Curcumin induces p53-independent inactivation of Nrf2 during oxidative stress–induced apoptosis

Abstract

The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a master regulator of a battery of antioxidant and detoxificant genes with cytoprotective function. Since Nrf2 inactivation is necessary for the complete execution of apoptosis in the presence of extensive cellular damage caused by oxidative stress, constant activation of Nrf2 may protect tumoral cells from apoptosis. The tumor suppressor gene p53 has been suggested to participate in apoptosis-related repression of Nrf2. Thus, we studied the inactivation of Nrf2 during oxidant-induced apoptosis in a p53 dysfunctional cellular model. Using curcumin dose–response assay and time–response assay in an immortalized lymphoblastoid cell line (control line 45), we observed a time-dependent increase in apoptotic markers such as deoxyribonucleic acid (DNA) fragmentation, phosphatidylserine exposure, and caspase-3, caspase-9 and poly (ADP-ribose) polymerases (PARP) cleavage. Interestingly, at early times of exposure to a proapoptotic dose of curcumin (15 μM), we observed nuclear accumulation of Nrf2 and the expression of Nrf2 target genes, whereas at late exposure times we found a reduction of total and nuclear protein levels of Nrf2 as well as downregulation of Nrf2 target genes in the absence of p53 activation. These data suggest that apoptosis-related inactivation of Nrf2 could occur in a p53 dysfunctional background, opening the possible occurrence of p53-independent mechanism to explain Nrf2 inactivation during apoptosis.

Introduction

The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is the central mediator of a prominent signaling pathway involved in cellular protection against oxidative stress and electrophilic compounds. ¹ Under basal conditions, Nrf2 is ubiquitinated and degraded through its binding to a substrate adaptor protein by a Cul3 ubiquitin ligase complex, called Keap1 (Kelch-like ECH-associated protein 1). ^2,3 However, in the presence of environmental or intracellular hazards, Keap1-mediated degradation of Nrf2 is inhibited, which allows Nrf2 accumulation in the nucleus and the expression of cytoprotective genes, such as heme oxygenase 1 (HMOX1), NAD(P)H quinone dehydrogenase 1 (NQO1), as well as the catalytic and modulatory subunits of glutamate-cysteine ligase (GCLC and GCLM, respectively). ⁴

The importance of Nrf2 as a cytoprotective molecule has been thoroughly studied. For instance, NRF2 transcriptional deficient mice exhibited high susceptibility to developing cancer in various organs, such as stomach, skin, and bladder, after exposure to specific carcinogens. ^{5 –7} Accordingly, genetic or pharmacological activation of Nrf2 exerts cellular protection against different environmental insults including arsenic, smoke-related carcinogens, ultraviolet radiation, and aflatoxins. ^{8 –11} Moreover, the anticarcinogenic effects of natural compounds such as curcumin, resveratrol, and sulforaphane are greatly diminished in the absence of Nrf2. ^{12 –14}

Further studies have reported the aberrant activation of Nrf2 signaling pathway as a common alteration in cancer. ¹⁵ For instance, somatic mutations in the genes coding for Keap1 and Nrf2 proteins, which abrogate Keap1-mediated inhibition of Nrf2, have been described in lung, breast, gastric, colorectal, and prostate cancer. ^{16 –19} Additionally, overexpression of Nrf2 protein has been found in different tumors such as lung, breast, cervical, hepatocellular, and gastric cancer. ^{20 –24}

Persistent activation of Nrf2 in cancer was initially perceived as an adaptation of cancer cells to a highly oxidative microenvironment and as a protective mechanism against antineoplastic compounds. ²⁵ Accordingly, Nrf2 activation in cancer-derived cell lines enhanced resistance to several antineoplastic drugs including 5-fluorouracil, cisplatin, paclitaxel, and bleomycin. ^{24,26 –28} Nevertheless, recent studies suggest the participation of Nrf2 in the regulation of apoptosis as a relevant function in tumorigenesis. For instance, Nrf2 activation delayed apoptosis initiation in mouse embryonic fibroblasts, human liver carcinoma (HepG2), and retinal pigment epithelium cells. ^{29 –31} Conversely, repression of Nrf2 in neural crest cells, human bronchial epithelial cells, and cerebral cortical neurons induced apoptosis. ^{32 –34}

Although inactivation of Nrf2 seems to be important to accomplish apoptosis, the underlying mechanism has not been described yet. However, the tumor suppressor gene p53 has been proposed as a negative regulator of Nrf2 during apoptosis initiation. In fact, p53 is able to bind downstream to antioxidant response element (ARE) in the promoter region of Nrf2 target genes and suppress Nrf2-dependent transcription. ³⁵ In lung cancer cells, p53 was able to interfere Sp1 binding to the Nrf2 promoter, reducing its activity. ³⁶ However, the inactivation of Nrf2 during apoptotic process in a p53-independent manner has not been explored yet. Thus, in this study we analyzed the inactivation of Nrf2 in a p53 defective background using the indirect oxidant curcumin to induce Nrf2 activation and apoptosis.

Material and methods

Cell culture and treatments

The human lymphoblastoid cell line control line 45 (CL-45) was immortalized with Epstein–Barr virus (EBV) as previously described. ³⁷ CL-45 cells were grown in Roswell Park Memorial Institute supplemented with 10% fetal bovine serum, 50 µg/mL streptomycin, 50 U/mL penicillin, 1% (v/v) nonessential aminoacids, 1% (v/v) pyruvate, and 2 mM L-glutamine (GIBCO, New York, New York, USA) at 37°C in a humidified atmosphere containing 5% CO₂. For dose–response assays, CL-45 cells (250,000 cells/mL) were grown for 24 h, and then treated with 5, 10, 15, 20, and 30 µM of curcumin (Sigma Aldrich, St Louis, Missouri, USA) during 24 h. For time–response assays, cells were treated with 15 µM of curcumin during 6, 12, 18, and 24 h. Cells treated with 0.1% (v/v) dimethyl sulfoxide (DMSO) (curcumin vehicle) during 24 h were used as untreated control (Sigma Aldrich).

Cell viability assay

After the corresponding treatment, the number of viable cells was determined with the trypan blue exclusion test by direct counting of unstained cells on a light microscope Zeiss Axiovert 40 CFL (Göttingen, Germany). Data were expressed as a percentage relative to control cell culture, which was considered as a 100% of viability.

Cell death and apoptosis assays

Cell death was determined using LIVE/DEAD^® Fixable Dead Cell Stain Kit (Thermo Fisher, Waltham, Massachusetts, USA), according to manufacturer instructions. Briefly, 1 × 10⁶ CL-45 cells were resuspended in 1 mL of phosphate buffered saline (PBS) 1×, stained with 1 µL of red fluorescent reactive dye, and incubated on ice for 30 min in the dark. Then, cells were pelleted by centrifugation and fixed with 3.7% formaldehyde at room temperature for 15 min. Finally, cells were washed and resuspended in 1 mL of PBS with 1% bovine serum albumin. Samples were analyzed on a BD FACSAria III flow cytometer system (Beckman Coulter, San Jose, California, USA). At least 10,000 events were recorded for each sample. Data analysis was performed with BD FACSDiva Software v.6.1.3 (Beckman Coulter).

To indirectly evaluate cell death, we determined sub-G1 deoxyribonucleic acid (DNA) content employing propidium iodide staining. Briefly, after curcumin treatment, cells were washed with 1 mL of cold PBS 1× and fixed overnight at −20°C with 1 mL of ice-cold 70% ethanol. Later, cells were washed with cold PBS 1×, resuspended in 250 µL of PBS 1×, treated with RNase A (0.5 mg/mL) for 1 h at 37°C, and incubated with 10 µg/mL of propidium iodide (Sigma Aldrich) for 30 min on ice. Samples were analyzed using the BD FACSAria III flow cytometer system (Beckman Coulter); at least 20,000 events were captured. Cell cycle histograms were performed using ModFit LT 3.2 software (Verity Software House, Topsham, Maine, USA).

To evaluate apoptosis, we used Annexin V-fluorescein-5-isothiocyanate (FITC) Apoptosis Detection Kit (Abcam, Cambridge, MA, USA), according to manufacturer protocol. Briefly, after the indicated treatments cells were washed with PBS 1×, resuspended in 100 µL of Annexin-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂, pH 7.4), with 5 µL of Annexin V conjugate and incubated at room temperature for 15 min. After incubation, samples were supplemented with 400 µL of Annexin-binding buffer, mixed gently, and kept on ice until analysis on a BD FACSAria III flow cytometer system (Beckman Coulter). At least 10,000 events were recorded for each sample, and BD FACSDiva Software v.6.1.3 (Beckman Coulter) was used for data analysis.

Western blot analysis

After time response treatments, cells were harvested, washed with cold PBS 1×, and pelleted by centrifugation. Whole cell lysates were obtained using ProteoJET™ mammalian cell lysis reagent (Fermentas Life Sciences, USA), according to manufacturer instructions. Briefly, cell lysis buffer was supplemented with protease (PIERCE, Rockford, Illinois, USA) and phosphatase cocktail inhibitors (Roche, Indianapolis, Indiana, USA). Equal amounts of protein (35 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes (Hybond-P Amersham Biosciences, GE Healthcare, Buckinghamshire, UK), and blocked with 5% nonfat dry milk in TBS 1× plus 0.1% Tween 20 at room temperature for 60 min. Membranes were probed overnight at 4°C using specific antibodies against Nrf2 (H-300, dilution 1:1000), and p53 (FL-393, dilution 1:200) obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA), as well as caspase-9 (#9502, dilution 1:1000), caspase-3 (#9662, dilution 1:1000), and poly (ADP-ribose) polymerases (PARP) (#9542, dilution 1:1000) acquired from Cell Signaling Technology (Boston, Massachusetts, USA). After incubation, the membranes were washed with TBS/Tween 20 (0.1%) and incubated with rabbit or mouse horseradish peroxidase–conjugated secondary antibody (Invitrogen, Camarillo, California, USA) diluted 1:5000 at room temperature for 90 min. Signals were detected using SuperSignal West Pico or Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, Illinois, USA) employing a ChemiDoc (Bio-Rad, Hercules, California, USA), an imaging capture system. All membranes were treated with stripping buffer (100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 62.5 mM Tris-HCl, pH 6.8), incubated at 65°C for 30 min with agitation, and re-probed with anti-β-actin mouse monoclonal antibody in dilution 1:500, as a loading control (kindly gifted by Dr JM Hernández; CINVESTAV-IPN).

Immunofluorescence assays

CL-45 cells (1 × 10⁶) were grown in glass coverslips previously treated with 0.1% poly-L-lysine. After the indicated treatment, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich), washed twice with PBS 1×, and permeabilized with PBS/Triton X-100 (0.1%; AppliChem, Germany) for 5 min. Then, cells were blocked with PBS/bovine serum albumin (BSA) (2%) for 15 min. Slides were incubated, first with anti-Nrf2 (H-300), or anti-p53 (FL-393) polyclonal antibodies (dilution 1:100) for 90 min, washed with PBS/BSA (2%), and then incubated with anti-rabbit coupled FITC (Invitrogen) diluted 1:500 for 1 h at room temperature. For actin cytoskeleton staining, coverslips were incubated with 1:40 rhodamine–phalloidin dilution in PBS/BSA (1%; Life Technologies, Foster, California, USA) for 20 min and for DNA staining cells were incubated with 1 μg/mL 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) (Molecular Probes, Invitrogen) for 5 min. Slides were mounted using Vectashield (Roche, Marlborough, Massachusetts, USA) and visualized in a confocal microscopy, Zeiss LSM 510, chamber Axiovert 200M (Carl Zeiss, Germany). At least 200 positive cells were evaluated for subcellular localization analysis of Nrf2 and p53 proteins in CL-45 cells.

Reverse transcription real-time quantitative PCR

To evaluate transcriptional activity of Nrf2, we determined expression levels of Nrf2 target genes HMOX1, GCLC, GCLM, thioredoxin (TXN), and NQO1, as well as p53 activation, analyzing P21 expression by reverse transcription real-time quantitative PCR (RT-qPCR) using sinergy brands (SYBR) green as detection system and GAPDH as endogenous gene. After the specific treatments, total RNA was extracted using TRIzol (Invitrogen, Carlsbad, California, USA) method and synthesis of cDNA was performed with the TaqMan Reverse Transcription kit (Applied Biosystems, Foster, California, USA) following the manufacturer’s specifications. PCR amplification was carried out in 7900HT Fast Real-Time PCR System (Applied Biosystems), using 200 ng of cDNA and the following specific PCR primers:

HMOX1

Forward: 5′-TCCCGCAGTCAGGCAGAGG-3′

Reverse: 5′-ACGGGGGCAGAATCTTGCAC-3′

GCLC

Forward: 5′-GGGGCGATGAGGTGGAATACA-3′

Reverse: 5′-ACTCTGGTCTCCAAAGGCTAGGATG-3′

GCLM

Forward: 5′-TGCCTCCTGCTGCTGTGTGATGCC-3′

Reverse: 5′-CAGTAGC CACAGC GGCACCC-3′

TXN

Forward: 5′-GTGAAGCAGATCGAGAGCAAG-3′

Reverse: 5′-CGTGGCTGAGAAGTCAACTAACTA-3′

NQO1

Forward: 5′-ATGGTCGGCAGAAGAGCACT-3′

Reverse: 5′-AGTTCGCAGGGTCCTTCAG TTTAC-3′

P21

Forward: 5′-TGTCCGTCAGAACCC ATGC-3′

Reverse: 5′-AAAGTCGAAGTTCCATCGCTC-3′

GAPDH

Forward: 5′-CATCTCTGCCCCCTCTGCTGA-3′

Reverse: 5′-GGATGACCTTGCCCACAGCCT-3′

Statistical analysis

Data represent the average of at least three independent experiments. The ΔΔCt method was used for calculation of relative expression of induced genes according to a previous report. ³⁸ A Shapiro test was performed to determine the normality of the data followed by a Student’s t-test to determine significant differences. Differences were considered significant when p < 0.05. The tests were performed in the terminal R 3.5.1.

Results

Curcumin decreases cell viability in CL-45 cells

To establish the proapoptotic conditions of curcumin in lymphoblastoid cells, we first determined the number of viable cells through trypan blue exclusion test after dose–response assays with the phytochemical. We found a dose-dependent reduction in cellular viability after curcumin treatment for 24 h, starting at curcumin 5 µM (80%) and reaching the lowest percentage of cellular viability (10%) with curcumin 30 µM (Figure 1(a)). Then, we evaluated the percentage of cell death using a commercial fluorescent dye (LIVE/DEAD) to assess the integrity of cell membrane. Consistently, we observed a dose-dependent increase in the percentage of cell death in the cultures treated with curcumin, which started in response to 15 µM (25%) and peaked at 30 µM (65%; Figure 1(b)). In accordance with curcumin-induced cell death, we also observed the presence of a sub-G1 phase in flow cytometry assays after treatment with curcumin 15 µM (17%), 20 µM (20%), and 30 µM (20%) for 24 h (Figure 1(c) and (d)).

Figure 1.

Curcumin induces cell death in lymphoblastoid cell line. CL-45 cells were treated with different curcumin doses during 24 h. (a) Cell viability was determined employing trypan blue exclusion assay. CL-45 cells exposed to DMSO were considered as 100% of viability. (b) Percentage of cell death determined using LIVE/DEAD® system that measures membrane loss integrity. (c) Representative histograms of cell cycle profiles of CL-45 cells treated with different curcumin doses during 24 h. (d) Graphic bars showing percentage of CL-45 cells with sub-G1 DNA content from panel C. All experiments were repeated at least three times. Data are presented as mean + SD. *p < 0.05 compared with the respective control (DMSO). CL-45: control line 45.

Curcumin activation of apoptosis in CL-45 cells

Next, we evaluated the presence of apoptotic markers in time–response assays using curcumin 15 µM. We first observed a time-dependent increase of a sub-G1 phase, a strong marker of apoptosis, beginning at 12 h of treatment and reaching its maximal level after 24 h (Figure 2(a) and (b)). Similarly, exposure to curcumin induced a significant increase in the percentage of cells positive for Annexin V, starting at 12 h of exposure (Figure 2(c)). Accordingly, processing of caspase-9 and caspase-3 was clearly detected after 12 h of exposure to curcumin (Figure 2(d)). Consistent with the activation of caspases, we also observed the cleavage of PARP protein, a known substrate of caspase-3, at equivalent times of exposure than caspase-3 (Figure 2(d)).

Figure 2.

Curcumin induces apoptosis in CL-45 cells. Lymphoblastoid cell line was treated with 15 µM curcumin during 6, 12, 18, and 24 h. (a) Cell cycle profile was measured using propidium iodide staining to evaluate sub-G1 peak. (b) Graphic bars showing percentage of CL-45 cells with sub-G1 DNA content. (c) Graphs showing percentage of Annexin V positive cells determined by flow cytometry. (d) Caspase-9 and caspase-3 processing as well as PARP cleavage was analyzed by Western blot. Actin was used as a loading control. Experiments were repeated at least three times. Data are presented as mean + SD. *p < 0.05 compared with the respective control (DMSO). CL-45: control line 45.

Curcumin-induced activation of Nrf2 is repressed by apoptosis triggering

Once established the proapoptotic conditions of curcumin in lymphoblastoid cells, we evaluated the effect of apoptosis initiation on the curcumin-mediated activation of Nrf2. As expected, early exposure to curcumin induced a significant elevation in the protein levels of Nrf2. However, at late times of exposure, we observed an evident reduction in the protein levels of the transcription factor (Figure 3(a)). Accordingly, we observed an initial accumulation of Nrf2 protein in the nuclear compartment at 6 and 12 h of exposure to curcumin, followed by a sharp reduction at 18 and 24 h (Figure 3(b)). Consistently, Nrf2 target genes HMOX1, GCLC, GCLM, and TXN showed a time-dependent increase in their expression levels up to 12 h of exposure, whereas at 18 and 24 h of treatment showed a significant reduction (Figure 3(c)). Interestingly, the expression of NQO1, an Nrf2 target gene related to apoptosis, showed a sustained increase in its expression, even after the induction of apoptosis. Our data indicate that repression of curcumin-induced activity of Nrf2 occurred during apoptosis engagement.

Figure 3.

Nrf2 activation is repressed in response to curcumin treatment. CL-45 cells were exposed to 15 µM curcumin for the indicated time periods. (a) Nrf2 protein levels were evaluated in whole cell extracts by Western blot. Actin was used as a loading control. (b) Subcellular localization of Nrf2 was determined by immunofluorescence employing anti-Nrf2 (green), rhodamine–phalloidin (red) for actin cytoskeleton staining, and DAPI (blue) for DNA staining. Scale represents 5 µm. (c) Expression levels of Nrf2 target genes HMOX1, GCLC, GCLM, TXN, and NQO1 were measured through RT-qPCR. Data are shown as fold change with respect to 18 s gene and represent the mean of at least three replicates ±SEM. All experiments were repeated at least three times. *p < 0.05 compared with the respective control (DMSO). CL-45: control line 45; Nrf2: nuclear factor erythroid 2-related factor 2; RT-qPCR: reverse transcription real-time quantitative PCR; HMOX1: heme oxygenase 1; NQO1: NAD(P)H quinone dehydrogenase 1; GCLC: catalytic subunit of glutamate-cysteine ligase; GCLM: modulatory subunit of glutamate-cysteine ligase.

Apoptosis-related Nrf2 repression occurs in absence of p53 induction in CL-45 cells

Previous studies have described the ability of p53 to repress Nrf2 in the presence of DNA damage. ³⁵ Thus, we evaluated the involvement of p53 in the apoptosis-mediated inactivation of Nrf2. When we evaluate p53 activation in response to curcumin treatment in CL-45 cells, we found no significant changes in its protein levels with respect to untreated cultures, even during late times of apoptosis activation (Figure 4(a)). Similarly, nuclear accumulation of p53, a clear marker of activation in a transcription factor, was not evident at any time after curcumin exposure (Figure 4(b)). In accordance with a lack of p53 activation, we detected a very slight increase of P21 expression, a p53 target gene, only at 6 and 12 h of treatment, but not at late times of exposure, when Nrf2 inactivation and apoptosis induction take place (Figure 4(c)). These data suggest that in CL-45 cells, Nrf2 repression might occur through p53-independent mechanisms.

Figure 4.

Lack of p53 induction in CL-45 cells after curcumin treatment. Lymphoblastoid cell line was treated with 15 µM curcumin during 6, 12, 18, and 24 h. (a) Protein levels of p53 were evaluated in whole cell extracts by Western blot. Actin was used as a loading control. (b) Subcellular localization of p53 protein was determined by immunofluorescence using anti-p53 (green), rhodamine–phalloidin (red), and DAPI (blue) for actin cytoskeleton and DNA staining. Scale represents 5 µm. (c) Expression levels of CDKN1A (p21^Cip1/Waf1), a typical p53 target gene, were measured employing RT-qPCR. Data are shown as fold change with respect to 18 s gene and represent the mean of at least three replicates ±SEM. All experiments were repeated at least three times. *p < 0.05 compared with the respective control (DMSO). CL-45: control line 45; RT-qPCR: reverse transcription real-time quantitative PCR.

Discussion

The transcription factor Nrf2 has been recognized as the regulator of the main cytoprotective pathway against a myriad of environmental and intracellular hazards. ³⁹ Nevertheless, recent studies have demonstrated the participation of Nrf2 in the oncogenic process, by increasing cell survival under the presence of high levels of reactive oxygen species (ROS), promoting cellular proliferation, and importantly repressing apoptosis. ^40,41 Several studies have described the importance of inhibiting the antioxidant function of Nrf2 for apoptosis engagement after DNA damage in normal cells, and the tumor suppressor gene p53 has been proposed as an essential molecule for this inactivation. ^35,36 However, it is still unknown if mechanisms others than p53 are involved in the apoptosis-related inhibition of Nrf2. Thus, to determine the presence of alternative mechanisms to p53 for Nrf2 inactivation during oxidant-induced apoptosis, an EBV-immortalized lymphoblastoid cell, as a p53 dysfunctional model, and the phytochemical curcumin were used in this study.

Curcumin is a known inducer of apoptosis in different types of normal and tumoral cells. ^{42 –49} Importantly, previous studies showed that curcumin may induce apoptosis in EBV-immortalized lymphoblastoid cells. ⁵⁰ Thus, we first corroborated the proapoptotic properties of curcumin in CL-45 cells and found that the phytochemical induced cell death in a dose-dependent manner. This cell death showed typical apoptotic markers such as DNA fragmentation, phosphatidylserine exposure, and caspase cleavage.

We also observed that the initial curcumin-related increase in both the protein and the transcriptional activation of Nrf2 was lost in the presence of apoptosis. These data indicate that Nrf2 inactivation occurs during apoptosis. In accordance with our data, previous reports have found the inactivation of the antioxidant function of Nrf2 as a necessary step for apoptotic progression. For instance, inhibition of Nrf2 activity using the natural compound brusatol enhances ROS accumulation and induces apoptosis in pancreatic cancer cells. ⁵¹ Accordingly, constitutive activation of Nrf2 by Keap1 ablation reduced the rate of apoptosis in human cell lines. ⁵²

It is worth noting that NQO1 was the only one out of the five tested Nrf2 target genes to show no decreased expression under curcumin proapoptotic conditions. The protein NQO1 participates in quinone oxido-reduction reactions, but it is also involved in apoptosis promotion through the upregulation of the expression of both p53 and p63, which could explain its continued presence under apoptotic conditions even in the absence of p53. ^53,54

At the present, p53 has been proposed as the most important negative regulator of the antioxidant function of Nrf2 in the context of apoptosis activation. In response to extensive DNA damage, p53 binds near to the ARE region on Nrf2 target genes and suppresses Nrf2-dependent transcription. ³⁵ Additionally, p53 may interfere binding of transcription factor Sp1 to the Nrf2 promoter, reducing its promoter activity. ³⁶ However, the EBV-mediated immortalization process of our cellular model involved the disruption of p53 transcriptional activity, ⁵⁵ suggesting that the observed inactivation of Nrf2 during apoptosis may occur in a p53-independent manner. We corroborated the inactivation of p53 in the CL-45 cell line, since no augmentation of total levels of p53 protein or its nuclear translocation was observed during the apoptotic process induced by curcumin. Accordingly, the mRNA levels of p21, an important target of p53, showed no response to curcumin exposure. Taken together, these data indicated that Nrf2 inactivation in immortalized lymphoblastoid cells occurs through a p53-independent mechanism.

Further studies are needed to fully understand the cellular mechanisms involved in the apoptosis-related repression of Nrf2. However, the glycogen synthase kinase 3β (GSK3β) and the Keap1 proteins could be suitable candidates. The GSK3β kinase is a proapoptotic protein capable of promoting Nrf2 degradation after direct phosphorylation. ⁵⁶ Regarding Keap1, it has been shown to modulate a range of proapoptotic mechanisms such as the degradation of the antiapoptotic protein Bcl-2, as well as the recruitment of the proapoptotic protein Bax and the release of cytochrome C from mitochondria. ⁵⁷ It is also important to analyze in other cellular models and in the complete absence of p53 the participation of this protein in the control of apoptosis-related inactivation of Nrf2.

In conclusion, our data showed that curcumin-induced apoptosis in CL-45 cells was correlated with Nrf2 inactivation in the context of p53 dysfunctional response, suggesting the occurrence of p53-independent mechanisms to explain Nrf2 inactivation during apoptosis.