteasome complex (Figure 3).

Figure 3.

Canonical Pathway of Nrf2- Under normal conditions, Nrf2 ETGE/DLG motifs interact with the KEAP1 Kelch domain. After this interaction with KEAP1, CUL3/RBX1 E3 interacts with KEAP1 and then targets Nrf2 degradation through 26 s proteasome complex. Due to oxidative and electrophilic attacks, cysteine residues get altered in KEAP1 protein leading to the disruption of Nrf2/KEAP1 interaction. After the disruption of Nrf2 with KEAP1, Nrf2 accumulation in the cytosol increases and then translocates into the nucleus to form heterodimer with sMAF and activates transcription. After translocation of Nrf2, Nuclear export of Nrf2 is possible due to phosphorylation in Src family proteins such as Fyn.

In non-canonical pathways, Nrf2 is independently regulated by KEAP1. In this manner, the ubiquitin-binding autophagy receptor p62/sequestosome-1 (p62/SQSTM1) is a positive regulator of ARE in an independent redox state. The P62/SQSTM1 binds with KEAP1 through its STGE motif and promotes further degradation to increase the level of Nrf2 protein expression [43]. The glycogen synthase kinase 3 beta (GSK-3β) is another positive regulator of Nrf2. The GSK-3β can directly involve phosphorylates β-TrCP to ubiquitination of Nrf2. It also phosphorylates Fyn, and thus in turn activate Nrf2 and it helps to return to cytosol, and further degradation takes place via proteasomal degradation in cytosol. The PI3/AKT pathway is also involved in this regulatory mechanism due to its ability to interact with GSK-3β [44]. HMG-CoA reductase degradation protein 1 (HRD1) is another Nrf2 E3 ubiquitin ligase which interacts with Nrf2 and causes further degradation in ER stress [45]. The cullin4/damaged DNA binding protein-1/WD Repeat Domain 23 (CUL4/DDB1/WDR23) is another E3 ligase of Nrf2 that interacts independently of cul3/KEAP1 in Nrf2/KEAP1 pathway (Figure 4).

Figure 4.

In non-canonical pathways, Nrf2 can independently activate by p62/SQSTM1 which further activates the KEAP1 autophagy degradation through STGE binding motifs. The CUL3/SKP1 complex initiates the degradation and ubiquitination of Nrf2. In ER stress pathway, HRD1 interacts with Neh4 and 5 domains and activates Nrf2. In a recent review, CUL/DDB1/WDR23 was identified as another form of E3 ubiquitin ligase to activate and control Nrf2, and its mode of action is still not well understood.

6. Possible link between inflammation and the cancer

Our novel approach in this chapter is to study the possible links between inflammation and cancer through Nrf2 signalling cascade by activation of MAPK in both cancer and inflammation-related pathways. The cytoprotective and antioxidant expressions are regulated by Nrf2, and they protect from electrophilic and oxidative stress and inhibit the accumulations of pro-inflammatory mediators, thereby contributing to the basic way to prevent inflammation-associated cancer [46]. Based on the requirement, our project is more interested in designing new novel molecules that are basically inhibitors to suppress the Nrf2 and KEAP1 mediated pathways that lead to the over-activation of cancer for their cellular growth and metastasis.

7. Schiff base and Nrf2

Novel Schiff base molecules are found to have a wide range of pharmacological activities and attract the researchers to work on this nucleus. Schiff base and its targets with Nrf2 are not well studied; hence we are more focused towards molecular interaction of Schiff base with KEAP1/Nrf2 pathway. Literature survey indicates that many novel Schiff base molecules have been synthesised with the aim of getting compounds of significant anti-inflammatory and anticancer activities with reduced adverse effects [47]. This chapter aims to compile the reports on novel Schiff base molecules possessing anti-inflammatory and anticancer activities. This chapter also includes the reports on Schiff base of existing anticancer medication in future days.

8. Chemistry and scheme of the proposed molecule for this project (d1) and (d2)

Based on the above observation, our group has designed Schiff base molecules to target the KEAP1 and Nrf2 pathway, and we are planning to work with this project in upcoming days. For the syntheses of compounds (d1) and (d2), to a solution of 2-methoxyaniline (100 mg, 0.813 mmol) in ethanol (15 ml) an equimolar amount of 5-bromo-2-hydroxy-3- methoxybenzaldehyde (187 mg, 0.813 mmol) for (d1) or 2-hydroxy-3- methoxybenzaldehyde (123 mg, 0.813 mmol) for (d2), was added dropwise with constant stirring, in the presence of a catalytic amount of glacial acetic acid. These solutions were heated under reflux for 4 h, after which the reactions were judged from TLC to be complete [48]. The mixtures were cooled to ambient temperature, and the resulting solid products were collected by filtration and then recrystallised from N, N-dimethylformamide for (d1) and from acetonitrile for (d1 and d2).

9. Conclusion

Attributable to their great pharmacological activities, Schiff base and their analogs are fundamental and essential drugs for disease treatment, particularly those wherein oxidative stress and inflammation have been involved. This is because of the way that the majority of these Schiff bases enact the NRF2 signalling pathway, which directs hindrance of Nrf2 and KEAP1, the critical go-between in the advancement of chemotherapeutic medications. Through their NRF2-intervened cell reinforcement and mitigating impacts, Schiff base constricts the slow decrease in disease related to Nrf2 and KEAP1 mutation. Besides, the ability of these Schiff bases to restrain the articulation levels of NRF2, for example, DLG and ETGE addresses a fundamental restorative technique for human disease. In rundown, in view of NRF2-interceded activities, pharmacological profile and SAR assessment, Schiff base and their analogs address great possibility for additional advancement for anticancer medication.

Acknowledgments

Our studies and preparations of this chapter were supported by the Food technology Department, Davangere University, India.

Conflict of interest

There is no conflict of interest.