NF-kappaB Regulates Redox Status in Breast Cancer Subtypes

Abstract

Oxidative stress (OS) is an indispensable condition to ensure genomic instability in cancer cells. In breast cancer (BC), redox alterations have been widely characterized, but since this process results from a chain of inflammatory events, the causal molecular triggers remain to be identified. In this context, we used a microarray approach to investigate the role of the main pro-oxidant transcription factor, nuclear factor-kappa B (NF-κB), in gene profiles of BC subtypes. Our results showed that NF-κB knockdown in distinct BC subtypes led to differential expression of relevant factors involved in glutathione metabolism, prostaglandins, cytochrome P450 and cyclooxygenase, suggesting a relationship between the redox balance and NF-κB in such cells. In addition, we performed biochemical analyses to validate the microarray dataset focusing on OS and correlated these parameters with normal expression or NF-κB inhibition. Our data showed a distinct oxidative status pattern for each of the three studied BC subtype models, consistent with the intrinsic characteristics of each BC subtype. Thus, our findings suggest that NF-κB may represent an additional mechanism related to OS maintenance in BC, operating in various forms to mediate other important predominant signaling components of each BC subtype.

1. Introduction

Breast cancer (BC) is a heterogeneous disease composed of multiple subtypes with distinct progressions, outcomes, and molecular features. This neoplasia is the leading cause of cancer-associated death among women worldwide. In the U.S., it is expected that approximately 270,000 new cases of female BC and more than 40,000 deaths will occur in 2018 [1]. Based on gene expression profiles, human BC may be classified in three major intrinsic groups: Luminal, HER2-enriched and triple-negative (TNBC) [2]. The Luminal subtype is characterized by the expression of estrogen and/or progesterone receptors (ER and PR, respectively); HER2-enriched overexpresses HER2/neu (human epidermal growth factor receptor 2); and TNBC shows negative expression for ER, PR and HER2 [2,3]. Recent advances have led to an understanding of the biological events in BC at the molecular level, but many issues must still be addressed. Molecular components from inflammatory networks have risen as major players in BC pathogenesis, especially nuclear factor (NF)-κB-driven signaling [4].

The NF-κB family consists of five conserved proteins, RelA (p65), RelB, c-Rel, p50, and p52. All of them share the conserved Rel homology domain (RHD) responsible for DNA binding, dimerization, and association with the repressor protein IκB. The NF-κB pathway comprises the canonical or noncanonical pathway. The classical pathway is activated by proinflammatory cytokine receptors, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, the Toll-like receptor (TLR) family, T cell receptor (TCR), and B cell receptor (BCR), ultimately leading to ubiquitin-dependent degradation of the repressor IκB through phosphorylation of the IKK complex (IKKα, IKKβ and IKKγ). The repressor protein IκB degradation releases the p65/p50 dimer to translocate into the nucleus and activate the transcription of target genes [5,6]. Noncanonical signaling occurs downstream of cluster of differentiation 40 ligand (CD40L) and B cell-activating factor receptor (BAFF-R) pathways that lead to NF-κB-inducing kinase (NIK) activation, resulting in phosphorylation of the homodimer IKKα and transfer of the phosphate group to the p100 subunit to be processed into p52. As a result, nuclear translocation of p52/RelB dimer occurs [5,7].

Since cancer-related inflammation (CRI) was recognized as a hallmark of cancer, NF-κB assumed new importance in cancer studies [8,9]. NF-κB is a regulator of innate immunity and is responsible for inducing the expression of cyclooxygenase (COX)-2 and nitric oxide synthase (NOS), inflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-α, and chemokines such as CCL2 and CXCL8 [4,5]. Regarding BC, constitutive activation of NF-κB contributes to several downstream pathways that regulate cellular proliferation, angiogenesis, evasion of apoptosis, induction of the cancer stem cell phenotype, oxidative stress (OS) and invasiveness [10,11,12,13,14,15,16,17]. Hence, the NF-κB pathway is a promising target for cancer therapy.

Despite wide knowledge about NF-κB in cancer, few studies have focused on understanding its role in the distinct molecular subtypes of BC. Here, we sought to examine the role of NF-κB in BC cells representing the main phenotypes of the disease (Luminal, HER2-enriched and TNBC). Therefore, we performed a series of microarray analyses after NF-κB silencing, aiming to unravel the main downstream pathways regulated by this transcription factor in each BC subtype. Our findings demonstrate that NF-κB has an important role in the redox balance in the studied BC subtype models, suggesting that NF-κB may represent an additional mechanism related to OS maintenance in BC, operating in various forms to mediate other important predominant signaling components of each BC subtype.

2. Materials and Methods

2.1. Cell Culture and Transfection

The human breast cancer cell lines MDA-MB-231 (ATCC HTB-26, TNBC), HCC-1954 (ATCC CRL-2338, HER2) and MCF-7 (ATCC HTB-22, Luminal) were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cells were cultured in a humidified 5% CO₂ atmosphere at 37 °C.

Specific short interference RNA (siRNA) (sc-29410, Santa Cruz Biotechnology, Dallas, TX, USA) was used to knock down the gene expression of NF-κB/p65. An oligonucleotide that did not match any human coding cDNA was used as a negative control (Scramble, sc-37007, Santa Cruz). Transfections were performed using Lipofectamine LTX with Plus Reagent as per the manufacturer’s instructions (Thermo Fisher, Waltham, MA, USA). As previously described [18], 4 × 105 cells were seeded in a 6-well plate and were then transfected with 50 nM of siNF-κB or Scramble for 72 h. The transfected cells were subsequently used for further experiments.

2.2. Chemicals

Dehydroxymethylepoxyquinomicin (DHMEQ) was used to inhibit NF-κB/p65. As previously described [18], 10 µg/mL of DHMEQ was used in all experiments. All chemicals used in the oxidative stress analysis were purchased from Sigma-Aldrich.

2.3. Expression Chip Array Data Analysis

Total RNA from NF-κB-silenced and non-silenced cells was obtained using the RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Next, 100 ng of each RNA was used to synthesize and biotinylate cRNA according to the GeneChip whole transcription (WT) sense target-labeling assay (Thermo Fisher). The biotinylated cRNA was hybridized to the GeneChip human gene 1.0 ST array (Thermo Fisher), washed and stained according to the manufacturer’s protocols. The GeneChip arrays were scanned using GeneChip^® Scanner 3000. Affymetrix Expression Console™ software v.1.0 (Thermo Fisher) was used to create summarized expression values (CHP-files). Data were analyzed using the Affymetrix Transcriptome Analysis Console (Thermo Fisher), whereby differentially expressed genes with ≥1.5-fold-change were used as criteria to define up-regulation or down-regulation compared with the corresponding Scramble.

In silico analysis of gene ontology, biological processes and signaling pathways, that were altered as a consequence of NF-κB knockdown, was performed using KEGG [19] and PANTHER software [20]. A pathway enrichment analysis was performed using MetaCore software v.6.35 (Thomson Reuters, Toronto, Canada) A Venn diagram was generated using the online software of Bioinformatics & Evolutionary Genomics [21].

2.4. Real-Time Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

Two micrograms of RNA were subjected to the DNAse Amplification Grade I Kit (Thermo Fisher) for removal of DNA contamination and reverse-transcribed into cDNA using the Superscript-III kit (Thermo Fisher) per the manufacturer’s protocol. Real-Time reverse transcription PCR (RT-qPCR) was performed with SYBR Green Master Mix (Thermo Fisher) in a Rotor-Gene Q (Qiagen), and the conditions were as follows: 95 °C for 10 min, followed by 45 cycles of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. The primers used are described in

Table 1. Primer sequences of the investigated genes.

Primer	Sequence
ACTB forward	5′-TTCCTTCCTGGGCATGGAGTC-3′
ACTB reverse	5′-AGACAGCACTGTGTTGGCGTA-3′
GAPDH forward	5′-ATTCCACCCATGGCAAATTC-3′
GAPDH reverse	5′-GGCGTGGATGGGTCTTTCA-3′
BRCA1 forward	5′-GACAGAGGACAATGGCTTCC-3′
BRCA1 reverse	5′-AGCTCCTGGCACTGGTAGAG-3′
BRCA2 forward	5′-GCCGTACACTGCTCAAATCA-3′
BRCA2 reverse	5′-TTTGAAGTCATCTGGGCTGA-3′
TP63 forward	5′-GAGGTTGGGCTGTTCATCAT-3′
TP63 reverse	5′-GAGGAGAATTCGTGGAGCTG-3′
PEX1 forward	5′-TGACTGCACTTGGTCACACA-3′
PEX1 reverse	5′-CTGTCCAGGTCGAAACATTG-3′
HPGD forward	5′-CCATTTGTCCAGGCTTTGTT-3′
HPGD reverse	5′-AATCAATGGTGGGTCCAAAA-3′
ARG2 forward	5′-GGCTGAGGTGGTTAGCAGAG-3′
ARG2 reverse	5′-ACAAAGGTCTGGGCAGTGTC-3′
GPX2 forward	5′-TTGCAACCAATTTGGACATC-3′
GPX2 reverse	5′-TTTTTGGACAAGGGTGAAGG-3′
COX8C forward	5′-GGAAATGGCTGTTGGACTTG-3′
COX8C reverse	5′-ACTGCTTCAGGTTGCCTAGC-3′
OXR1 forward	5′-TTGGTGCGTTAGCATCTGAG-3′
OXR1 reverse	5′-CAAATTCTCCTCCTCCACCA-3′
TMX1 forward	5′-GCAGATTGCCTTTGTCCTTC-3′
TMX1 reverse	5′-TTCTTCATCCGCCTCTTGTT-3′

. The mean of the housekeeping genes ACTB and GAPDH was used as the reference expression for the mRNA levels. Each sample was examined in triplicate. The fold expression was calculated according to the ΔΔCt method [22].

2.5. Biochemical Analysis

Based on the microarray results, we chose to investigate oxidative-stress related genes for the validation step. The oxidative status of the total content of cells and supernatant (5 × 105 cells/mL) was determined by the quantification of total thiol content and estimation of nitric oxide levels by nitrite (NO) and lipid peroxidation profiling. The thiol content was measured by the addition of 320 µL of Tris-EDTA buffer (0.25 M/0.02 M, pH 8.2) and 40 µL of DTNB (5,5′-dithiobis(2-nitrobenzoic acid)—0.01 M) to 50 µL of sample containing cells and supernatant. The resulting yellow compound was measured at 412 nm, and the results are represented as µM of thiol [23]. For NO levels, aliquots of 60 µL of sample containing cells and supernatant were deproteinized in 50 µL of ZnSO₄ (75 mM), centrifuged at 10,000 rpm for 2 min and then mixed with 70 µL of NaOH (55 mM). After centrifugation at 10,000 rpm for 5 min, 150 µL of supernatant was added to 50 µL of glycine buffer (45 g/L, pH 9.7) and incubated for 10 min with cadmium granules (Fluka, Sigma-Aldrich, St. Louis, MO, USA) previously activated by CuSO₄ (5 mM, 5 min) to convert nitrate to nitrite. Next, of 50-µL aliquots were mixed with Griess reagent for 10 min, and the absorbance was measured at 550 nm [24]. The results are expressed as µM of nitrite. Lipid peroxidation profiling was determined using 500 µL of sample containing cells and supernatant, supplemented with 500 µL of phosphate buffer (K₂HPO₄ 30 mM in KCl 1.15%, pH 7.4, 37 °C) and 20 µL of tert-butyl hydroperoxide (3 mM) [25]. Data were quantified using a Glomax luminometer (Promega, Madison, WI, USA), and the results were analyzed with OriginLab 7.5 (OriginLab) software and expressed as relative light units (RLUs) in 20 min.

2.6. Statistical Analysis

All data are expressed as the mean ± standard deviation (SD) of at least three independent experiments and were analyzed by a two-tailed Student’s t-test or ANOVA with GraphPad Prism v.5 (GraphPad Inc., San Diego, CA, USA). p-values < 0.05 were considered statistically significant.

3. Results

3.1. NF-κB/p65 Inhibition Altered the Gene Expression Profile of Redox Signaling

We have recently described the importance of NF-κB/p65 in the regulation of epithelial-to-mesenchymal transition (EMT) in breast cancer cells [18]. For that purpose, we silenced NF-κB/p65 using the siRNA-strategy and confirmed the knockdown at both transcript and protein levels, as previously reported [18]. Here, we questioned its role in other biological processes, focusing on understanding distinct molecular breast cancer subtypes.

Initially, we evaluated the endogenous expression of NF-κB/p65 in our models, which was detectable in all studied cell lines (Figure S2). Then, we conducted the same NF-κB-silencing protocol described in our previous article [18].

To determine the global gene expression profile in NF-κB-silenced human breast cancer cells, we performed a comparative transcriptome analysis by expression chip array assay. Using this approach, transcript levels of NF-κB knockdown in MDA-MB-231, HCC-1954 and MCF-7 cells were used and compared with their scramble counterparts from each cell line. As a consequence of NF-κB/p65 knockdown, considering a ≥1.5-fold change as the cut-off to define up- or down-regulation, 2017 genes were differentially expressed in NF-κB/p65-silenced (siNF-κB) MDA-MB-231, 564 genes in NF-κB/p65-silenced (siNF-κB) HCC-1954, and 2614 genes in NF-κB/p65-silenced (siNF-κB) MCF-7 cells. The total numbers of increased and decreased genes are shown in Figure 1. Notably, more down-regulated genes were found in MCF-7 cells, while the most up-regulated genes were found in MDA-MB-231 cells.

As each studied cell line corresponded to one specific BC subtype, different genes were altered in response to NF-κB silencing. Through a pathway enrichment analysis using MetaCore software v.6.35 (Thomson Reuters, Toronto, Canada), we found Oxidative stress cellular signaling among the Top 20 pathways most representative in all differentially expressed gene lists: siNFkB MDA-MB-231 (p-value = 1.180 × 10⁻⁷), siNFkB HCC-1954 (p-value = 4.294 × 10⁻³), and siNFkB MCF-7 (p-value = 4.557 × 10⁻⁴). We also identified pivotal members of the redox balance using the PANTHER and KEGG gene ontology tools, as described in Table 2. Thus, the focus of the validation dataset was on OS experiments to understand biological significance of OS for the intrinsic BC phenotypes during NF-κB silencing.

In silico analysis of these results showed that the genes that were altered in siNF-κB MDA-MB-231 were related to redox metabolism, such as aldo-keto reduction (AKR1B10, AKR1C2), glutathione metabolism (GPX2, GPX3, GSTM1, GSTT1, GSTT2, GSTK1, GSTP1), NADH dehydrogenase activity (NDUFB9, NDUFS6), the PI3K family (PIK3C2B, PIP4K2C, PITPNC1), prostaglandin signaling (PTGER4, PTGES, PTGFRN, PTGS2), and the cytochrome P450 family (POR, CYP2S1, CYP2UI, CYP4B1, CYP4Z1, CYB5A). In addition, the potent hydroxylase xanthine dehydrogenase (XDH) was up-regulated, revealing an antioxidant effect. The altered genes in siNF-κB HCC-1954 were mainly related to the COX family (COX11P1, COX8C) and fatty acid metabolism (FAR1, LIPK). Finally, we identified members of the PI3K family (PIK3C2A, PIK3CA) and phospholipid metabolism (PLCD1, PLA2G1B, PLA2G15, PLDG, PLCB4) as the main modified genes in siNF-κB MCF-7. In addition, important genes related to the response to OS were found among the altered genes in siNF-κB MCF-7, such as OXR1 and TMX1. Although none of the altered genes were common to all lists of redox metabolism-related genes, HPGD was found in the siNF-κB HCC-1954 and siNF-κB MDA-MB-231 lists, while PEX1 was found in the siNF-κB HCC-1954 and siNF-κB MCF-7 lists (as presented in the Venn diagram, Figure 2). Interestingly, DNA repair members related to the response to OS were identified in all three altered gene lists: up-regulation of TP63 and RAD51 in siNF-κB MDA-MB-231, up-regulation of BRCA1, RB1 and XRCC4 in siNF-κB HCC-1954, and down-regulation of BRCA2, ATM and ATR in siNF-κB MCF-7.

To confirm the microarray results, we selected some altered genes for mRNA level evaluation by RT-qPCR. As shown in Figure 3, we confirmed the microarray data: TP63, GPX2, HPGD were up-regulated and ARG2 was down-regulated in siNF-κB MDA-MB-231 (Figure 3A); BRCA1, PEX1, HPGD and COX8C were up-regulated in siNF-κB HCC-1954 (Figure 3B); and BRCA2, PEX1, OXR1 and TMX1 were down-regulated in siNF-κB MCF-7 (Figure 3C).

3.2. Oxidative Stress Analyses

To evaluate the relationship between NF-κB/p65 and OS in each BC subtype models, we treated the cells with the NF-κB inhibitor DHMEQ as described previously [18]. Initially, we evaluated the basal levels of lipid peroxidation, thiol, and NO in untreated cells (Figure S1). Regarding the thiol content, we observed that all BC cells presented distinct basal levels. DHMEQ treatment resulted in reduced thiol in MCF-7 cells after both durations of exposure (from 55.83 ± 1.5 µM to 43.05 ± 1.67 µM in 24 h, p = 0.0048, Figure 4A, and from 54.3 ± 2.5 µM to 39.72 ± 3.95 µM in 48 h, p = 0.0357, Figure 4B). For HCC-1954 cells, DHMEQ treatment led to an increased thiol content only after 24 h of exposure (from 47.7 ± 1.35 µM in controls to 53.16 ± 1.27 µM in treated cells, p = 0.0420, Figure 4C). For MDA-MB-231 cells, DHMEQ reduced the thiol content after 24 h (from 18.58 ± 0.72 µM in controls to 7.84 ± 0.55 µM in treated cells, p = 0.003, Figure 4E), while it augmented the thiol content after 48 h of exposure (14.4 ± 0.64 µM in controls and 20.45 ± 0.82 µM in the DHMEQ group, p = 0.0044, Figure 4F).

Concerning the lipid peroxidation profile, it was noted that after 24 h of DHMEQ treatment, MCF-7 cells exhibited higher lipid peroxidation than the control (Figure 5A, p < 0.001), and this profile was reversed after 48 h (Figure 5B, p < 0.001). In HCC-1954 cells, DHMEQ treatment reduced lipid peroxidation after both periods of exposure (Figure 5C,D, p < 0.001). In contrast, MDA-MB231 cells presented enhanced lipid peroxidation after DHMEQ exposure after both durations of treatment (Figure 4E,F, p < 0.001).

The NO measurements indicated a reduction after 24 h of DHMEQ inhibition for MCF-7 (from 80.64 ± 1.43 µM in the control to 43.17 ± 1.82 µM in the DHMEQ group, p < 0.001, Figure 6A), followed by augmentation after 48 h of exposure (from 60.51 ± 1.06 µM in controls to 80 ± 2.18 µM in the DHMEQ group, p = 0.0013, Figure 6B). In HCC-1954 cells, only the 24-h treatment with DHMEQ altered NO (from 47.7 ± 1.35 µM in the control to 53.16 ± 1.26 µM in DHMEQ, p = 0.0420, Figure 6C). MDA-MB 231 cells did not show altered NO levels under any condition (Figure 6E,F).

4. Discussion

Oxidative stress (OS) is an indispensable condition to ensure genomic instability in cancer cells [26,27,28,29]. Thus, a redox imbalance represents an important factor for BC carcinogenesis and prognosis and is frequently associated with pro-inflammatory conditions [24,30,31,32]. Although a correlation between NF-κB and OS has been reported in cancer [33,34], its role in BC intrinsic groups has not been reported to date.

In the present study, we showed that NF-κB silencing altered the expression of a large number of genes in distinct subtypes of BC cells, some of which were shown to be important members of the redox balance through pathway enrichment analysis and gene ontology information. Relevant members involved in glutathione metabolism, prostaglandins, cytochrome P450 and cyclooxygenase were differentially expressed after NF-κB silencing in the distinct BC subtypes, suggesting a relationship between the redox balance and this transcription factor in such cells.

When we analyzed individually each differentially expressed gene list, we observed that only peroxisomal biogenesis factor (PEX) 1 and 15-hydroxyprostaglandin dehydrogenase (HPGD) were differentially expressed in more than one list. PEX1 was down-regulated in NF-κB-silenced MCF-7 and up-regulated in NF-κB-silenced HCC-1954. In these findings, because PEX1 levels are in accordance with NF-κB levels, PEX1 may play a pro-tumorigenic role in MCF-7 cells, which is supported by Bartling et al. [35] and Pang et al. [36], in opposition to what is observed in NF-κB-silenced HCC-1954 cells, as proposed by Lee et al. [37]. Different from PEX1, there was an accordance between HPGD up-regulation in NF-κB-silencing for MDA-MB-231 and HCC-1954, showing that HPGD may play a tumor suppressor role, as reported by Pham et al. [38] and Thiel et al. [39]. Additionally, high expression of HPGD is pointed out as favorable prognostic marker in renal cancer by The Human Protein Atlas [40].

Consequently, we validated the microarray dataset focusing on OS. To achieve this goal, we performed biochemical analyses assessing the antioxidant capacity, lipid peroxidation profile and NO status in BC cell subtypes and correlated such parameters with the presence or absence of NF-κB. Among the current small molecules able to inhibit NF-κB, DHMEQ is known for its effectiveness and specificity [41,42]. It directly binds to NF-κB/p65 and represses its nuclear translocation as well as DNA-binding activity [43], in addition to showing interesting effects against several tumors, including breast cancer [11,18,44]. Thus, the activity of this molecule as an NF-κB/p65 inhibitor was utilized in our study.

First, we evaluated the basal levels of untreated cells. Lipid peroxidation was similar along time (24 h and 48 h). HCC-1954 cells were the most oxidized lineage when compared to both MCF-7 and MDA-MB-231, indicating that HER2 amplification may promote this event. Studies showed that cells overexpressing HER2 exhibit more lipid peroxidation than other subtype models [45], although patients carrying HER2-amplified tumors showed reduced lipid peroxidation in plasma. This contradiction has been discussed by other authors, and they suggested that the sustained oxidative stress may favor the change HER2-positive phenotype to a HER2-negative [46]. Both MCF-7 and MDA-MB-231 exhibited a very similar pattern of lipid peroxidation (Figure S1), although experimental and clinical studies have proposed that ER positive tumors are inducers of oxidative stress and lipid peroxidation [47,48,49], while others have reported that some oxidative stress-related markers are generally down-regulated in TNBC [50,51]. MDA-MB-231 also presented low basal levels of NO and thiol content when compared to the other cells, with enhanced depletion of antioxidant content after 48 h (Figure S1).

Regarding the NF-κB-inhibition effects, our data revealed completely distinct oxidation statuses of the three studied cell lines. MCF-7 cells, in which the main regulatory axis is ER signaling, displayed higher antioxidant capacity at basal levels when compared to the other cells, as observed by the attenuated lipid peroxidation profile and high thiol content. Such a profile may be related to the antioxidant activity of estrogen-induced signaling, which blocks reactive oxygen species (ROS) [52] and increases NO synthesis [53]. Furthermore, microarray data from MCF-7 cells revealed a few relevant members of the antioxidant response, indicating that ER signaling should explain the observed redox status. Despite the power of estrogen-induced signaling, our results indicated that the initial NF-κB inhibition observed after 24 h affected the redox status of MCF-7 cells, suggesting that the integrity of this axis might contribute to the redox stability of luminal cancer. Despite the mutual NF-κB/ER interaction in MCF-7 cells, ER signaling seemed to predominate in this case [54]. Sustained NF-κB inhibition seemed to potentiate the antioxidant capacity of MCF-7, since lipid peroxidation was even more attenuated after 48 h of treatment, in association with augmented NO. It has been demonstrated that NO can inhibit NF-κB binding to DNA by modifying the transcription factor structure [55], impairing its pro-oxidant capacity.

HER2-amplified cells demonstrated an intermediary oxidative profile when compared to the other BC cells investigated at basal levels, potentially because HER2 overexpression attenuates OS in breast cancer [32,49]. The 24-h NF-κB inhibition simultaneously augmented the thiol content and reduced lipid peroxidation, revealing a role for this transcription factor in the regulation of the OS of HER2-amplified cells. HER2 amplification is known to activate NF-κB in breast cancer through its canonical pathway [15], consequently triggering OS-related responses [56]. Thus, when we silenced NF-κB in these cells, this pro-oxidant arm was probably restricted, culminating in reduced OS. After 48 h, the attenuated oxidative profile was maintained, as noted by the reduced lipid peroxidation profile exhibited by DHMEQ-treated cells.

TNBC cells showed lower levels of antioxidants at basal levels, exhibiting a pro-oxidant status after 24 h of DHMEQ exposure that was completely reversed after 48 h of NF-κB inhibition. It is anticipated that TNBC cells would have a distinct response pattern compared with the other cells due to their different histological origin. Despite the lack of a known potent signaling pathway to deal properly with redox variations [50,51], NF-κB-silenced MDA-MB-231 exhibited important components of the antioxidant arm of the redox balance in the microarray analysis, such as glutathione peroxidase (GPX2), glutathione transferases (GSTM1, GSTK1, GSTP1, GSTT1 and GSTT2), ceruloplasmin (CP) and selenoprotein (SEPW1) (Table 2). Overexpression of ROS scavenger enzymes in TNBC has been associated with a good prognosis [57]. NF-κB inhibition for 48 h restored the thiol content and equilibrated the differences in the lipid peroxidation profile observed between the treated sample and its control, although the treated group retained elevated peroxidation. These findings suggest that after NF-κB inhibition, these cells may activate a late compensatory pathway that restores the antioxidant capacity. Thus, there may be some compensatory mechanism in cells that triggers increased thiol production likely to contain stress, which is in accordance with the findings of Kim et al. [57], who reported that increased expression of enzyme scavengers is associated with a better prognosis.

It is important to highlight that cancer cells are under continuous oxidative stress and, thus, present mechanisms to overcome and adapt to this situation. Since mild oxidative stress is a pivotal condition to ensure genomic instability, cells will utilize necessary compensatory mechanisms to ensure this condition. As we showed, different breast cancer cell types exhibit distinct patterns of redox-related genes. Furthermore, NF-κB may represent an additional mechanism related to oxidative stress maintenance in these cells, which operates in various forms to mediate other important predominant signaling components present in cancer cells.