Finding a resveratrol analogue as potential anticancer agent with apoptosis and cycle arrest

Article abstract of DOI:10.1016/j.jphs.2020.03.007

Resveratrol has been extensively studied as the anti-cancer agent. A variety of resveratrol analogues have been developed with structural modification to improve its bioactivity. In this work, resveratrol analogues, compound 1-4, were designed and synthesized with the Stille–Heck reaction. These results showed compound 1–4 had better anticancer effect than that of parent resveratrol. Especially compound 1 ((E)-4,4′-(ethene-1,2-diyl)bis(3-methylphenol)) displayed the excellent cytotoxicity and high selectivity. The mechanism research indicated compound 1 inhibited cell proliferation by binary paths of cell cycle arrest in S phase regulated by cyclin A1/A2 and apoptosis induction mediated by Bax/Bcl2 in a prooxidant manner.

Full text of DOI:10.1016/j.jphs.2020.03.007

Journal of Pharmacological Sciences xxx (xxxx) xxx
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Journal of Pharmacological Sciences
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Short Communication
Finding a resveratrol analogue as potential anticancer agent with
apoptosis and cycle arrest
Zhen-Hui Xin a, Hui-Hui Yang a, Yu-Han Gan a, Ya-Li Meng a, Ya-Peng Li b, Li-Ping Ge a,
,
*
Cun-Hui Zhang a, Lian-Na Liu a, Yan-Fei Kang a
a Hebei Key Laboratory of Quality & Safety Analysis-Testing for Agro-Products and Food and College of Laboratory Medicine, Hebei North University,
11 Diamond Street South, Zhangjiakou, 075000, Hebei, People's Republic of China
b Zhangbei Hospital, Guangchang Alley, Garden Street, Zhangbei Country, Zhangjiakou, 076450, Hebei, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Resveratrol has been extensively studied as the anti-cancer agent. A variety of resveratrol analogues have
been developed with structural modification to improve its bioactivity. In this work, resveratrol ana-
logues, compound 1-4, were designed and synthesized with the StilleeHeck reaction. These results
showed compound 1e4 had better anticancer effect than that of parent resveratrol. Especially compound
1 ((E)-4,40-(ethene-1,2-diyl)bis(3-methylphenol)) displayed the excellent cytotoxicity and high selec-
tivity. The mechanism research indicated compound 1 inhibited cell proliferation by binary paths of cell
cycle arrest in S phase regulated by cyclin A1/A2 and apoptosis induction mediated by Bax/Bcl2 in a
prooxidant manner.
Received 22 October 2019
Received in revised form
15 February 2020
Accepted 16 March 2020
Available online xxx
Keywords:
Resveratrol
Cell apoptosis
Cell cycle arrest
© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological
Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Carcinogenesis is a multistep and heterogeneous process, which
involves the DNA damage and imbalance of homeostasis resulting
in cells escaping the restraints into uncontrolled growth. Cancer
has been one of the most common non-communicable chronic
diseases, ranking as the leading cause of death worldwide.1
Therefore, the prevention and treatment of cancer is of great
importance.
Natural products, as potential sources of new drugs, provide
diversified and promising drug lead scaffolds, which have been the
hotspot in new drug discovery.2 Resveratrol (trans-3,5,40-trihy-
droxy-trans-stilbene, RES) with unique skeleton and pharmaco-
logical properties, has been increasing prominence.3 Many studies
have reported RES shown the anti/pro-oxidation, anti-inflamma-
tion, anti-microbial effects, especially anticancer activities via tar-
geting oxidative systems, inducing cell-cycle arrest, apoptosis or
autophagy.4,5 However, the poor cytotoxicity and low bioavail-
ability limit its practical application.6 Therefore, researchers often
modify the structure to improve its anticancer bioactivity.7
RES is consisted of two phenolic rings connected by a styrene
double bond with trans-stilbene skeleton. Up to now, the
modifications of RES mainly focus on the change fans-into cis-, the
reduction of carbon-carbon double bonds, the elongation of the
conjugated chains, and the substitution of amino, methoxyl, hy-
droxyl, or other groups on the benzene ring.7e11 Halogen, methyl
and trifluoromethyl played the important roles in improving drug
activities.9,11 Hence, in this work, we designed and synthesized the
compound 1e4 (Fig. 1) based on (1) structural modification of
methyl, chlorine, fluorine, trifluoromethyl and (2) group introduc-
tion at benzene ring in the adjacent position of the double bond
with increase of dihedral angle for improving ability of recognition
and bond between drug and proteins. We anticipated to obtain
molecules with better anticancer activities through the above
structural modification strategies. Interestingly, These results
showed the synthesized analogues of resveratrol have better anti-
cancer effect than that of parent resveratrol, and compound 1 ((E)-
4,40-(ethene-1,2-diyl)bis(3-methylphenol)) displayed the best
cytotoxicity with excellent selectivity. Consequently, the action
mechanism research demonstrated the compound 1 inhibited cell
proliferation by cell cycle arrest and apoptosis.
Firstly, the compounds 1e4 were synthesized based on the
classic one-step StilleeHeck tandem reaction (Fig. 1B, for details see
the Supporting Information). With the compounds 1e4 in hand, the
cytotoxic activities of the resveratrol and compound 1e4 were
firstly investigated by MTT assay in cancer cell lines (HeLa, MCF-7
* Corresponding author. Fax: þ86 313 4029275.
E-mail address: kangyanfei172@163.com (Y.-F. Kang).
Peer review under responsibility of Japanese Pharmacological Society.
https://doi.org/10.1016/j.jphs.2020.03.007
1347-8613/© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Xin Z-H et al., Finding a resveratrol analogue as potential anticancer agent with apoptosis and cycle arrest, Journal of
Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.03.007

2
Z.-H. Xin et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
Fig. 1. The structure and the synthesis of compounds 1e4.
and H1299) and normal cells (MRC-5). Exhilaratingly, compound
1e4 exerted more obvious cytotoxicity in HeLa and MCF-7 cells
compared to RES, which meant the drug modification strategy was
effective (Table S1). Compound 1 showed the most excellent
cytotoxicity among the remain compounds, especially in HeLa cells
Firstly, the cell cycle arrest with incubation of compound 1 was
investigated in HeLa. Flow cytometry result revealed that S phase
proportion increased significantly at 10 mM after treatment for 24 h/
48 h (Fig. 2A and B), which confirmed that compound 1 arrested
cell cycle progression in S phase. The precis and strict regulation of
the key checkpoints of the cell cycle, G1/S, intra-S, and G2/M, is
closely related to the cyclins. Based on the fact that cyclin A
mediated S phase progression, the western blot was performed to
detect its level. After incubation with compound 1 for 24 h, the
expression of cyclin A1 and cyclin A2 decreased dramatically at
with the IC50 value of 10.72 mM, and showed markedly less cyto-
toxicity in normal cells simultaneously (Figure S1). However, the
compound 2 didn't exhibit the excellent cytotoxicity and selectivity
like that of compound 1. Therefore, the change of dihedral angle of
molecular structure and the increase of methyl group's lipid solu-
bility played an important role for improving biological activities of
drug molecule.
Favorable activity and good selectivity of compound 1 encour-
aged us to further investigate its antiproliferation mechanism. As
we know, carcinogenesis is a complex and multi-stages process,
and may be associated with multiple mechanisms, especially the
disorder of cell cycle or the deregulation of apoptosis in response to
DNA damage or cellular perturbations, resulting in the abnormal
proliferation.12,13
concentration 16
mM (Fig. 2C), which validated compound 1
induced cell cycle arrest in S phase to inhibit cellular proliferation
by down-regulation the expression of cyclin A1 and cyclin A2 in
HeLa cells.
Besides, the induction of apoptosis is another valid mechanism
to inhibit the cell proliferation. To further explore the contribution
of apoptosis to the inhibition process, the apoptotic effect of com-
pound 1 was detected. As shown in Fig. 2D, the apoptotic effect was
found at concentration 8 mM and showed concentration-dependent
Fig. 2. Compound 1 inhibited tumor cell proliferation by both arresting cell cycle in S phase and apoptosis. (A) HeLa cells were treated with gradient concentration (control, 5
and 10 M, respectively) for 24 h. (B) HeLa cells were treated with gradient concentration (control, 4 M and 8 M, respectively) for 48 h. (C) Compound 1 down-regulated the
expression of cyclin A1 and cyclin A2. (D) HeLa cells were incubated with indicated concentration (control, 2 M, 4 M, 8 M, 16 M and 24 M, respectively) for 48 h. The
mM
m
m
m
m
m
m
m
m
percentage shown in each quadrant 4e1e2e3 represents the proportion of early apoptotic cells, late apoptotic cells, necrotic and normal cells, respectively. (E) DAPI fluorescence
observation showed apoptosis induction by compound 1 in HeLa. HeLa cells were treated with compound 1 at different concentration for 24 h, the apoptosis characteristic was
observed using fluorescent microscope after nuclear staining with DAPI. The white arrow indicates nuclear chromatin condensation and the formation of apoptotic bodies.
Please cite this article as: Xin Z-H et al., Finding a resveratrol analogue as potential anticancer agent with apoptosis and cycle arrest, Journal of
Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.03.007

Z.-H. Xin et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
3
characteristics as incubation time extended to 48 h. In addition,
apoptosis is characterized by cell shrinkage, chromatin condensa-
tion, nuclear fragmentation and the formation of apoptotic bodies,
which were observed by fluorescence microscopy with DAPI
staining. After 24 h of incubation with compound 1, DAPI staining
showed typical apoptosis characteristics with chromatin conden-
sation and nuclear fragmentation (arrow) of HeLa cells at above
investigate the source of ROS, we determined the expression of
mitochondrial complex V. The results displayed its expression was
decreased with incubation of compound 1, which lead to ROS level
increasement with reduction of ATP synthesis (Fig. 3D). In addition,
increased intracellular Ca2þ and elevated ROS levels promote each
other.15 Therefore, we tested the intracellular free Ca2þ with the dye
Fluo-3 AM. The result showed that the fluorescence increased
steadily in a compound 1 concentration dependent manner, which
indicated that the intracellular free Ca2þ was associated with
compound 1-induced apoptosis (Fig. 3E). Thus, this is the other
important reason for increase of ROS levels.
To further investigate the mechanism of apoptosis, we analyzed
the expression of apoptosis-related proteins including p53, p21, Bax
and Bcl-2 by western blot. After compound 1 treatment for 24 h, the
pro-apoptotic protein Bax up-regulated, while the anti-apoptotic
protein Bcl-2 down-regulated accordingly (Fig. 3F). Obviously, the
increased Bax/Bcl-2 ratio promoted apoptosis. Subsequently, the
Bax inhibitor peptide V5 (BIP V5), which also increased the
expression of Bcl-2, was utilized to further verified the role of Bax in
apoptosis induced by compound 1. The result showed that the
apoptosis effect started to reverse (Fig. 3G). However, the level of
p53, the key regulator in apoptosis induction, was not altered
significantly (Fig. 3F). Meanwhile, the cell apoptosis state didn't
concentration 8 mM (Fig. 2E). The result confirmed compound 1
inhibited HeLa cells proliferation by inducing apoptosis.
Excessive ROS accumulation may cause oxidative damage in
response to chemotherapeutic stimuli, which may lead to DNA
damage and cell death by apoptosis induction.14 Therefore, the ROS
level was detected to explore the apoptosis mechanism. Interest-
ingly, the DCF fluorescent intensity increased obviously with the
increase of compound 1 concentration (Fig. 3A). In addition, the cell
survival rate was improved after ROS scavengers, including gluta-
thione (GSH) and N acetyl L cysteine (NAC), which were separately
added in the cells combined with compound 1 (Fig. 3B). Next, the
O2ꢀꢁ-specific fluorescence probe dihydroethidium (DHE) and
dihydrorhodamine 123 (DHR 123 detects H2O2) were utilized to
analyze the ROS species. As shown in Fig. 3C, the fluorescence
intense of DHE increased about 23.0% after compound 1 treatment
at concentration 24 mM for 6 h, whereas the fluorescence of DHR
123 showed little variation. Obviously, ROS, especially O2ꢀꢁ, accu-
mulation ascribed to compound 1 which may enhance cytotoxicity
by oxidative stress leading to a disruption of cell signaling. To
relieve after compound 1 cotreatment with pifithrin-
inhibitor of p53 protein (Fig. 3G), which further evidenced that the
apoptosis induced by compound was p53-independent.
b (PFTb), an
1
Fig. 3. Apoptosis induction by compound 1 was attribute to the triangular interaction among increased ROS, complex V inhibition and intracellular free Ca2þ stimulation as well as
the increased Bax/Bcl-2 ratio in p21 dependent manner. (A) ROS level obviously increased after compound 1 treatment for 6 h. (B) The cell viability increased after treatment of ROS
scavengers (GSH 10 mM and NAC 10 mM) combined with compound 1 (10
24
m
M and 20
m
M, respectively) for 28 h. (C) The O2ꢀꢁ level increased after compound 1 treatment for 6 h at
m
M, but there was no difference in H2O2 between.treated and untreated samples. (D) The activity of mitochondrial complex V decreased significantly after compound 1 (36 mM)
treatment for 5 h. (E) The concentration of intracellular free Ca2þ increased remarkably in a concentration-independent manner after compound 1 treatment for 8 h. (F) The
expression of tumor suppressors p53, p21, pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 were analyzed. (G) Bax inhibitor BIP V5 relieved compound 1-induced
apoptosis, while the p53 inhibitor pifithrin-b didn't change the state. The asterisks (*) represents the significant differences (P < 0.05) between compared groups. The differences
analyzed by ANOVA using SPSS 22.0.
Please cite this article as: Xin Z-H et al., Finding a resveratrol analogue as potential anticancer agent with apoptosis and cycle arrest, Journal of
Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.03.007

4
Z.-H. Xin et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
Nonetheless, the tumor suppressor p21 was significantly increased
(Fig. 3F). Based on the above study, the apoptosis induction by
compound 1 may be attributed to p21 in p53-independent upre-
gulation of Bax and downregulation of Bcl-2. On the other hand,
p21 is also a negative regulator of the cell cycle by inhibiting
cyclinecyclin dependent kinase (CDK) complexes in response to
various cellular signals to inhibit cells proliferation.16 Considering
that compound 1 blocked cell cycle in S phase by down-regulation
of cyclin A1/A2 in HeLa cell as our testing shows, it is suspected that
compound 1 induced cell cycle arrest in S phase in a p21-dependent
manner. Therefore, compound 1 inhibited tumor cell proliferation
by binary paths of cell cycle arrested in S phase and apoptosis.
In summary, in this work, we synthesized a series of drug
molecules with resveratrol as the parent molecule based on methyl,
halogen and trifluoromethyl modification. The results showed
compound 1 possessed the best cytotoxicity and highest selective
anticancer potential in our test. Therefore, the tactics is effective for
improving biological activities based on structural modification of
methyl, chlorine, fluorine, trifluoromethyl and group introduction
at benzene ring in the adjacent position of the double bond with
increase of dihedral angle. The mechanism research displayed tu-
mor cell death was mediated by a binary pathway including cell
cycle arrest in S phase and apoptosis. On one hand, compound 1
cause cycle arrest mainly by regulating expression of cyclin and
CDK in a p21-dependent manner. On the other hand, the compound
1 can inhibit the expression of mitochondrial complex V causing a
decrease in ATP synthesis to lead increase of ROS levels. Meanwhile,
the increase of intracellular Ca2þ and the increase of ROS promote
each other, which had contributed significantly to the increase in
ROS levels. Up-expression of p21 protein with higher levels of ROS
can obviously increase Bax/Bcl-2 ratio, which lastly causes
apoptosis. The related detailed molecular mechanism is still under
further study in our laboratory. This study provides basic research
for designing effective anti-tumor drugs.
Education of Hebei province (Z2019028) and Hebei North Univer-
sity Fund (No. YB2018002; No. QN2018008; No. QN2018012).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jphs.2020.03.007.
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This work was supported by the Natural Science Foundation of
Hebei Province (No. B2016405026), Young Elitist Foundation of
Hebei Province (No. BJ2016003), Hebei North University PhD
startup fund (No. 12995556), Guide projects of Department of
Please cite this article as: Xin Z-H et al., Finding a resveratrol analogue as potential anticancer agent with apoptosis and cycle arrest, Journal of
Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.03.007