Toxicology in Vitro
Trigonelline inhibits Nrf2 via EGFR signalling pathway and augments
efficacy of Cisplatin and Etoposide in NSCLC cells
Chandrani Fouzder a
, Alpana Mukhuty a
, Sandip Mukherjee b,c
, Chandan Malick a
,
Rakesh Kundu a,*
a Cell Signaling Laboratory, Visva-Bharati University, Santiniketan 731 235, India b Molecular Endocrinology Laboratory, Department of Zoology, Siksha- Bhavana, Visva-Bharati University, Santiniketan 731 235, India c Department of Pharmacology and Physiology, Saint Louis University, School of Medicine, Saint Louis, Missouri 63104, USA
ARTICLE INFO
ABSTRACT
Constitutive high expression of Nrf2 (Nuclear factor erythroid 2-related factor 2) is an important contributor of
proliferation and chemoresistance in Non-small cell lung cancer (NSCLC). The aim of this present study was to
investigate the Nrf2 inhibitory effect of Trigonelline, its mechanism of action and its possible use in combinatorial treatment with anti- lung cancer drugs, Cisplatin and Etoposide. Our immunofluorescence, western blot
and real time PCR data showed that Trigonelline prevented nuclear accumulation of pNrf2 (four folds) and
downregulated Nrf2 targeted genes in both A549 and NCIH460 cells. Trigonelline inhibited Nrf2 via reduced
activation of EGFR signalling pathway and its downstream effector ERK 1/2 kinase. Trigonelline in combination
with Cisplatin/Etoposide abrogated proliferation of NSCLC cells (A549, NCIH460 and NCIH1299) without
showing any visible cytotoxicity to the normal lung epithelial cell (L132). Combinatorial treatment of Trigonelline with Cisplatin/Etoposide showed strong synergism at a sufficiently low concentration than the IC50
values of these drugs. Nrf2 knockdown experiment in NSCLC cells obliterated the effect of Trigonelline- Cisplatin
and Trigonelline-Etoposide combination, indicating the role of Nrf2 inhibition in augmenting drug sensitivity.
Our study demonstrated that Trigonelline blocks Nrf2 activation and its nuclear translocation via inhibition of
EGFR signalling pathway. It has improved responsiveness of NSCLC cells for Cisplatin and Etoposide and could be
a promising choice for lung cancer therapy.
1. Introduction
Non-small cell lung cancer (NSCLC) is the leading cause of cancerrelated death worldwide. 75–80% of lung cancer cases constitute
NSCLC and every year, more than 1 million new NSCLC cases are
diagnosed (Siegel et al., 2015). Conventional chemotherapy and radiotherapy continue to be the standard regime for NSCLC patients who lose
their chances of surgery (Hirsch et al., 2013; Tian et al., 2016a).
Recently, several novel chemotherapeutic agents have been used for
lung cancer therapy before or after surgery. Even so, the overall 5-year
survival rate is only 20% due to poor prognosis and development of drug
resistance (Wang et al., 2015). According to The Cancer Genome Atlas,
alterations in the Nrf2-Keap1 pathway occur in several types of cancer
including NSCLC (Padmanabhan et al., 2006; Shibata et al., 2009).
Nuclear factor erythroid 2-related factor 2 (Nrf2), a basic leucine zipper
transcription factor, primarily resides in the cytoplasm and interacts
with its actin-associated inhibitory cytosolic protein Keap1 (Kelch-like
ECH-associated protein 1). Keap1 correspondingly functions as a substrate adaptor protein for Cul3-Rbx1-dependent E3 ubiquitin ligase
complex which ubiquitinates and degrades Nrf2 for maintaining its
steady- state level in cells (Jaramillo and Zhang, 2013). Several studies
stated that A549 and NCIH460 (NSCLC) cell lines have homozygous
point mutation in Keap1 which reduced Nrf2 degradation. Although
mRNA expression of Nrf2 remains unaltered, Keap1 mutation persuaded
Nrf2 nuclear translocation and increased transcription of AREresponsive Keap1/Nrf2 pathway downstream genes (Gao et al., 2020;
Tian et al., 2016b). Activation of EGFR (epidermal growth factor receptor), PI3K (phosphatidylinositol-3-kinase), PKC δ, JNK and ERK 1/2
leads to Nrf2 phosphorylation at serine 40 residue (Lin, 2017; Xu et al.,
2013). Phosphorylated Nrf2 then translocate to the nucleus and orchestrates the transcription of a battery of antioxidant genes, drugmetabolizing enzymes and drug efflux pumps by binding to the ARE
* Corresponding author.
E-mail address: [email protected] (R. Kundu).
Contents lists available at ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
https://doi.org/10.1016/j.tiv.2020.105038
Received 17 April 2020; Received in revised form 22 September 2020; Accepted 22 October 2020
Toxicology in Vitro 70 (2021) 105038
2
(antioxidant response element) region situated in the promoter of these
genes (McMahon et al., 2001). In addition, NSCLC cells with elevated
levels of Nrf2 become less sensitive to common chemotherapeutic agents
such as Cisplatin, Carboplatin, Etoposide, 5-Fluorouracil or Doxorubicin
during chronic treatments (Jaramillo and Zhang, 2013).
A pivotal cause of this drug insensitivity is the occurrence of multidrug resistance (MDR), for which NSCLC become resistant to a broad
spectrum of chemotherapeutic agents (Furfaro et al., 2016). Moreover,
these commonly prescribed drugs have several side effects in a variety of
patients when administered as single drug chemotherapy. These adverse
effects can be overcome by combination of two or more drugs and is a
new effective strategy to strike multiple cancer targets.
In search of new therapeutic regimens for certain cancers, Nrf2 inhibitors are recently gaining attention for their anticancer effect. It has
been postulated that Nrf2 inhibitors could make cancer cells susceptible
to chemotherapeutic drugs by reducing nuclear accumulation of Nrf2
and subsequent downregulation of Nrf2 target genes. In this present
report, we have demonstrated the Nrf2 inhibitory effect of Trigonelline,
a plant alkaloid that could effectively reduce Nrf2 nuclear accumulation
and expression of its target genes in NSCLC cells. Trigonelline is the
major bioactive component present in fenugreek and green coffee beans
and has been reported to exert antibacterial, antidiabetic and antitumor
effects (Zhou and Zhou, 2012).
A study on pancreatic carcinoma cell lines (Panc1, Colo357 and
MiaPaca2) indicated the Nrf2 inhibitory effect of Trigonelline and its
anti-cancer drug induced apoptosis. The study also revealed greater antitumor responses of Trigonelline towards anti-cancer drug treatment in
tumor bearing mice (Arlt et al., 2013). Further, Trigonelline has also
been found to inhibit hepatocellular carcinoma cell migration following
Raf/ERK/Nrf2 signalling pathway (Liao et al., 2015). Nrf2 silencing by
Trigonelline induces Artesunate mediated sensitivity in head and neck
cancer cells (Roh et al., 2017). However, anti- lung cancer effect of
Trigonelline and its efficacy in combinatorial treatment with commercially available drugs has not yet been reported and thus our aim of
interest.
The present study demonstrated that Trigonelline effectively inhibits
EGFR signalling pathway activation and Nrf2 nuclear translocation.
Nrf2 inhibition by Trigonelline, reduced proliferation of NSCLC cells and
induced apoptosis when incubated with Cisplatin/Etoposide. The effects
of combinatorial treatment in different schedules had been tested to
assess the efficacy of the regimen. Besides, our results indicated that
Trigonelline has no visible toxicity to the normal human lung cells
(L132). These findings thus enhance the possibility of Trigonelline as
therapeutic regimen for its safe and effective use in treatment of nonsmall cell lung cancer.
2. Materials and methods
2.1. Chemicals and reagents
Cell culture materials and were purchased from Gibco-BRL/Life
Technologies, USA. Primary antibodies for Nrf2, A10 (sc-365,949),
Keap1, H-190 (sc-33,569), HO1 (sc-7695), Phospho Src, Tyr 419 (sc-
81,521), phospho EGFR, Tyr 1173 (sc-377,547), BAX (sc-6236), BCl2
(sc-492), Caspase 3 (sc-1225), PARP1 (sc-25,780) and H3 (sc-56,616)
were obtained from Santa Cruz Biotechnology, Dallas, Texas, USA. The
phospho Nrf2, Ser 40 (PA5-67520), NQO1 (PA5-21290), EGFR (PA1-
1110) and α-Tubulin (236–10,501) were procured from Thermo Fisher
Scientific, USA. ERK ½ (BB-AB0135) and β-actin (BB-AB0024) were
from BioBharati LifeScience Pvt. Ltd., India and phospho ERK ½, Thr
202/Tyr 204 (9101S) was purchased from Cell Signalling Technology,
USA. Alkaline phosphatase conjugated anti-rabbit (A3687), anti-mouse
(A3562), anti-goat (A4062) secondary antibodies and all other chemicals were obtained from Sigma-Aldrich, St. Louis, USA.
2.2. Cell culture
The lung cancer adenocarcinoma cell lines A549, NCIH460, NCI
H1299 and human embryonic pulmonary lung epithelial cells (L132)
were procured from the National Centre for Cell Science, Pune, India.
NCCS performs authentication of cell lines via short tandem repeat
(STR) profile analysis. So, we did not carry out additional testing to
authenticate the cell lines, but its morphology and behavior were
consistent with NCCS descriptions. Cells were cultured in advanced
DMEM containing Earle’s salts and non-essential amino acids in addition
with 10% (v/v) FBS (fetal bovine serum), penicillin (100 units/ml) and
streptomycin (100 μg/ml) in a humidified 5% CO2 atmosphere at 37 ◦C.
Confluent cells were sub-cultured by trypsinization and were subsequently seeded in culture plates containing DMEM with essential supplements (Mukhuty et al., 2017).
2.3. Drugs and treatments
Trigonelline (T5509, Sigma-Aldrich) was dissolved in molecular
biology grade water to a stock concentration of 1 M and stored at
− 20 ◦C. Cisplatin (P4394, Sigma-Aldrich) was dissolved in 0.15 M NaCl
to a stock concentration of 1 mg/ml and stored at 4 ◦C. The Cisplatin was
freshly prepared before use and not preserved for greater than 1 week.
Etoposide (E1383, Sigma-Aldrich) and PP2 (P0042, Sigma-Aldrich) was
dissolved in DMSO and then diluted to the required concentrations, with
incomplete cell culture medium. The final concentration of DMSO was
not greater than 0.1%. This concentration of solvent (DMSO) had no
effect on cell viability (measured by MTT assay).
2.4. Cell viability assay
To evaluate the efficacy of antineoplastic drugs on the survivability
of A549, NCIH460, NCI H1299 and L132 cell lines, MTT assays were
performed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazoliumbromide (MTT) colorimetric assay kit (Milipore, Temecula, CA,
USA) following manufacturer’s instructions. The cells were seeded in
96-well plates at a concentration of 1× 104 cells per well in a volume of
200 μl of cell culture medium. After 24 h, Trigonelline, Cisplatin and
Etoposide were added alone and the plates were kept in the incubator for
72 h (IC50 determination). In a separate experiment, Trigonelline was
added in combination with Cisplatin/Etoposide and incubated for 48 h
(combinatorial dose determination). The insoluble purple formazan
crystals were dissolved in acidified isopropanol (0.04 N HCl in isopropanol) and absorbance was quantified by measuring at 490 nm in a
microplate reader.
2.5. Combination index (CI) measurement
To evaluate the combinatorial effects of Trigonelline and/or
Cisplatin and Etoposide on survivability of A549 and NCIH460 cell lines,
the Combination Index (CI) was calculated using CompuSyn software
(ComboSyn, Inc., Paramus, NJ, USA). The calculated CI value is the
indicator of the degree of interaction between different drugs. CI values
<1 indicate synergistic effect, CI values = 1 indicate additive effect,
while CI values >1 indicate antagonistic effect.
2.6. Sub-cellular fractionation
Nuclear and Cytosolic fractions were prepared following Millipore
nuclear extraction kit (2900).
2.7. Electrophoresis and immunoblotting
Immunoblot analysis was performed by following Pal et al. 2012.
After the treatment, cells were lysed, centrifuged for 10 min at 10,000g
at 4 ◦C and the supernatant was collected. Protein contents of the
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supernatant were determined following Lowry et al. method. 80μg of
control and treated cell lysates protein were resolved using 10% SDSPAGE and then transferred to PVDF membrane (Millipore, Bedford,
USA) by Wet/Tank Blotting System (Bio-Rad Laboratories Inc., Hercules,
USA). The membranes were incubated with different primary antibodies
at 1:1000 dilutions overnight at 4 ◦C followed by secondary antibodies
conjugated with alkaline phosphatase at 1:2000 dilutions for 2 h. The 5-
bromo 4-chloro 3-indolyl phosphate/nitrobluetetrazolium (BCIP/NBT)
was used as a substrate to develop the protein bands. The intensity of the
protein bands was analysed by Image Lab Software (Bio-Rad Gel
DocTMXR+, Philadelphia, PA, USA) (Mukhuty et al., 2017; Seal et al.,
2012).
2.8. Immunofluorescence study
A549 and NCIH460 cells were seeded on sterile lysine coated glass
coverslips in a 6 well cell culture plate. At the end of the treatment, an
immunofluorescence study was conducted following Abcam’s protocol.
Cells were fixed with chilled methanol, permeabilized with Triton-X-100
containing PBS and were then incubated with pNrf2 (Ser 40) or pEGFR
(Tyr 1173) primary antibody (1:100) for 2 h followed by incubation
with FITC-conjugated secondary antibody (1:500) for another 1 h at
room temperature. The cells were counterstained with DAPI nuclear
stain, mounted with Dabco 33-LV mounting medium and observed
under confocal laser scanning microscope [Leica TCS SP8].
2.9. siRNA transfection
Human Nrf2 siRNA (Santa Cruz Biotechnology) was transfected in
cells using LipofectamineTM 2000 (Invitrogen) following the manufacturer’s protocol for 48 h.
2.10. Quantitative real-time polymerase chain reaction (qRT-PCR)
analysis
Total RNA isolation and RT-PCR analysis were performed as
described previously (Seal et al., 2012). For measurement of cDNA
corresponding to human Nrf2 mRNA, forward primer was 5′
-ATAGCTGAGCCCAGTATC-3′
, the reverse primer was 5′
-CATGCACGTGAGTGCTCT-3′
; Keap1 forward primer was
CATCCACCCTAAGGTCATGGA, reverse primer was GACAGGTTGAAGAACTCCTCC, NQO1 forward primer was AGGCTGGTTTGAGCGAGT,
reverse primer was TTGAATTCGGGCGTCTGCTG, HO1 forward primer
was AACTTTCAGAAGGGCCAGGT, reverse primer was
CTGGGCTCTCCTTGTTGC. gapdh (glyceraldehyde-3-phosphate dehydrogenase) was simultaneously amplified in separate reactions. The Ct
value was corrected using corresponding gapdh controls.
2.11. DNA fragmentation study by DAPI staining
Both A549 and NCIH460 cells grown on coverslips, incubated with or
without Trigonelline, Cisplatin, Etoposide were fixed in chilled methanol for 10 min and stained with DAPI (4′
,6-diamidino-2-phenylindole,
D9542) (10 μg/ml) for 20 min in dark at room temperature (RT). The
cells with fragmented nucleus were observed under confocal laser
scanning microscope [Leica TCS SP8] and counted along with cells with
an intact nucleus to calculate the number of apoptotic cells concerning
normal cells.
2.12. JC-1 mitochondrial membrane potential assay
The mitochondrial membrane potential (MMP) of the cells was
determined by JC-1 dye, JC-1 mitochondrial membrane potential assay
kit, Cayman Chemical Company (Ann Arbor, MI, USA) as per manufacturer’s protocol. Cells were counterstained by Hoechst 33258 and the
shift of fluorescence of JC-1 dye from red to green was observed under
confocal laser scanning microscope [Leica TCS SP8].
2.13. Apoptosis study by HOECHST/PI staining
The cells were trypsinized, centrifuged at 200 ×g and supernatants
were stained with Hoechst 33342 (B2883) followed by Propidium Iodide
(P4170) before observing under inverted fluorescence microscope
[Leica Dmi8]. Cell apoptosis was determined by comparing the PIpositive cells against Hoechst-positive cells.
2.14. Clonogenic assay
To investigate the effects of chemotherapy on anchorage independent growth of NSCLC cell lines, clonogenic assay was performed after
seeding the cells in new plates (after treatment) and incubated further
with drug free medium for 14 days thereafter. Colonies were stained
with methanol/crystal violet and were counted manually. All the experiments were performed in triplicates.
2.15. Apoptosis analysis
Apoptosis of A549 and NCIH460 cells were performed by following
manufacturer’s instruction. The Annexin V-FITC apoptosis detection kit
(BD Biosciences, San Jose, CA, USA) was used, apoptotic cells were
evaluated using flow cytometer (BD Accuri) and analysed.
2.16. Statistical analysis
Data were derived from at least three independent experiments and
the statistical analysis was performed with GraphPad prism 4.0
(GraphPad Software Inc. La Jolla, CA, USA). All values are means±s.e.
m. The p values ˂0.05 were regarded as statistically significant.
3. Results
3.1. Trigonelline inhibits Nrf2 nuclear import, but not its export
Constitutive Nrf2 activation is known to play a key role in NSCLC
cells proliferation and progression (Singh et al., 2006). Here, we have
studied the expression level of Nrf2 and its target gene NQO1 (NAD(P)H
quinine Oxidoreductase 1) and HO1 (Heme oxygenase 1) in NSCLC cells
(A549 and NCIH460) and our results showed their higher expression in
comparison to normal lung cells, L132 (Fig. S1A). The Real-Time PCR
results also demonstrated increased levels of Nrf2, NQO1 and HO1
mRNA in NSCLC cells than L132 cells (Fig. S1B). Treatment of both A549
and NCIH460 cells with different concentrations of Trigonelline (10, 25,
50 and 100 μM) for 12 h showed localization of pNrf2 (Ser 40) in the
nucleus at initial time points, but gradually declined at 50 μM dose
compared to the untreated ones. Decreasing level of nuclear pNrf2
corresponded with the increasing level of pNrf2 in the cytoplasm
(Fig. S1C-F). Further, treatment of cells with tBHQ (tert- Butylhydroquinone), a known Nrf2 inducer, greatly enhanced the nuclear level
of pNrf2 at 12 h. However, pre-treatment of Trigonelline at 50 μM dose
for 1 h showed a decline in nuclear pNrf2 level even on tBHQ stimulation
(12h) (Fig. 1A-D). Next, immunoblot analysis proved that Trigonelline
(50 μM) treatment for 24 h did not cause any change in total cellular
Nrf2 protein level. However, the protein levels of Nrf2 target genes
NQO1 and HO1 clearly declined after Trigonelline treatment (Fig. 1E),
indicating non-availability of nuclear Nrf2 was the cause for their subdued expression. Again, cells treated with Trigonelline (50 μM) for 12 h
showed reduced pNrf2 protein level in the nucleus compared to the
cytosolic fraction (Fig. 1F). Trigonelline pre-treatment for 1 h followed
by tBHQ stimulation for another 12 h showed reduced pNrf2 level in the
nucleus (Fig. 1G-H). It is known that nuclear export of Nrf2 has been
mediated through exportin 1 (XPO1) also known as Chromosomal
maintenance 1 (crm-1) protein transporter (Jaramillo and Zhang, 2013).
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Now the question comes, whether these reduced level of pNrf2 inside
nucleus was due to inhibition of Nrf2 nuclear import or increased nuclear export? To address this question, cells were pre-treated with crm-1
protein inhibitor Leptomycin B to assess whether Trigonelline functions
through nuclear export mechanism, a decline in the pNrf2 level in the
nucleus has been observed even after tBHQ stimulation (Fig. 1I). However, a rise of pNrf2 in the cytoplasm of Leptomycin B and tBHQ treated
cells suggested that Trigonelline is not involved in the Nrf2 export system from the nucleus. In all the cases nuclear and cytosolic Nrf2 level
was found unaltered. Moreover, Real-Time PCR data of Nrf2, Keap1 and
Nrf2-target genes, NQO1 and HO1 revealed that except Nrf2, the mRNA
levels of NQO1 and HO1 declined by more than 5-folds in both A549 and
NCIH460 cells. The mRNA level of Keap1 remained unaltered
throughout the experiments (Fig. 1J). Collectively the above findings
illustrated that Trigonelline specifically hampers Nrf2 nuclear import in
NSCLC cells.
3.2. Trigonelline inhibits Nrf2 activation and cell proliferation via EGFR
signalling pathway
Several upstream kinases like EGFR, ERK1/2 and Src are known to
regulate Nrf2 phosphorylation and nuclear accumulation in NSCLC cells
(Xu et al., 2013). We have compared the phosphorylation status of EGFR
in A549 and NCIH460 cells in Trigonelline incubation in both dose (10,
25 and 50 μM, 12 h) and time (50 μM for up to 24 h) dependent manner.
Western blot analysis indicated that Trigonelline reduced the level of
phospho- EGFR (Tyr 1173) and its downstream component phospho
ERK (Thr 202/Tyr 204) with a corresponding reduction of phospho-Nrf2
(Ser 40) in the above treated cells. (Fig. 2A and B). Similar results were
obtained in epidermal growth factor (EGF) stimulated cells where
Trigonelline treatment continued to deactivate EGFR signalling
pathway, as could be seen from reduction in its phosphorylation status
and also from the phosphorylation status of the downstream kinases,
ERK and Nrf2 (Ser 40), in both the A549 and NCIH460 cells (Fig. 2C). Src
functions as upstream kinase of EGFR and transmits activating signal to
Nrf2 (Ishii and Warabi, 2019). Pharmacological inhibition of Src kinase
by PP2 treatment diminished the tyrosine phosphorylation of EGFR and
ERK kinase in A549 and NCIH460 cells (Fig. 2D). We have used EGFR
specific inhibitor AG1478 (10 μM) in combination with Trigonelline (50
μM) for more robust inhibition of Nrf2 phosphorylation in EGF (GibcoBRL) stimulated cells. Immunoblot and immunofluorescence data
showed that induction of EGFR, ERK 1/2 and Nrf2 were reduced in the
above treatment (Fig. 2E-G). Taken together, these results suggested that
Trigonelline blocks EGFR activation and impairs nuclear translocation
of Nrf2.
.
3.3. Suppression of Nrf2 promotes apoptosis and increase the efficacy of
Cisplatin and Etoposide
Suppression of Nrf2 by Nrf2 siRNA resulted in apoptosis of NSCLC
cells. Cell viability assay, DNA fragmentation analysis (DAPI staining)
and western blot of PARP1 confirmed this apoptotic phenomenon
(Fig. S1G-J). To evaluate the anti-proliferative effects of Trigonelline,
Cisplatin and Etoposide, we performed a series of MTT assays in A549,
NCIH460 and NCI H1299 cells. All the cell lines were treated with
Fig. 1. Trigonelline treatment reduced the nuclear accumulation of Nrf2 protein in NSCLC cells. (A and B) Both A549 and NCIH460 cells were treated with 50 μM of
Trigonelline (Trig) for 12 h, 50 μM tBHQ for 12 h alone or after pre-incubating the cells with 50 μM of Trigonelline for 1 h and were subjected to immunofluorescence
staining with anti-phospho Nrf2 (Ser 40) antibody. (C and D) Quantification of nuclear pNrf2 in control versus different conditions treated cells (*P < 0.05 vs
untreated A549 and #P < 0.05 vs untreated NCIH460). (E) After incubating both A549 and NCIH460 cells with 50 μM of Trigonelline (24 h), total cell lysates from
both the treatments were immunoblotted for Keap1, Nrf2 and NQO1 proteins. α-tubulin was used as loading control. The cells were treated with 50 μM tBHQ (G) for
12 h, 50 μM Trigonelline (F) for 12 h alone or 12 h tBHQ administration after pre-incubating the cells with 50 μM of Trigonelline (Trig) for 1 h (H). (I) Both of the
NSCLC cells were treated with/without 50 μM of Trigonelline for 1 h and were then further incubated with 50 μM tBHQ and 20 ng/ml of LMB (crm1 inhibitor) in
combination for an additional 12 h. Cytosolic and nuclear extract were prepared from all these experiments and submitted to western blot analysis of pNrf2, Nrf2, H3,
and α-Tubulin. Representative results out of four was shown here. (J) Trigonelline reduced the mRNA level of Nrf2 target genes. A549 and NCIH460 cells were
treated with 50 μM of Trigonelline for 12 h and subjected to qRT-PCR (§P < 0.05 vs untreated A549 and $P < 0.05 vs untreated NCIH460).
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Fig. 2. Trigonelline inhibits activation of the EGFR-ERK1/2 kinase signalling pathway that positively regulates Nrf2 nuclear translocation. (A) A549 and NCIH460
cells were incubated with Trigonelline at varying concentration (0, 10, 25 and 50 μM) for 12 h or with 50 μM for up to 24 h (B). the cells were also treated with
indicated concentration of Trigonelline for 12 h after stimulation with EGF (20 ng/ml) for 30 mint (C) and the cell lysates were subjected to western blot analysis
using antibodies specific for phospho-EGFR, EGFR, phospho ERK 1/2 (Thr 202/Tyr 204), total ERK, phospho Nrf2 (Ser 40), Nrf2 and α-Tubulin. Both cells were
treated with or without Src inhibitor PP2, 10 μM (D) or a combination of EGFR inhibitor AG1478 (10 μM for 1 h) followed by Trigonelline for 12 h alone (E) or in EGF
stimulated cells (F). Immunoblot level of pSrc, EGFR, pEGFR, ERK, pERK 1/2, Nrf2, pNrf2 and α-Tubulin was detected. (G) NCIH460 cells were exposed to EGF, EGF
+ Trigonelline, AG1478 + Trigonelline or EGF + AG1478 + Trigonlelline for 12 h and expression of membrane bound phospho EGFR levels were determined by
immunofluorescence analysis. All data are representative of three independent experiments that show a similar pattern.
Fig. 3. Anti-proliferative effect of Cisplatin/Etoposide combined with Trigonelline is dose dependent. A549 (A and D), NCIH460 (B and E) and NCI H1299 (C and F)
cells were treated with different doses of Cisplatin/Etoposide for 36 h followed by Trigonelline pre-treatment for 12 h and a series of MTT assays were conducted.
Data are represented as the mean + s.e.m. of three individual experiments done in triplicate. The values were compared with respective free drugs at P < 0.05 level
(*, **, ***, ****, *****).
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Trigonelline, Cisplatin or Etoposide at indicated doses for 72 h and IC50
values of the drugs were determined subsequently (Fig. S2A–I).
Calculated IC50 values of Trigonelline, Cisplatin and Etoposide have
been listed in Table S1 and was used to analyse and fix the ratios for
subsequent combination studies. Although A549 and NCI H1299 cells
showed less sensitivity towards Cisplatin and Etoposide, NCIH460 cell
had higher sensitivity towards these drugs.
Next, the impact of Nrf2 suppression on the anticancer drug
(Cisplatin, Etoposide) induced apoptosis was studied in all these cell
lines. A549 (Fig. 3A and D), NCIH460 (Fig. 3B and E) and NCI H1299
(Fig. 3C and F) cells were treated with Cisplatin and Etoposide for 36 h in
combination with Trigonelline (12 h pre-treatment) to study their
growth inhibitory effect. The drug combinations gave an altered cell
inhibition profile when compared to the single drug treatment. Trigonelline at 50 μM concentration augments the effect of Cisplatin and
Etoposide at a very low dose compared to its IC50 value.
3.4. Trigonelline–Cisplatin and Trigonelline-Etoposide sequence results in
dose dependent synergistic cytotoxicity in NSCLC cells
Cells were treated with 50 μM Trigonelline along with different
concentrations of Cisplatin and Etoposide in separate incubations and
then Fa vs CI graphs of A549 and NCIH460 cells were obtained from
CompuSyn software. The median effect analysis and calculation of the
Combination Index (CI) of Trigonelline and Cisplatin incubation indicated that Trigonelline and Cisplatin produced varying results at
different concentrations. In A549 cells, Trigonelline at 50 μM concentration produced strong synergistic effect when used with Cisplatin at
0.5, 1 and 2.5 μg/ml doses (Fig. 4A), whereas Trigonelline at 25 μM dose
exerts additive effect with Cisplatin at 2.5 and 5 μg/ml concentrations
(not shown here). As Trigonelline concentration was fixed at 100 μM, a
synergistic effect is observed at lower concentrations of Cisplatin i.e. at
0.5 and 1 μg/ml (Table S2). This confirmed that effective combination of
two or more drugs governed by their respective concentrations. A
similar trend was observed in NCIH460 cells when the same combinations of Trigonelline and Cisplatin (Table S3) were used (Fig. 4C). For
Trigonelline and Etoposide combination, in both A549 (Fig. 4B) and
NCIH460 cells (Fig. 4D), higher concentrations of Trigonelline (50 and
100 μM) produced strong synergism with Etoposide (0.5,1,2.5 μg/ml).
For this solid synergistic effect of Trigonelline with Cisplatin and Etoposide, a combined concentration of 50 μM Trigonelline with 1 μg/ml
Cisplatin or 0.5 μg/ml Etoposide were selected for further studies.
3.5. Combinatorial treatment of Trigonelline along with Cisplatin/
Etoposide had no cytotoxic effects on normal lung cells, but induces
apoptosis in A549 and NCIH460
To elucidate the cytotoxic effect of Trigonelline alone (Fig. S3 A) or
in combination with Cisplatin/Etoposide (Fig. S3B and S3C) MTT assay
was performed on normal lung epithelial cell line, L132. There was no
significant difference in cell viability between control and treated cells
in all the treatment conditions. However, from the earlier experiments it
was proved that combination of Cisplatin/Etoposide with Trigonelline
caused a decrease in cell viability in both A549 and NCIH460 cell lines.
To confirm that Trigonelline potentiate Cisplatin and Etoposide efficacy
in NSCLC cells, we treated both A549 and NCIH460 cells with Trigonelline (50 μM) for 12 h and additionally with Cisplatin (1 μg/ml)/
Etoposide (0.5 μg/ml) for another 36 h. Hoechst-PI staining of above
incubated cells showed that percentage of PI (+) ve cells (apoptotic
cells) was greatly increased after Trigonelline+Cisplatin and Trigonelline+Etoposide treatment by two folds compared to the drugs alone
(Fig. 5A). The nuclear specific dye DAPI staining exhibited similar results indicating DNA fragmentation, a characteristic feature of apoptosis
(Fig. 5B). Further, more experiments were carried out to examine the
expression of apoptosis related proteins. As shown in Fig. 5C, the protein
levels of Bcl2, Bax active PARP1 and caspase 3 were markedly varied in
the combinatorial treatment group than in the control or Trigonelline
treated group. The mitochondrial membrane potential (MMP), an
important aspect to assess mitochondrial function, was further quantified by JC-1 staining (Cayman). Co-treatment of Trigonelline with
Cisplatin/Etoposide showed high intensity of green fluorescence, corresponds to dead cells, indicating loss of MMP (Fig. 5D). Collectively
these results suggested that apoptosis induced by the combinatorial
treatment of Trigonelline with Cisplatin/Etoposide followed mitochondrial pathway. Flow cytometry analysis with Annexin V-FITC and PI
staining showed significant number of apoptotic cells in combinatorial
Fig. 4. Trigonelline–Cisplatin and Trigonelline-Etoposide sequence resulted in dose dependent synergistic cytotoxicity in NSCLC cells. A549 (A and B) and NCIH460
(C and D) cells were treated with 50 μM of Trigonelline with different concentrations of Cisplatin/Etoposide and then Fa vs CI graphs of were obtained from
treatment after 48 h (Fig. 5E). The colony formation assay was conducted as a way to measure potency of cell growth post drug treatment.
Combination treatment of Trigonelline with Cisplatin/Etoposide
demonstrated significant inhibition of colony forming capacity of cells
post drug treatment compared to the drugs when treated alone or
against the untreated control in both A549 and NCI H460 cells (Fig. 5F).
The results suggested that Trigonelline enhanced the efficacy of
Cisplatin and Etoposide considerably in NSCLC cells.
3.6. The apoptosis sensitizing effect of Trigonelline depends on inhibition
of Nrf2 nuclear accumulation
Inhibition of Nrf2 and subsequent downregulation of cytoprotective
genes, as has been shown in our results, could be the possible reason
behind enhanced sensitivity of NSCLC cells to the chemotherapeutic
drugs. To confirm this hypothesis, A549 and NCIH460 cells were
transfected with control or Nrf2 siRNA for 48 h and then treated with
Cisplatin (1 μg/ml) and Etoposide (0.5 μg/ml) alone for 36 h or in
combination with Trigonelline (50 μM) as described in Fig. 6. To
quantify apoptotic cells, Hoechst-PI staining showed high percentage of
PI (+) ve cells (apoptotic) in combinatorial treatment compared to the
cells treated with Cisplatin or, Etoposide alone in control siRNA transfected group. In the Nrf2 siRNA transfected group, similar incubations
did not produce any additive effect of Trigonelline suggesting that it
functions through Nrf2 mediated pathway (Fig. 6A and B). Comparable
results were obtained in the DAPI stained nuclei for identification and
quantification of chromatin fragmented apoptotic cells (Fig. 6C and D).
Confocal image of JC-1 stained cells showed significantly high number
of apoptotic cells with green fluorescence in combinatorial incubation
than the cells treated with Cisplatin or Etoposide alone in control siRNA
transfected group (Fig. 6E). In the Nrf2 siRNA transfected group, Trigonelline treatment again did not make any additional effect compared to
the Cisplatin or Etoposide alone confirming the hypothesis that Trigonelline functions through Nrf2 signalling pathway and its inhibition
augments drug sensitivity of NSCLC cells.
4. Discussion
Aberrant activation of Nrf2/ARE pathway is an important mechanism for protecting NSCLC cells from ROS mediated damages (Nioi and
Nguyen, 2007). Besides, it has been verified to play an important role in
the development of resistance to a broad spectrum of chemotherapeutic
drugs (Ohta et al., 2008). These constitutively active Nrf2 provides a
great advantage for cell survival, proliferation and progression (Padmanabhan et al., 2006; Singh et al., 2006; Zhang, 2006). Our observation indicated that both expression and accumulation of Nrf2 protein
inside the nucleus were considerably high in A549 and NCIH460 cells
compared to the normal human lung epithelial cell line, L132. High
amount of nuclear Nrf2 reduces ROS overload via antioxidant response
and provides cytoprotection in NSCLC cells (Liao et al., 2015). Interestingly, suppression of Nrf2 using Nrf2 siRNA induces significant
apoptosis in NSCLC cells. This indicates that use of Nrf2 inhibitors could
improve the treatment regimen for lung cancer, but this warrants
adequate amount of research and human trial to confirm the hypothesis.
In our investigation we have used Trigonelline, a plant alkaloid from
Fenugreek, to test its ability of Nrf2 nuclear translocation inhibition in
NSCLC cells and efficacy in combinatorial treatment with Cisplatin and
Etoposide. Our results showed that Trigonelline at 50 μM dose decreased
Fig. 5. Effect of Trigonelline (Trig), Cisplatin (Cis) and Etoposide (Eto) alone or in combinations in NSCLC cells. Both A549 and NCIH460 cells were treated with 50
μM Trigonelline for 12 h. The cells were further treated with/without Cisplatin (1 μg/ml) or Etoposide (0.5 μg/ml) for an additional 36 h. (A) At the end of the
incubation, the PI (+) cells were quantified to measure the rate of apoptosis after various treatments. (B) The apoptotic cells with fragmented nucleus were also
quantified and plotted after DAPI staining (*P < 0.05 compared to Cis treated A549, #p < 0.01 compared to Eto treated A549, **p < 0.01 compared to Cis treated
NCIH460, ##p < 0.01 compared to Eto treated NCIH460) (C) In a similar experiment, expression of PARP1 and Caspase 3 (Cas 3) were detected by western blot
analysis. α-tubulin was used as loading control. (D) Mitochondrial membrane potential (MMP) of the above said incubated cells were detected by JC1 staining. (E)
Flow cytometric analysis of Annexin V-FITC and PI stained A549 and NCIH460 cells from the above said treatments represents apoptotic cells. (F) The colony
formation assay was performed to determine the colony forming ability of human lung cancer cells. Data are represented by three individual experiments.
C. Fouzder et al.
Toxicology in Vitro 70 (2021) 105038
8
nuclear accumulation of pNrf2 without altering Nrf2 level in NSCLC
cells. Stimulation with tBHQ, a known Nrf2 inducer, showed significant
amount of pNrf2 in the nucleus of untreated cells. However, Trigonelline
treatment sufficiently blocked pNrf2 nuclear translocation in tBHQ
stimulated cells suggesting its overwhelming capacity against Nrf2
activation. Use of leptomycin B, a crm1 protein inhibitor, in NSCLC
incubation suggested that Trigonelline interferes with the Nrf2 import
mechanism but not with the export system. Our results were in agreement with a previous study in pancreatic carcinoma cells (Panc1,
Colo357 and MiaPaca2) where Trigonelline efficiently decreased basal
and tBHQ-induced Nrf2 activity in all cell lines, thereby reduced nuclear
accumulation of the Nrf2 protein. Along with Nrf2 inhibition, Trigonelline blocked the Nrf2-dependent expression of proteasomal genes
(for example, s5a/psmd4 and a5/psma5) and decreased proteasome
activity. These blocking effects were absent after treatment with Nrf2
siRNA suggesting that Trigonelline functions through inhibiting Nrf2
activation (Arlt et al., 2013).
Several recent reports support a role for the EGFR-ERK1/2 pathway
in regulating nuclear accumulation of Nrf2 in response to oxidative
stress (Xu et al., 2013). Our results stated that Trigonelline decreased the
phosphorylated forms of EGFR and ERK 1/2, which correlated with the
diminished Nrf2 phosphorylation and nuclear localization. Besides, we
found that Src inhibition by PP2 attenuated EGFR and Nrf2 phosphorylation in NSCLC cells. These findings suggested that Trigonelline
inhibited Nrf2 activation through suppression of EGFR signalling
cascade and thereby block its way to the nucleus. EGF induced EGFR
activation plays an important role in cell growth, proliferation and
survival in NSCLC and thus small molecule inhibitors of EGFR are
gaining attention. Thus, our study opens a new window concerning
NSCLC inhibition.
Presently used anti lung cancer drugs, Cisplatin and Etoposide,
possess several side effects such as nausea and vomiting (severe), diarrhoea, hair loss etc. (html, 2016). Moreover, Cisplatin induced EGFR
phosphorylation and also Nrf2 activation discouraged its chemotherapeutic use in NSCLC. We therefore presumed that use of Nrf2 inhibitors
in combination with the scheduled lung cancer drugs could be a choice
to enhance their efficacy and to avoid drug induced adverse effects if
used at sufficiently low doses. We have observed that inhibition of nuclear pNrf2 by Trigonelline enhanced ROS accumulation in NSCLC cells
(Fig. S4A and S4B). Thus, Trigonelline in combination with Cisplatin/
Etoposide greatly enhanced mitochondria mediated apoptosis when
used at very low dose compared to their individual IC50 values.
Oral and subcutaneous median lethal doses (LD50) of Trigonelline in
rats are very high and are also non mutagenic to several bacterial strains
in the presence or absence of metabolic activation (Zhou and Zhou,
2012). This provides evidence that Trigonelline is a pharmacologically
safe compound to be used in vivo. However, one major concern for the
use of Trigonelline as a Nrf2 inhibitor is that it could inhibit Nrf2 in
normal non-cancerous cells and might augment cytotoxic effects of anticancer drugs. In our study Trigonelline in combination with Cisplatin/
Etoposide did not show any visible toxicity in the normal lung epithelial
cells, L132. Although the true reason behind this is unknown, but we can
believe that NSCLC cells with high expression of Nrf2 become obsessed
with high ROS level and corresponding expression of antioxidant genes
that fuel their survival and proliferation. Therefore, any small change in
the ROS mediated genes might cause greater change in cellular response
to drug and the same thing is not viable to normal non-cancerous cells.
Thus, in our case the same regimen of drug could efficiently kill lung
cancer cells but largely ineffective to normal lung cells.
In conclusion, high expression of Nrf2 makes the NSCLC cells resistant to some of the commercially available anti-cancer drugs. Here, we
have presented that Trigonelline inhibits Nrf2 via EGFR signalling
pathway which could restore the use of well-established anticancer
drugs. Combinatorial anticancer effect of Trigonelline with Cisplatin/
Etoposide enhanced efficacy of combinations via inhibiting Nrf2 signalling pathway. We presume that Trigonelline could effectively reduce
Fig. 6. Induction of apoptosis by Trigonelline depends on Nrf2 in NSCLC cells. Both of the A549 and NCIH460 cells were transfected with control or Nrf2 siRNA for
48 h. After transfection, cells were treated with/without 1 μg/ml of Cisplatin (Cis) or 0.5 μg/ml of Etoposide (Eto) for 36 h after 12 h pre-treatment with 50 μM of
Trigonelline (Trig). At the end of the incubation, the rate of apoptosis was determined by propidium iodide staining and DAPI staining indicates nuclear fragmentation. The PI (+) cells and cells with fragmented nuclei (DAPI stained) were quantified and plotted for A549 (A and C) and NCIH460 (B and D). Each bar
represents results of three independent experiments (*P < 0.05 vs Cis + control siRNA, #p < 0.01 vs Eto + control siRNA, **p < 0.01 vs Cis + control siRNA, ##p <
0.01 vs Eto + control siRNA). (E) JC1 staining determines the Mitochondrial membrane potential of the above treated cells and indicates apoptosis.
C. Fouzder et al.
Toxicology in Vitro 70 (2021) 105038
9
the adverse side effects of Cisplatin and Etoposide in vivo and expect to
widen the treatment options to enhance the survival of NSCLC patients.
However, our in vitro study undeniably requires further validation in
animal models before proceeding to clinical trials.
Author contribution
C.F. performed and designed all the experiments, analysed the data
and wrote the ERK inhibitor manuscript. S.M did the flow cytometry analysis. A.M and
C.M analysed the data and wrote the manuscript. R.K. conceived and
designed the experiments, supervised this study, analysed data and
wrote the manuscript.
Declaration of Competing Interest
The authors declare no conflict of interest associated with this work.
Acknowledgments
The authors appreciate the use of facilities as extended by Prof. Samir
Bhattacharya and the Head, Department of Zoology (Supported by UGC
CAS-II and DST-FIST level II), Visva-Bharati University. This study is
primarily supported by UGC-BSR SStart-Up-Grant, University Grants
Commission, Govt. of India (Grant No. F. 30-7/2014(BSR)) and a partial
support from Science & Engineering Research Board (SERB), Department of Science & Technology, Govt. of India (Grant No. ECR/2017/
001028/LS). AM is thankful to SERB for her JRF fellowship.
Appendix A. Supplementary data
Supplementary data to this article can be found online
References
Arlt, A., Sebens, S., Krebs, S., Geismann, C., Grossmann, M., Kruse, M.L., Schreiber, S.,
Sch¨
afer, H., 2013. Inhibition of the Nrf2 transcription factor by the alkaloid
trigonelline renders pancreatic cancer cells more susceptible to apoptosis through
decreased proteasomal gene expression and proteasome activity. Oncogene 32 (40),
4825–4835. https://doi.org/10.1038/onc.2012.493.
Furfaro, A.L., Traverso, N., Domenicotti, C., Piras, S., Moretta, L., Marinari, U.M.,
Pronzato, M.A., Nitti, M., 2016. The Nrf2/HO-1 Axis in Cancer Cell Growth and
Chemoresistance. Oxidative Medicine and Cellular Longevity, 2016(Table 1). https://
doi.org/10.1155/2016/1958174.
Gao, F., Yu, X., Li, M., Zhou, L., Liu, W., Li, W., Liu, H., 2020. Deguelin suppresses nonsmall cell lung cancer by inhibiting EGFR signaling and promoting GSK3 β / FBW7-
mediated Mcl-1 destabilization. Cell Death Dis. https://doi.org/10.1038/s41419-
020-2344-0.
Hirsch, F.R., J¨
anne, P.A., Eberhardt, W.E., Cappuzzo, F., Thatcher, N., Pirker, R.,
Choy, H., Kim, E.S., Paz-Ares, L., Gandara, D.R., Wu, Y.L., Ahn, M.J., Mitsudomi, T.,
Shepherd, F.A., Mok, T.S., 2013. Epidermal growth factor receptor inhibition in lung
cancer: status 2012. J. Thorac. Oncol. 8 (3), 373–384. https://doi.org/10.1097/
JTO.0b013e31827ed0ff.
Ishii, T., Warabi, E., 2019. Mechanism of rapid nuclear factor-e2-related factor 2 (Nrf2)
activation via membrane-associated estrogen receptors: roles of NADPH oxidase 1,
neutral sphingomyelinase 2 and epidermal growth factor receptor (EGFR).
Antioxidants 8 (3), 1–17. https://doi.org/10.3390/antiox8030069.
Jaramillo, M.C., Zhang, D.D., 2013. The emerging role of the Nrf2-Keap1 signaling
pathway in cancer. Genes Dev. 27 (20), 2179–2191. https://doi.org/10.1101/
gad.225680.113.
Liao, J.C., Lee, K.T., You, B.J., Lee, C.L., Chang, W., Te, Wu, C, Y., Lee, H.Z., 2015. Raf/
ERK/Nrf2 signaling pathway and MMP-7 expression involvement in the trigonellinemediated inhibition of hepatocarcinoma cell migration. Food Nutr. Res. 59, 1–10.
https://doi.org/10.3402/fnr.v59.29884.
Lin, T., 2017. Induction of Cbl-dependent epidermal growth factor receptor degradation
in Ling Zhi-8 suppressed. Lung Cancer 2607, 2596–2607. https://doi.org/10.1002/
ijc.30649.
McMahon, M., Itoh, K., Yamamoto, M., Chanas, S.A., Henderson, C.J., McLellan, L.I.,
Wolf, C.R., Cavin, C., Hayes, J.D., 2001. The cap “n” collar basic leucine zipper
transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and
inducible expression of intestinal detoxification and glutathione biosynthetic
enzymes. Cancer Res. 61 (8), 3299–3307.
Mukhuty, A., Fouzder, C., Mukherjee, S., Malick, C., Mukhopadhyay, S.,
Bhattacharya, S., Kundu, R., 2017. Palmitate induced Fetuin-A secretion from
pancreatic β-cells adversely affects its function and elicits inflammation. Biochem.
Biophys. Res. Commun. https://doi.org/10.1016/j.bbrc.2017.08.022.
Nioi, P., Nguyen, T., 2007. A mutation of Keap1 found in breast cancer impairs its ability
to repress Nrf2 activity. Biochem. Biophys. Res. Commun. 362 (4), 816–821. https://
doi.org/10.1016/j.bbrc.2007.08.051.
Ohta, T., Iijima, K., Miyamoto, M., Nakahara, I., Tanaka, H., Ohtsuji, M., Suzuki, T.,
Kobayashi, A., Yokota, J., Sakiyama, T., Shibata, T., Yamamoto, M., Hirohashi, S.,
2008. Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer
cell growth. Cancer Res. 68 (5), 1303–1309. https://doi.org/10.1158/0008-5472.
CAN-07-5003.
Padmanabhan, B., Tong, K.I., Ohta, T., Nakamura, Y., Scharlock, M., Ohtsuji, M.,
Kang, M. Il, Kobayashi, A., Yokoyama, S., Yamamoto, M., 2006. Structural basis for
defects of Keap1 activity provoked by its point mutations in lung cancer. Mol. Cell 21
(5), 689–700. https://doi.org/10.1016/j.molcel.2006.01.013.
Roh, J.L., Kim, E.H., Jang, H., Shin, D., 2017. Nrf2 inhibition reverses the resistance of
cisplatin-resistant head and neck cancer cells to artesunate-induced ferroptosis.
Redox Biol. 11 (November 2016), 254–262. https://doi.org/10.1016/j.
redox.2016.12.010.
Seal, S., Chatterjee, P., Bhattacharya, S., Pal, D., Dasgupta, S., Kundu, R., Mukherjee, S.,
Bhattacharya, S., Bhuyan, M., Bhattacharyya, P.R., Baishya, G., Barua, N.C.,
Baruah, P.K., Rao, P.G., Bhattacharya, S., 2012. Vapor of volatile oils from Litsea
cubeba seed induces apoptosis and causes cell cycle arrest in lung cancer cells. PLoS
One 7 (10), 1–11. https://doi.org/10.1371/journal.pone.0047014.
Shibata, T., Ohta, T., Tong, K.I., Kokubu, A., Odogawa, R., Tsuta, K., Asamura, H.,
Yamamoto, M., Hirohashi, S., 2009. Cancer related mutations in NRF2 impair its
recognition by Keap1-Cul3 E3 ligase and promote malignancy (Proceedings of the
National Academy of Sciences of the United States of America (2008) 105, 36,
(13568-13573) DOI: 10.1073/pnas.0806268105). Proc. Natl. Acad. Sci. U. S. A. 106
(25), 10392–10393. https://doi.org/10.1073/pnas.0905305106.
Siegel, R.L., Miller, K.D., Jemal, A., 2015. Cancer statistics, 2015. CA Cancer J. Clin. 65
(1), 5–29. https://doi.org/10.3322/caac.21254.
Singh, A., Misra, V., Thimmulappa, R.K., Lee, H., Ames, S., Hoque, M.O., Herman, J.G.,
Baylin, S.B., Sidransky, D., Gabrielson, E., Brock, M.V., Biswal, S., 2006.
Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med. 3
(10), 1865–1876. https://doi.org/10.1371/journal.pmed.0030420.
Tian, Y., Liu, Q., He, X., Yuan, X., Chen, Y., Chu, Q., Wu, K., 2016a. Emerging roles of
Nrf2 signal in non-small cell lung cancer. J. Hematol. Oncol. 9 (1), 1–9. https://doi.
org/10.1186/s13045-016-0246-5.
Tian, Y., Wu, K., Liu, Q., Han, N., Zhang, L., Chu, Q., Chen, Y., 2016b. Modification of
platinum sensitivity by KEAP1 / NRF2 signals in non-small cell lung cancer.
J. Hematol. Oncol. 1–14. https://doi.org/10.1186/s13045-016-0311-0.
Wang, M.C., Liang, X., Liu, Z.Y., Cui, J., Liu, Y., Jing, L., Jiang, L.L., Ma, J.Q., Han, L.L.,
Guo, Q.Q., Yang, C.C., Wang, J., Wu, T., Nan, K.J., Yao, Y., 2015. In vitro synergistic
antitumor efficacy of sequentially combined chemotherapy/icotinib in non-small
cell lung cancer cell lines. Oncol. Rep. 33 (1), 239–249. https://doi.org/10.3892/
or.2014.3583.
Xu, Y., Duan, C., Kuang, Z., Hao, Y., Jeffries, J.L., Lau, G.W., 2013. Pseudomonas
aeruginosa Pyocyanin activates NRF2-ARE-mediated transcriptional response via the
ROS-EGFR-PI3K-AKT/MEK-ERK MAP kinase signaling in pulmonary epithelial cells.
PLoS One 8 (8), 1–13. https://doi.org/10.1371/journal.pone.0072528.
Zhang, D.D., 2006. Mechanistic studies of the Nrf2-Keap1 signaling pathway. Drug
Metab. Rev. 38 (4), 769–789. https://doi.org/10.1080/03602530600971974.
Zhou, J.Y., Zhou, S.W., 2012. Isorhynchophylline: a plant alkaloid with therapeutic
potential for cardiovascular and central nervous system diseases. Fitoterapia 83 (4),