Curcumin analog, WZ37, promotes G2/M arrest and apoptosis of HNSCC cells through Akt/mTOR inhibition

Article abstract of DOI:10.1016/j.tiv.2019.104754

Head and neck squamous cell carcinoma (HNSCC) is a leading form of malignancy arising from the head and neck region. Existing conventional therapies are toxic and induce resistance to advanced HNSCC, therefore, new highly efficient therapeutic agents are urgently needed. The present study investigated the anti-cancer efficacy of WZ37, a curcumin analog, in HNSCC cell lines, and defined the mechanism of this activity. Results indicated that WZ37 inhibited proliferation of several HNSCC cell types by G2/M cycle arrest, promoted expression of a pro-apoptotic protein profile, and induced ROS-dependent mitochondrial injury and ER stress. Pre-treatment with NAC, an ROS scavenger, lowered the anti-cancer activity of WZ37 in HEP-2 cells. Long-term treatment of WZ37 (24 h) decreased Akt/mTOR phosphorylation which was accompanied by increased expression of BAD and PTEN. Moreover, co-treatment of WZ37 with MK-2206 (Akt inhibitor) promoted cancer cell apoptosis. Our findings indicated that the anti-cancer potential of WZ37 was attributed to ROS-dependent cell cycle arrest, mitochondrial injury, and ER stress, leading to apoptosis. The basis of the HNSCC cell apoptosis was through a mechanism of inhibition of the oxidant-sensitive Akt/mTOR pathway. We conclude that WZ37 can be a promising anti-cancer agent for the treatment of HNSCC.

Full text of DOI:10.1016/j.tiv.2019.104754

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Curcumin analog, WZ37, promotes G2/M arrest and apoptosis of
HNSCC cells through Akt/mTOR inhibition
Ziheng Zhang, Renyu Lin, Zhoudi Liu, Tao Yan, Yiqun Xia,
Leping Zhao, Feng Lin, Xi Zhang, Chenglong Li, Yi Wang
PII:
S0887-2333(19)30325-X
https://doi.org/10.1016/j.tiv.2019.104754
TIV 104754
DOI:
Reference:
To appear in:
Toxicology in Vitro
Received date:
Revised date:
Accepted date:
26 April 2019
2 December 2019
17 December 2019
Please cite this article as: Z. Zhang, R. Lin, Z. Liu, et al., Curcumin analog, WZ37,
promotes G2/M arrest and apoptosis of HNSCC cells through Akt/mTOR inhibition,
Toxicology in Vitro(2019), https://doi.org/10.1016/j.tiv.2019.104754
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2019 Published by Elsevier.

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Curcumin analog, WZ37, promotes G2/M arrest and apoptosis of HNSCC cells through
Akt/mTOR inhibition
1
,2,3,#
1,2,#
1
1
2
3
Ziheng Zhang
, Renyu Lin,
, Zhoudi Liu , Tao Yan , Yiqun Xia , Leping Zhao , Feng
2
3
1
1,
Lin , Xi Zhang , Chenglong Li , Yi Wang *
1
Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China
2
The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325035,
China
3
Department of Pharmacy, the Affiliated Yueqing Hospital of Wenzhou Medical University,
Wenzhou, Zhejiang 325000, China
#
These authors contribute equally to this work.
Running title: W37 inhibits Akt/mTOR in HNSCC
*
Corresponding author
Yi Wang, Ph.D, Associate Professor
Address: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou
Medical University, Wenzhou 325035, China
Tel: +86-577-85773060; Fax: +86-577-85773060;
E-mail: yi.wang1122@wmu.edu.cn
1

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Abbreviations
DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; HNSCC, head and neck
squamous cell carcinoma; AM, acetoxymethyl; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyltetrazolium bromide; NAC, N-acetyl cysteine; Δψm, mitochondrial membrane
potential; PI, propidium iodide; PVDF, polyvinylidene difluoride; ROS, reactive oxygen
species; TEM, transmission electron microscopy; UPR, unfolded protein response
2

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ABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is a leading form of malignancy arising
from the head and neck region. Existing conventional therapies are toxic and induce
resistance to advanced HNSCC, therefore, new highly efficient therapeutic agents are
urgently needed. The present study investigated the anti-cancer efficacy of WZ37, a curcumin
analog, in HNSCC cell lines, and defined the mechanism of this activity. Results indicated
that WZ37 inhibited proliferation of several HNSCC cell types by G2/M cycle arrest,
promoted expression of a pro-apoptotic protein profile, and induced ROS-dependent
mitochondrial injury and ER stress. Pre-treatment with NAC, an ROS scavenger, lowered the
anti-cancer activity of WZ37 in HEP-2 cells. Long-term treatment of WZ37 (24 h) decreased
Akt/mTOR phosphorylation which was accompanied by increased expression of BAD and
PTEN. Moreover, co-treatment of WZ37 with MK-2206 (Akt inhibitor) promoted cancer cell
apoptosis. Our findings indicated that the anti-cancer potential of WZ37 was attributed to
ROS-dependent cell cycle arrest, mitochondrial injury, and ER stress, leading to apoptosis.
The basis of the HNSCC cell apoptosis was through a mechanism of inhibition of the
oxidant-sensitive Akt/mTOR pathway. We conclude that WZ37 can be a promising anti-
cancer agent for the treatment of HNSCC.
KEYWORDS
HNSCC; curcumin analog WZ37; reactive oxygen species; calcium homeostasis; Akt/mTOR
pathway
3

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1
. Introduction
Head and neck squamous cell carcinoma (HNSCC), including laryngocarcinoma and
nasopharyngeal carcinoma, is a leading form of malignancy arising from the head and neck
region (Rayess et al., 2018). HNSCC accounts for approximately 350,000 annual deaths
globally (Rayess et al., 2018). The prognosis of HNSCC patients has improved markedly in
past decades, with most being treated by surgical resection and radiotherapy for the early
stage of the HNSCC (Yamazaki et al., 2017). At advanced stages, treatment may be
multimodal with surgery combined with chemotherapy. Despite advances in surgery,
chemotherapy, and radiotherapy, the five-year survival rate has remained essentially low (Ho
et al., 2017). However, inherent and acquired resistance to the therapy limits its clinical
effectiveness (Nicco and Batteux, 2017), which is reflected by poor patient survival rate.
Chemoresistant cells can evade cell death signals through drug efflux, metabolic change,
alteration in DNA damage repair mechanisms and/or by other unknown mechanisms (Kupsco
and Schlenk, 2015). Therefore, new approaches are needed to improve the therapeutic
options to decrease HNSCC incidence and severity associated with this kind of cancer.
Oxidative stress, defined as excessive amounts of cellular reactive oxygen species
(ROS), is implicated in a number of diseases including cancer (Senft and Ronai, 2015).
Cancer cells exhibit an higher ROS content than normal cells, developing resistance to pro-
inflammatory and pro-apoptotic molecules in order to safeguard their own survival (Shpilka
and Haynes, 2017). Moreover, uncontrolled high levels of ROS activate pro-apoptotic
signalling pathways, particularly endoplasmic reticulum (ER) stress, mitochondrial
dysfunction, ultimately leading to cellular apoptosis. The ER is a central organelle in which
proteins are synthesized and properly folded. In cancer cells, these protein processing
activities are particularly active under conditions of uncontrolled or impaired signalling
2
+
pathways, such as the intracellular content of ROS and Ca
(Hou et al., 2017). During
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protein processing (in a normal or pathological milieu), an accumulation of unfolded or
misfolded proteins in the ER lumen activates the unfolded protein response (UPR), a highly
conserved cellular stress response occurring in mammals as well as yeast and worm
organisms (Zou et al., 2015). The UPR is a cell survival response, attempting to restore
normal cell function by halting protein translation and cell cycle progression, degrading
unfolded/misfolded proteins, and activating signals to promote proper protein folding
(Poorghorban et al., 2015). However, if these objectives are not achieved within a certain
time, the UPR activates steps for apoptosis (Leone et al., 2016). Therefore, the regulation of
apoptosis and cell cycle arrest are potential key cellular pathways which can be exploited for
anti-cancer mechanisms.
Curcumin is a naturally occurring hydrophobic polyphenol isolated from the rhizome
of the herb Curcuma longa, which has a wide spectrum of pharmacological activities
including anti-cancer effects by targeting a multitude of signalling pathways (Capodanno et
al., 2017). To-date, three different phase-I clinical trials demonstrated that curcumin at a high
dose of 12 g/day is relatively safe and well-tolerated. However, despite its biological efficacy
and safety, curcumin has yet to be approved for cancer therapy due to its low bioavailability
caused by poor solubility and short biological half-life. In recent years, our laboratory has
focused on development of curcumin derivatives with more favorable in vivo bioavailability
and therapeutic efficacy. We have synthesized stable mono-carbonyl analogs of curcumin by
deletion of the β-diketone moiety, and one of these is WZ37, which displayed improved anti-
inflammatory activity (Wang et al., 2015). The present study investigated the anti-cancer
efficacy of WZ37 in HNSCC cell lines, and defined the mechanism of this activity. Our
findings indicated that WZ37 induced ROS-dependent apoptosis through mitochondrial
collapse, and ER stress, which was regulated by inactivation of Akt/mTOR signalling.
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2
. Material and methods
2
.1 Chemicals and reagents
Curcumin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT),
dimethyl sulfoxide (DMSO), N-acetyl cysteine (NAC), and AKT inhibitor MK-2206 were
purchased from Sigma (St. Louis, MO). Apoptosis Detection Kit Annexin V FITC and
propidium iodide (PI) were purchased from BD Pharmingen. Antibodies, anti-MDM-2, anti-
CDC2, anti-Cyclin B1, anti-p53, anti-Bcl-2, anti-Bax, anti-BAD, anti-caspase-3p30/17, anti-
cleaved PARP, anti-PTEN, anti-p-PERK, anti-eIF2α, anti-ATF6, anti-XBP-1, anti-p-Akt,
anti-p-mTOR, anti-GAPDH, goat anti-mouse IgG-HRP (horse-radish peroxidase), donkey
anti-rabbit IgG-HRP and donkey anti-goat IgG-HRP, were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Anti-CHOP and anti-ATF4 antibodies were
purchased from Cell Signaling Technology (Danvers, MA). Caspase-3 activity kit, ROS
detection kit, JC-1 fluorescence probe and membrane-permeable calcium indicator fluo-3-
acetoxymethyl (AM) ester were obtained from Beyotime Institute of Biotechnology
(Shanghai, China).
2
.2 WZ37 synthesis
The curcumin analog, WZ37, was synthesized by our laboratory and structurally
1
identified by using mass spectrometry and H nuclear magnetic resonance analysis (Wang et
al., 2015). Briefly, the vanillin and 3,4-dihydro-2H-pyran are stirred in the dichloromethane,
with PPTS as a catalyst, at room temperature for 5 h. This reaction mixture was purified by
chromatography on silica gel to afford 3-methoxy-4-((tetrahydro-2H-pyran-2-
yl)oxy)benzaldehyde. Aqueous NaOH solution (20% w/v, 5 ml) was added to a solution of 3-
methoxy-4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde in acetone (20 ml). The reaction
mixture was stirred at room temperature for 5-6 h to obtain the second intermediate, which
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further reacted with 2,4,6-trimethyl-benzaldehyde in mixed suspension (10 ml) containing
sodium hydroxide powder and dioxane. The reaction mixture was stirred at room temperature
for 6 h, then purified by the chromatography on silica gel to afford (1E,4E)-1-(3-methoxy-4-
((tetrahydro-2H-pyran-2-yl)oxy)phenyl)-5-(2’,4’,6’-trimethylphenyl) penta-1,4-dien-3-one.
Finally, 5% HCl (1 ml) was added dropwise with stirring, and the end product, WZ37, was
filtered and washed with water and dried in vacuum. Before using, compounds were
recrystallized from CHCl3/EtOH. HPLC evaluation determined the purity at >99%. The
structure of WZ37 is shown in Figure 1A.
2
.3 Cell lines and cell culture
The HNSCC cell lines, HEP-2 (Cat#: 3131C0001000700021), CNE-1 (Cat#:
tw029125), CNE-2 (Cat#: 3142C0001000001300), CNE-2Z (Cat#: 3111C0001CCC000151)
and 5-8F (Cat#: CM-H305), were purchased from Cell Bank of Shanghai Institutes
[September, 2016; Biological Sciences, Chinese Academy of Sciences (Shanghai, China)].
The cells have been authenticated and tested for mycoplasma contamination by the Cell Bank
of Shanghai Institutes using PCR assay. The cell lines were maintained in RPMI 1640
medium (Gibco, Eggenstein, Germany) supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (Hyclone, Logan, UT) and 0.2% of antibiotic solution (100 units/ml penicillin
and 100 µg/ml streptomycin, Gibco, Invitrogen) at 37°C in a humidified atmosphere of 95%
air and 5% CO2 in an incubator (Thermo scientific).
2
.4 Cell viability
Cell viability was assessed with the MTT assay. Briefly, the HEP-2, CNE-1, CNE-2,
CNE-2Z and 5-8F cells were seeded at a density of 2500 cells per well in 96-well plates and
allowed to attach overnight. On the following day, the cells were treated with increasing
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concentrations of WZ37, curcumin, cisplatin (0.6, 1.25. 2.5, 5, 10, 20 and 40 μM) or with
DMSO alone as solvent control for a period of 24 h. A 20 μl aliquot of MTT (5 mg/ml in
PBS) was added to each well and incubated for 4 h. The medium was discarded, and 150 μl
of DMSO was added to each well to dissolve the colored formazan crystals. After shaking for
1
0 min, the absorbance was measured at 495 nm in a microplate reader (Biotek Instruments,
Inc., USA). Cell viability was expressed as a percentage of control groups and the IC50 was
calculated as the concentration of drug required to obtain 50% maximal inhibition of cell
viability.
2
.5 Intracellular ROS measurement
The intracellular ROS content was determined using the dichlorofluorescein diacetate
5
(
DCFH-DA) reagent. In brief, HEP-2 (3×10 cells/well) were seeded in 6-well plates and
allowed to attach overnight. After treatment with increasing concentrations of WZ37 (5, 15
and 25 μM) or DMSO alone as solvent control, cells were washed with serum-free RPMI
1
640 and incubated with l μl (5 μM) of DCFH-DA at 37℃ for 30 min. In some experiments,
HEP-2 cells were pre-treated with 5 mM NAC for 2 h prior to WZ37 exposure. The cells
were harvested, washed with PBS, re-suspended in 1 ml PBS, and the fluorescent intensity
was quantified by using FACS calibur flow cytometer (BD Biosciences, CA) analysed using
FlowJo software (TreeStar, Ashland, OR, USA).
2
.6 Determination of mitochondrial membrane potential (Δψm)
Mitochondrial transmembrane potential (Δψm) was detected using the JC-1
mitochondrial membrane potential assay kit. The HEP-2 cells were seeded at a density of 2×
5
1
0 cells/dish in 30-mm culture dishes. After treatment with WZ37 (25 μM, 0-24 h), the cells
were incubated with an equal volume of JC-1 staining solution (5 μg/ml) at 37°C for 20 min
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and washed twice with JC-1 staining buffer. Fluorescence microscopy was used to monitor
and record red emission of the fluorescent dye (indicating potential-dependent aggregation in
mitochondria) and green fluorescence (indicating monomeric JC-1, appearing in the cytosol
after mitochondrial membrane depolarization). Mitochondrial membrane potentials were
determined by ratio of the green/red fluorescence intensity.
2
+
2
.7 Measurement of free intracellular Ca concentration
2
+
2+
2+
The free intracellular [Ca ] ([Ca ]i) in HEP-2 cells was measured using a Ca -
sensitive fluorescence indicator, fluo-3-AM. Cells were treated with increasing
concentrations of WZ37 (5, 15 and 25 μM) for 24 h or with WZ37 (25 μM) for different
times (3, 9, or 24 h). Fluo-3 AM (5 μg/ml) was added to the cells and incubated for 30 min at
3
7°C. Then the cells were trypsinized, rinsed with PBS, re-suspended in 1 ml PBS, and the
fluorescence intensity was quantified by using flow cytometry and analyzed using FlowJo
software.
2
.8 Cell cycle analysis
The cell-cycle distribution was determined by flow cytometric DNA analysis. HEP-2
cells were seeded at a density of 300,000 cells/well in 6-well plates and incubated overnight.
On the following day, the cells were treated with increasing concentrations of WZ37 (5, 15
and 25 μM), curcumin (25 μM) or DMSO alone and incubated for 24 h. After treatment, the
cells were harvested, washed twice with ice-cold PBS, fixed with 70% ethanol (2 h at ﹣
2
0°C). The cells were centrifuged to remove the ethanol, washed with cold PBS, suspended
in 500 μl 1x PI staining solution and incubated at room temperature in the dark for 10 min.
Flow cytometric analysis of cell cycle was conducted using FACS calibur flow cytometer and
the data were analysed using FlowJo software.
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2
.9 Apoptosis determination
Apoptosis was determined using the Annexin V-FITC/PI apoptosis detection kit. The
HEP-2 cells were plated as described for the cell cycle analysis. In some experiments, the
cells were pre-treated with either 5 mM NAC for 2 h prior to WZ37 exposure, or with MK-
2
206 (10 μM) alone or in combination with WZ37 (25 μM) for 12 h. After treatment, cells
were harvested and washed twice with ice-cold PBS, re-suspended in 500 μl binding buffer,
stained with 3 μl FITC-conjugated anti-Annexin V for 10 min, and with 1 μl PI for 5 min.
Apoptotic cells were measured using the FACS calibur flow cytometer and the data were
analyzed using FlowJo software.
2
.10 Caspase-3 activity assay
Caspase-3 activity was measured using Caspase-3 Activity Kit according to the
manufacturer’s instructions. Briefly, cells were treated using the same protocol as described
for the cell cycle analysis. A cell lysis was made, which was centrifuged at 200,000×g for 15
min, and the protein concentration of the supernatant was determined by BCA protein assay.
Ten μl of the supernatant was incubated with 10 μl Ac-DEVD-pNA (2 mM) for 2 h at 37°C
in a 96-well microtitre plate. The release of p-nitroanilide (pNA) was quantified by
determining the absorbance at 405 nm with the Multiskan Spectrum (Thermo) and the
relative caspase-3 activity was calculated as a ratio of absorbance of treated cells to untreated
cells.
2
.11 Transmission electron microscopy (TEM)
The HEP-2 cells were seeded at a density of 6×10 cells/dish in 60-mm culture
dishes and allowed to attach overnight. On the following day, the cells were treated with
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WZ37 (25 μM), DMSO alone for 3 h or 24 h as solvent control. In some experiments, cells
were pre-treated with 5 mM NAC for 2 h prior to WZ37 exposure. The cells were
trypsinized, centrifuged and fixed in 2% glutaraldehyde in 0.1 M PBS (pH 7.4). Post-fixation
was performed by incubating cells with 1% osmium tetroxide for 1 h at 4°C, followed by
dehydration through a graded ethanol series, and embedded in epon. The embedded blocks
were sectioned into semi-thin sections and stained with toluidine blue. Ultrathin sections
were collected onto formvar-coated slot copper grids and counterstained with uranyl acetate
and Reynolds lead citrate. Sections were observed by TEM (H-7500, Hitachi, Ibaraki, Japan).
2
.12 Western blotting analysis
After indicated treatment protocol, the cells were washed with PBS, suspended in a
protein lysis buffer under cold conditions, and cell debris was removed by centrifugation at
2,000 rpm for 10 min at 4 °C. Protein concentration of the samples was determined by the
Bradford protein assay kit (Bio-Rad, Hercules, CA). The samples were separated on precast
-15% SDS polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF)
1
8
membrane. The membranes were blocked with 5% non-fat dry milk in tris-buffered saline
containing 0.1% Tween 20 (TBST) for 90 min at room temperature, and incubated with the
specific primary antibody in TBST overnight at 4°C. The membranes were washed with
TBST three times at 5 min intervals, incubated with HRP-conjugated secondary antibodies
(1:4000) for 1 h at room temperature, and washed three times with TBST. Protein bands were
detected using an enhanced chemiluminescence detection system (ECL, GE Healthcare), with
detection of GAPDH as a loading control.
2
.13 Statistical analysis
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The experiments were made in triplicates (n=3), and the data are presented as means ±
SEM. The statistical analyses were performed using GraphPad Pro. Prism 5.0 (GraphPad, San
Diego, CA), with statistical differences between two groups assessed by Student's t test. A P
value <0.05 was considered statistically significant.
3
. Results
3
.1 WZ37 inhibits cell viability of HNSCC
We have synthesized WZ37 with structural optimization for stability and improved
anti-inflammatory potential (Materials and Methods). We first evaluated the effects of WZ37
on cell viability of panel of head and neck cancer cell lines (5-8F, CNE-1, CNE-2Z, CNE-2,
HEP-2) using MTT assay. These cell lines are widely used for in vitro studies of HNSCC,
including new drug discovery and identification of cellular mechanisms (Borges et al., 2017;
Pozzi et al., 2015; Shen et al., 2011; Sun et al., 2015). We investigated the growth inhibitory
potential of WZ37 by measuring its effects on viability of these HNSCC cell lines (Fig. 1B-
1
F). The parental compound, curcumin, and the clinical anti-cancer drug, cisplatin, were
included for comparison with WZ37. As shown in Fig.1B-F, treatment with WZ37 for 24 h
significantly inhibited viability of HNSCC in a dose-dependent manner. IC50 for WZ37 was
consistently lower in all the HNSCC cell lines compared to those of curcumin and cisplatin.
Interestingly, WZ37 inhibited viability most effectively in HEP-2 cells (IC50=1.9±0.15 μM)
among the cell lines studied, suggesting cell-selective sensitivity of WZ37 (Fig. 1F). Based
on this greater sensitivity of HEP-2 cells to WZ37, the subsequent studies were made using
HEP-2 cells.
3
.2 WZ37 promotes cell cycle arrest and apoptosis
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We investigated the effects of WZ37 on cell cycle arrest and apoptotic cascade as
potential anti-cancer mechanisms in inhibiting HEP-2 cell growth. Analysis of the flow
cytometric profile of Annexin V/PI staining indicated that WZ37 treatment for 24 h induced
dose-dependent accumulation of apoptotic HEP-2 cells at early and late stages of apoptosis
(Fig. 2A-B). WZ37 at 25 µM induced >40% of apoptotic cells, whereas curcumin at the
same concentration, induced 20%, indicating the greater potency of WZ37. DMSO control
cells showed low or negative staining with Annexin V FITC and PI, indicating viable cells
(Fig. 2A-B). WZ37 treatment also increased the percentage of HEP-2 cells arrested at G2/M
phase (Fig. 2C-D). Further, we determined the effects of WZ37 on apoptosis-related protein
expressions by Western blotting. Results showed that WZ37 treatment for 24 h significantly
increased expression of Bax, cleaved PARP and concomitantly decreased Bcl-2 and pro-
caspase-3 (Fig. 2E), indicating pro-apoptotic changes. The pro-apoptotic profile of proteins
was consistent with significant increases in caspase-3 activity in HEP-2 cells treated with
WZ37 (Fig. 2F). Similarly, Western blot analysis of the effects of WZ37 on key proteins
related to G2/M cell cycle arrest indicated decreased expression of MDM2, CDC2 and cyclin
B1 (Fig. 2G). The effects of WZ37 (25 µM) on expression of apoptosis-related proteins and
G2/M arrest were greater than that of curcumin at the same concentration (Fig. 2E and 2G),
consistent with the observed greater potency of WZ37.
3
.3 WZ37 induces ROS accumulation in HEP-2 cells
We next investigated whether the WZ37-inhibited cellular growth was attributed to
intracellular ROS generation. ROS was measured by the common fluorescent probe DCFH-
DA, which increases in fluorescence intensity in the presence of ROS. The treatment with
WZ37 resulted in dose-dependent increases in the intracellular content of ROS in HEP-2 cells
relative to DMSO control cells (Fig. 3A-B). Moreover, when cells were pre-treated with 5
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mM NAC (ROS scavenger) for 2 h, the WZ37-induced intracellular accumulation of ROS
was prevented (Figure 3C-D), confirming that WZ37 induced ROS production. More
significantly, NAC pre-treatment abrogated the WZ37-induced apoptosis (Figure 3E-F) as
well as the changes in expression of pro-apoptotic proteins (Figure 3G). The findings
supported ROS as a mechanism by which WZ37 exerted its anti-cancer activity.
3
.4 WZ37 induces endoplasmic reticulum (ER) stress in HEP-2 cells
Elevated intracellular levels of ROS are known stresses to the ER, which can lead to
apoptosis. We investigated the involvement of potential ER stress in the WZ37-mediated
anti-cancer activity. Cultured HEP-2 cells were treated with various time points (Fig. 4A) and
doses (Fig. 4B), and expression of proteins associated with UPR was analysed by Western
blotting analysis. Results indicated that WZ37 increased expression of UPR-associated
proteins, i.e., p-PERK, p-EIF-2α, ATF-4, ATF6, XBp-1 and CHOP, with time- and dose-
dependency (Fig. 4A-B), indicating activation of the UPR response by ER stress. WZ37 at 25
µM increased expression of the proteins to a greater extent than curcumin at the same
concentration (Fig. 4B). We further evaluated the effects of WZ37 on the ultrastructure of
ER. The results indicated that WZ37 induced enlargement and dilation of ER (Figure 4D),
indicating ER injury and ER stress. Moreover, pre-treatment with NAC prevented the WZ37-
induced increases in expression of UPR-associated proteins (Figure 4C) as well as the ER
injury (Figure 4D). These results indicated that WZ37 induced ER stress as a consequence of
ROS generation.
2
+
3
.5 WZ37 increases [Ca ]i, injures mitochondria, and impairs Akt /mTOR signaling
Early indices of cell stress, such as that in response to high levels of ROS, are
2
+
2+
mitochondrial injury and increased [Ca ]i. We investigated the effects of WZ37 on [Ca ]i in
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HEP-2 cells using the Fluo-Am fluorescent indicator. Results indicated that WZ37 (15 or 25
2
+
µM) treatment for 24 h significantly increased [Ca ]i (Figure 5A-B). Moreover, the WZ37
treatment induced mitochondrial membrane depolarization, which corresponded to the
increased green fluorescence of the JC-1 probe (Figure 5C), indicating mitochondrial
membrane depolarization and injury. In contrast, cells treated with WZ37 up to 12 h showed
red fluorescent mitochondria, indicating intact mitochondrial membrane potential (Figure
5
C). Ultrastructural examination of HEP-2 cells following 3 h of WZ37 treatment revealed
mild ER swelling and normal mitochondria (Figure 5D). However, with 24 h of treatment,
the extent of ER swelling increased which was accompanied by mitochondrial swelling with
loss of crista (Figure 5D). Evidence indicates that the Akt/mTOR signalling cascade is
important for normal mitochondrial homeostasis, as well as regulation of cell survival and
apoptosis by targeting Bcl-2 family of proteins and PTEN, a negative regulator of Akt. We
investigated whether Akt/mTOR was a target of regulation by WZ37 in HEP cells. Western
blotting analysis indicated that WZ37 (25 µM) treatment for 24 h increased expression of
BAD, a regulator of mitochondria-dependent apoptosis, as well as PTEN, and significant
reduction of phosphorylation of Akt and mTOR (Figure 5E). The findings indicated that
although WZ37 activated the Akt/mTOR pathway at early time points (3 h), the activation
decayed by 24 h, indicating inhibition of the pathway. This latter effect of WZ37
corresponded with the peak expression of PTEN/BAD. The findings suggest that the later
decreased Akt/mTOR activity likely contributed to the promotion of apoptosis.
3
.6 Inhibition of oxidant-sensitive Akt/mTOR promotes apoptosis
We investigated the role of Akt/mTOR pathway in regulation of WZ37-induced
apoptosis. Since our data indicated that WZ37 can induce production of ROS, we first
determined whether ROS activated Akt/mTOR. Results indicated that pre-treatment of HEP-
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2
cells with NAC for 2 h prevented the WZ37-induced increases of p-Akt and p-mTOR
(Figure 6C). As expected, pre-treatment with MK-2206, an allosteric Akt inhibitor with broad
preclinical antitumor activity, also inhibited the WZ37-induced increases of p-Akt and p-
mTOR (Figure 6C), suggesting loss of important signals for regulating normal mitochondrial
homeostasis and cell survival. Therefore, we hypothesized that the reduced Akt/mTOR
signalling, such as that in response to MK-2206, would synergized with WZ37, leading to
heightened apoptotic activity. The results showed that a combined treatment of WZ37 with
MK-2206 caused 2-fold greater increase in apoptosis relative to either treatment alone
(Figure 6A-B). These results support the notion that the oxidant-sensitive Akt/mTOR plays a
significant pro-survival role in cancer cells.
4
. Discussion
HNSCC contributes globally to a great number of deaths and morbidity. Despite
continuous improvements in cancer detection techniques and management, HNSCC patients
are still predominantly incurable due to the metastatic potential and drug resistance (Dunn et
al., 2018). Therefore, there is much interest in developing targeted therapeutic agents for
treatment of HNSCC. Curcumin is a multifunctional bioactive natural product with relatively
safe and well-tolerated properties. In this study, we found that WZ37, a novel curcumin
analog, effectively suppressed proliferation of several HNSCC cells lines. Our laboratory
originally designed and discovered WZ37 as an anti-inflammatory compound based on
structural modification of curcumin (Wang et al., 2015). Since curcumin has been shown to
exhibit excellent anti-cancer effects in vivo and in vitro, we also tested WZ37 for its anti-
cancer effects. Interestingly, the WZ37 IC50 values were lower than that of either the parental
compound curcumin or cisplatin in almost all examined cell lines, indicating the greater
potency of WZ37 in cancer treatment. These were new findings pointing to WZ37 as a
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potential highly effective anti-cancer agent for treatment of HNSCC, therefore, we
investigated the cellular and molecular mechanisms by which WZ37 inhibited cancer cell
growth.
There is a great need of new drugs that are therapeutically effective to HNSCC.
Curcumin, which is the principal active ingredient of the traditional Chinese herb Curcuma
Longa, has inhibitory effects on proliferation and survival of HNSCC. Curcumin induces
cytotoxicity, apoptosis, and cell cycle arrest in G2/M phase in HSNCC cell lines. Recently,
Hu et al. (Hu et al., 2017) found that curcumin induces G2/M cell cycle arrest and apoptosis
of HNSCC in vitro and in vivo through ATM/Chk2/p53-dependent pathway. This group also
showed that anticancer activity of curcumin was associated with SIRT1 activation in HNSCC
cell lines (Hu et al., 2015). While our study did not test the reported signalling pathway by
which curcumin exerted anti-HNSCC activity, we observed that WZ37 had lower IC50 values
compared to curcumin, as evaluated by the MTT assay (Figure 1), indicating higher anti-
cancer ability of WZ37 than that of curcumin. Thus, we further investigated the anti-cancer
mechanism of WZ37 in HNSCC cells.
Several cellular stresses are known to activate the ER stress response, UPR, that could
lead to apoptosis (Song et al., 2017). We found that WZ37 induced a rapid production of
ROS in HEP2 cells, in which an elevated and persistent intracellular ROS content was
maintained for up to 24 h. This was accompanied by increased expression of PERK, ATF6
and XBP-1 (downstream of IRE), indicating activation of UPR. Studies have reported that the
PERK/eIF2α/ATF4 axis is likely a predominant participant of ER stress-induced apoptosis
via up-regulating the pro-apoptotic transcription factor, CHOP (Nabavi et al., 2018). Our data
indicated that WZ37 treatment was associated with increased CHOP expression, implicating
that elevated ROS induced cell death through a mechanism of ER stress.
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Mitochondria are dynamic organelles whose proper function is fundamentally
important for the viability of cells. Mitochondrial dysfunction can also lead to mitochondrial-
dependent apoptosis initiated by interactions of CHOP with pro-apoptotic proteins in the
BCL-2 family, such as BAD and BAX. The result of this complex interaction impairs the
integrity of the mitochondrial outer membrane, leading to increased mitochondrial membrane
2
+
permeability, release of free Ca , activation of aspartate-specific proteases and the caspases,
and ultimately apoptosis (Deegan et al., 2015; Shen et al., 2017; Vavilis et al., 2016). We
2
+
observed that WZ37 treatment in HEP-2 cells increased the cytosolic [Ca ] by 24 h, which
was associated with mitochondrial membrane depolarization, an indication of mitochondrial
injury. Subsequent TEM results demonstrated that cells treated with WZ37 developed
mitochondrial swelling, corroborating the loss of membrane potential. These data provide
evidence that ROS-induced mitochondrial dysfunction contributed to apoptosis in response to
WZ37 treatment.
The Akt/mTOR pathway is an important cellular communication cascade that cross-
talks with a variety of other signalling pathways to regulate cell survival (Wani et al., 2016;
Xu et al., 2017). Studies indicated that the Akt/mTOR pathway is highly activated and over-
expressed in most cancers (Huang et al., 2017). Evidence indicates that AKT/mTOR
decreases expression of pro-apoptotic BAD, thereby together with the anti-apoptotic
members of the BCL-2 family, promotes a protective effect on mitochondrial membrane for
cell survival (Zhang et al., 2017). Our data indicated that the WZ37-induced apoptosis of
HEP2 cells was through decreased activity of AKT/mTOR at 24 h, which was accompanied
by elevated expression of BAD. The results suggested that decreased AKT/mTOR signalling
contributed to the regulation of apoptosis in response to WZ37. This possibility was further
supported by the finding that when WZ37 treatment was combined with MK-2206, the Akt
inhibitor, apoptosis of HEP2 cells was significantly enhanced compared to WZ37 alone.
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Moreover, NAC pre-treatment prevented the WZ-37-induced activation of Akt/mTOR,
indicating oxidant-sensitivity. The findings support an oxidant-dependent mechanism of
apoptosis regulated by ER stress signals, mitochondrial dysfunction, and loss of Akt/mTOR
survival signalling. Thus, WZ37 in combination with Akt/mTOR-selective inhibitors could
provide a promising new therapeutic regimen for cancers such as HNSCC.
5
. Conclusions
In summary, results from our study indicated that the curcumin analog, WZ37, was
highly effective in inducing apoptosis of several HNSCC cell lines. Moreover, the apoptosis
was oxidant-dependent and regulated by ER stress signals, mitochondrial dysfunction, and
loss of Akt/mTOR survival signalling. Since this work was focused only on the cellular
mechanism of WZ37, further studies are necessary to validate this concept at the animal level
and to evaluate the preclinical pharmacodynamics and safety of WZ37 as an anti-cancer
candidate. These mechanistic insights of WZ37, along with improved stability and enhanced
anti-cancer potency over the parental compound, curcumin, provide strong support for the
novel WZ37 as a potential candidate for treatment of HNSCC.
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Supplementary file
Supplemental information includes 2 figures and can be found with this article online.
Acknowledgements
We kindly thank Professor Hazel Lum (Rush University, Chicago, IL) for language
editing of the manuscript.
This study was supported by Science Foundation of Zhejiang Province (LR18H160003 to
Y.Wang; LQ18H280007 to F. Lin), and National Natural Science Foundation of China
(81803781 to F. Lin; 81672627 to X. Zhang; 21572167 to C. Li; 81603153 to Y. Xia).
Conflict of interest
The authors declare that they have no competing interests.
Authors’ contributions
All authors have read and approved the submission of this manuscript; Z.Z., R.L., Y.X.,
L.Z., F.L., and X.Z. performed the research; G.L. and B.H. designed the research study; Y.Z.,
C.L., and L.W. contributed essential reagents or tools; L.W., B.H., and L.Z. analyzed the data;
G.L. and B.H. wrote the paper.
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Figure Legends
Figure 1. WZ37 inhibits cell viability of HNSCC in a dose-dependent manner
A) The structure of WZ37 and curcumin (CUR). Cell viability was assayed using the MTT
(
reagent (Methods) on several HNSCC cell lines: (B) 5-8F, (C) CNE-1, (D) CNE-2Z, (E)
CNE-2 and (F) HEP-2. The cells were treated with a range of concentrations (0.625, 1.25,
2
.5, 5, 10, 20, 40 μM) of WZ37, curcumin or cisplatin for 72 h, and viability reported as %
cell survival and IC50. Values were presented as means ± SEM from three individual
experiments.
Figure 2. WZ37 promotes apoptosis and cell cycle arrest in HEP-2 cells
HEP-2 cells were treated with WZ37 (5, 15, or 25 μM) or curcumin (CUR; 25 μM) for 24 h,
and analyzed as described below. (A) The treated cells were stained with Annexin V-
FITC/propidium iodide (Methods); shown are representative cytometric graphs of apoptotic
cells. (B) Quantification of the number of dead cells obtained in (A). (C) For cell cycle arrest
analysis, treated cells were stained with propidium iodide; shown are representative graphs of
number of HEP-2 cells arrested at G2/M as analyzed by flow cytometry. (D) Quantification
of arrested cells obtained in (C). (E) Treated HEP-2 cells were prepared for Western blot
analysis for detection of apoptosis-related proteins. Shown are representative blots from 3
separate determinations; GAPDH was used as internal control. (F) The treated HEP2 cells
were measured for caspase-3 activity using the substrate Ac-LEVD-pNA (Methods): the
relative caspase-3 activity was calculated as a ratio of treated cells to untreated cells. (G)
Shown is a representative Western blot analysis for detection of proteins related with G2/M
cell cycle regulation; 3 separate determinations; GAPDH was used as internal control. B, D,
and F: values reported as mean + SEM; n = 3; *P﹤0.05, ** P﹤0.01, and ***P﹤0.001.
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Figure 3. WZ37 induces ROS accumulation in HEP-2 cells
(A) The effects of WZ37 on ROS production in HEP-2 cells were determined using the
DCFH-DA fluorescent probe and detection by flow cytometry analysis (Methods); shown is a
representative cytometric graph of DCFH fluorescence (indicator of ROS content) in HEP2
cells treated with WZ37 at the indicated concentrations. (B) Quantification of the DCFH
fluorescence obtained from (A); values represented as the medium of relative fluorescence
intensity. (C) The effects of pre-treatment with NAC (5 mM for 2 h), an anti-oxidant, on
WZ37-induced ROS production were determined; shown is representative cytometric graph
of DCFH fluorescence in HEP-2 cells. (D) Quantification of the flow cytometric analysis
obtained in (C); values were calculated and represented as the medium of relative
fluorescence intensity. (E) The effects of NAC pre-treatment (5 mM for 2 h) on WZ37-
induced apoptosis were determined with Annexin V-FITC/propidium iodide stain and the
rates of apoptotic cells were detected by flow cytometry; shown are representative graphs of
apoptotic cells from treatment groups. (F) Quantification of the number of dead cells from
analysis in (E); values represented as % apoptotic cells. (G) Representative Western blot
analysis of the effects of NAC pre-treatment (5 mM for 2 h) of HEP-2 cells on expression of
apoptosis-related proteins; 3 separate determinations. B, D, and F: values reported as mean +
#
#
SEM; n = 3; *P﹤0.05, ** P﹤0.01 vs DMSO group; P﹤0.01 vs WZ37 group.
Figure 4. WZ37 induces endoplasmic reticulum (ER) stress in HEP-2 cells
Effects of WZ37 on ER stress response was determined in HEP-2 cells. Cells were treated
with WZ37 or curcumin (Cur) at the indicated time (A) and doses (B), or HEP-2 cells were
pre-treated with NAC (5 mM for 2 h) before exposure to WZ37 (25 μM) for 6 h (C), and ER
stress-related proteins were detected by Western blot analysis; shown is representative blot
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from 3 separate determinations; GAPDH was loading control. (D) The treated HEP-2 cells
were also evaluated for effects of WZ37 on ultrastructural changes of ER and mitochondria.
Black arrowheads indicate mitochondria and red arrowheads the ER; magnification ×
2
0,000; 3 separate determinations.
2
+
Figure 5. WZ37 impairs Ca homeostasis, mitochondrial function, and the Akt /mTOR
survival cascade
2
+
The effects of WZ37 on Ca homeostasis and mitochondrial function were determined in
HEP2 cells treated with WZ37 at the indicated concentrations and times. (A) Intracellular
2
+
2+
2+
Ca concentration ([Ca ]i) was measured using the Ca indicator fluorescent dye, Fluo
3
/AM (5 μg/ml) (Methods) and analyzed by flow cytometry; shown are representative graphs
2
+
2+
of Fluo fluorescence, indicating changes of [Ca ]i. (B) Quantification of the [Ca ]i in HEP-2
cells; values represent as the medium of relative fluorescence intensity; n = 3; values reported
as mean ± SEM; **P﹤0.01 compared to DMSO control. (C) Mitochondrial membrane
potential was determined to evaluate the effects of WZ37 on mitochondrial function. The
treated cells were stained with the fluorescent JC-1 membrane potential probe, and evaluated
by fluorescent microscopy. Red fluorescence represents a potential-dependent mitochondrial
aggregation, indicating intact mitochondrial membrane. Green fluorescence indicates
monomeric form of JC-1, indicating mitochondrial membrane depolarization; shown is
representative images from 3 separate determinations. (D) Mitochondrial ultrastructural
changes were evaluated from HEP-2 cells treated with WZ37 (25 μM) for 3 h or 24 h; black
arrowheads indicate mitochondria, and red arrowheads indicate ER; TEMs are representative
of 3 separate determinations; magnification ×20,000. (E) Effects of WZ37 or curcumin
(Cur) on expression of proteins of the AKT/mTOR and pro-apoptotic UPR pathways were
determined by Western blot analysis of HEP-2 cells treated with WZ37 (25 μM) at the
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indicated times or indicated doses for 24 h; shown is representative blot from 3 separate
determinations; GAPDH was used as loading control.
Figure 6. Inhibition of oxidant-sensitive Akt/mTOR promotes apoptosis
The role of AKT/mTOR signaling in regulation of the WZ37-induced apoptosis was
investigated with the Akt inhibitor, MK-2206, in HEP2 cells. Cells were treated with a
combination of WZ37 (25 μM) and MK-2206 (10 μM), or either reagents alone for 24 h. (A)
Shown is representative cytometric analysis of apoptotic cells from each treatment group. (B)
Quantification of apoptotic cells determined from (A); values reported as % apoptotic cells as
mean ± SEM; n = 3; * P<0.05, ** P<0.01, compared to DMSO control. (C) The treated HEP-
2
cells were analysed for expression of proteins of the AKT/mTOR pathway; shown is
representative Western blot analysis from 3 separate determinations; GAPDH was used as
loading control.
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Curcumin analog, WZ37, promotes G2/M arrest and apoptosis of HNSCC
cells through Akt/mTOR inhibition
1
,2,3,#
3
1,2,#
1
1
1
2
3
Ziheng Zhang
, Renyu Lin,
, Zhoudi Liu , Tao Yan , Yiqun Xia , Leping Zhao , Feng
2
1,
Lin , Xi Zhang , Chenglong Li , Yi Wang *
1
Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China
2
The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325035,
China
3
Department of Pharmacy, the Affiliated Yueqing Hospital of Wenzhou Medical University,
Wenzhou, Zhejiang 325000, China
Highlights:
1. New targeted therapies are in urgent needed for NHSCC treatment.
2. WZ37 inhibited proliferation of several HNSCC cell types by G2/M cycle arrest.
3.
WZ37 induced ROS-dependent mitochondrial injury and ER stress.
4
.
The anti-cancer effect of WZ37 was due to inhibition of the Akt/mTOR pathway.
WZ37 can be a promising anti-cancer agent for the treatment of HNSCC.
5.
28

Figure 1