Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits bladder cancer cell growth via the mitochondrial apoptosis and JNK pathways

Article abstract of DOI:10.1016/j.taap.2019.03.027

Erianin, a component extracted from the traditional Chinese herbal medicine Dendrobium, has shown significant anti-tumour activity in various cancers but not in bladder cancer. In this study, we assessed the effects of Erianin on bladder cancer growth and elucidated the related mechanisms. First, Erianin was synthesized with high yields, and markedly suppressed EJ and T24 cell proliferation. It induced G2/M-phase arrest in vitro. Furthermore, Erianin triggered apoptosis via caspase cascades activation and the mitochondrial-mediated apoptotic pathway. Bim up-regulation and Bcl-2 down-regulation as the symbol of apoptosis which were found to play the dominant role in the effects of Erianin. We further showed that JNK pathway activation is necessary for the Erianin-mediated anti-proliferation and apoptotic response. Finally, Erianin exhibited anti-tumour activity and induced apoptosis in tumour tissue in vivo. Collectively, these results suggest that Erianin induced cell cycle G2/M-phase arrest and apoptosis via the JNK signalling pathway in bladder cancer, indicating the potential usefulness of Erianin for the therapy of bladder cancer.

Full text of DOI:10.1016/j.taap.2019.03.027

Accepted Manuscript
Erianin, a novel dibenzyl compound in Dendrobium extract,
inhibits bladder cancer cell growth via the mitochondrial
apoptosis and JNK pathways
Qiyu Zhu, Yuwen Sheng, Wenhua Li, Jing Wang, Yulin Ma,
Baowen Du, Yaxiong Tang
PII:
S0041-008X(19)30117-6
https://doi.org/10.1016/j.taap.2019.03.027
YTAAP 14555
DOI:
Reference:
To appear in:
Toxicology and Applied Pharmacology
Received date:
Revised date:
Accepted date:
14 November 2018
29 March 2019
30 March 2019
Please cite this article as: Q. Zhu, Y. Sheng, W. Li, et al., Erianin, a novel dibenzyl
compound in Dendrobium extract, inhibits bladder cancer cell growth via the
mitochondrial apoptosis and JNK pathways, Toxicology and Applied Pharmacology,
https://doi.org/10.1016/j.taap.2019.03.027
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ACCEPTED MANUSCRIPT
Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits
bladder cancer cell growth via the mitochondrial apoptosis and JNK
pathways
a,1
a,1,*
a
a
Qiyu Zhu , Yuwen Sheng
shengyuwen0326@163.com, Wenhua Li , Jing Wang ,
a
a
a,*
Yulin Ma , Baowen Du , Yaxiong Tang tangyx@cib.ac.cn
a
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041,
China
b
University of Chinese Academy of Sciences, Beijing 100000, China
*
Corresponding authors.
Abstract
Erianin, a component extracted from the traditional Chinese herbal medicine
Dendrobium, has shown significant anti-tumour activity in various cancers but not in
bladder cancer. In this study, we assessed the effects of Erianin on bladder cancer
growth and elucidated the related mechanisms. First, Erianin was synthesized with
1
These authors contributed equally to this work.

ACCEPTED MANUSCRIPT
high yields, and markedly suppressed EJ and T24 cell proliferation. It induced
G2/M-phase arrest in vitro. Furthermore, Erianin triggered apoptosis via caspase
cascades activation and the mitochondrial-mediated apoptotic pathway. Bim
up-regulation and Bcl-2 down-regulation as the symbol of apoptosis which were
found to play the dominant role in the effects of Erianin. We further showed that JNK
pathway activation is necessary for the Erianin-mediated anti-proliferation and
apoptotic response. Finally, Erianin exhibited anti-tumour activity and induced
apoptosis in tumour tissue in vivo. Collectively, these results suggest that Erianin
induced cell cycle G2/M-phase arrest and apoptosis via the JNK signalling pathway in
bladder cancer, indicating the potential usefulness of Erianin for the therapy of
bladder cancer.
Keywords: Erianin, Bladder cancer, Apoptosis, JNK pathway, In vivo.
1
. Introduction
Bladder cancer is the most common and uncontrollable heterogeneous tumour
worldwide (Jin et al., 2015). According to American cancer statistics, an estimated
9,030 new cases and 16,870 deaths in the United States in 2017 will be due to
bladder cancer (Siegel et al., 2017). At present, surgical resection and chemotherapy
7

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are the most common bladder cancer treatments and are widely used throughout the
world (Marta et al., 2012). However, due to poor diagnosis, these treatments have
little effect on 70% of bladder cancer patients presenting with superficial tumours
(Zhang et al., 2016). The five-year recurrence rate for bladder cancer patients remains
at 50~70% (Jones et al., 2000). Targeted therapy, which is known to exhibit low
toxicity and high efficiency, has become an area of great interest for cancer treatment.
Some targeted drugs, such as Atezolizumab and Avelumab (Krishnamurthy et al.,
2
017; Vandeveer et al., 2016), have shown promising results in bladder cancer
treatment. However, targeted therapy is limited in bladder cancer by unclear targets.
Therefore, more chemopreventive and chemotherapy drug candidates are urgently
needed for controlling bladder cancer progression.
Apoptosis is an evolutionary process that occurs after physiological and
pathological stimulation and induces programmed cell death (Elmore, 2007). This
process involves morphological and biochemical changes, such as membrane
blebbing, DNA fragmentation, chromatin condensation, protein cleavage, et al
(Kalimuthu and Se-Kwon, 2013). Furthermore, apoptosis is accompanied by the
activation of the extrinsic and intrinsic apoptosis pathways and other pathways. The
extrinsic pathway, also called the death receptor pathway, is initiated when the death
receptor Fas is activated and initiated caspase cascades (Kantari and Walczak, 2011).
The intrinsic pathway, which is also known as the mitochondria-dependent pathway,
is primarily regulated by the Bcl-2 protein family and mediated apotosis by a release
of mitochondrial cytochrome c (Sawada et al., 2000). In addition, the JNK pathway is

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the crucial role in cell survival and apoptosis (Son et al., 2011). In response to an
apoptosis signal, JNK rapidly moves to the mitochondrial membrane and induces
cytochrome c release (Tobiume et al., 2001). In addition, JNK can activate Bim and
then activate Bax or Bak, thus inducing apoptosis (Marani et al., 2002). Many
previous reports have shown that cell survival inhibition and apoptosis induction by
therapeutic agents might be correlated with the tumour response, and some drugs have
shown significant inhibition of bladder cancer by inducing apoptosis (e.g., Artesunate
and Naproxen) (Zuo et al., 2014; Kim et al., 2014). Thus, using agents to promote cell
apoptosis is a promising strategy for bladder cancer treatment.
The medicinal value of plants has been recognized by many cultures and most
human societies for millennia. The chemical diversity in the natural world are
unsurpassed by synthetic compounds ( Davison EK and Brimble MA, 2019). Many
phytochemicals have been used for cancer treatment in human clinical trials and have
shown promising anti-cancer effects due to their low toxicity, safety, and general
availability (Gupta et al., 2001). Modern pharmacological studies have found that
Dendrobium contains various chemical components with extensive pharmacological
effects (Huang et al., 2016; Li et al., 2018; Yuan et al., 2019). Erianin is a
low-molecular-weight natural product extracted from Dendrobium chrysotoxum (Ma
et al., 1994) and belongs to the stilbene dihydrostilbene derivative family named
combretastatins, which consistently exert anti-proliferative effects on carcinoma cell
lines (Ma et al., 1994). Similarly, Erianin has been reported to exert an obvious
inhibitory effect on cell proliferation and a promotive effect on apoptosis in liver

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cancer, colon cancer, gastric carcinoma and leukaemia HL-60 cells (Li et al., 2001).
However, to the best of our knowledge, neither the mechanism nor the effect of
Erianin on bladder cancer have been reported. Hence, in this study, we assessed the
anti-tumour potential and molecular mechanisms of Erianin in human bladder cancer
EJ and T24 cells and explored the potential value of Erianin application for
chemoprevention and bladder cancer therapy.
2
. Materials and methods
2
.1 Compounds
Erianin was synthesized from 3,4,5-trimethylbenzaldehyde. The intermediate
product bromotriphenyl(3,4,5-trimethoxybenzyl)-phosphane was obtained through

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borohydride reduction and the Michaelis-Arbuzov reaction and was then was reacted
with the hydroxyl groups of isovanillin through Wittig, borohydride reduction and
dehydroxylation reactions, yielding a white powder that was identified as Erianin
1
(
1.49 g, 95%, purity 98.5%). H NMR (400 MHz, CDCl3) δ 6.81 (d, J = 1.9 Hz, 1H),
6
.77 (d, J = 8.2 Hz, 1H), 6.64 (dd, J = 8.2, 1.9 Hz, 1H), 6.38 (s, 2H), 5.59 (s, 1H), 3.87
(s, 3H), 3.83 (d, J = 1.2 Hz, 9H), 2.82 (s, 4H).
Pure Erianin was dissolved in dimethyl sulfoxide (DMSO) and stored at -80°C.
2
.2 Reagents
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were
obtained from Sigma-Aldrich (St. Louis, MO, USA). 4,6-diamino-2-phenyl indole
DAPI), the JNK inhibitor SP600125, the caspase inhibitor Z-VAD-FMK, Annexin
(
V-FITC detection kit, a One-Step TUNEL Apoptosis Detection Kit were purchased
from Beyotime (Shanghai, China). The cell cycle assay kit, the mitochondrial
membrane potential kit (JC-1), the cytochrome c-releasing apoptosis assay kit were
purchased from KeyGEN BioTECH (Nanjing, China). Primary antibodies against
β-actin, p-Chk2, p-cdc2, p-H2A.X, p-cdc25C, PARP, caspase-9, caspase-7, caspase-3,
cytochrome c, COX IV, Bim, Bik, Bax, Bad, BID, Bcl-2, Mcl-1, p-JNK1/2, p-c-Jun
and p-Bcl-2 were purchased from Cell Signalling Technology (Beverly, MA, USA).
Chemiluminescence detection reagents were obtained from Millipore (Plano, TX,
USA). Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA).
2
.3 Cell culture

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The human bladder cancer cell lines EJ and T24 were obtained from State Key
Laboratory of West China Hospital Cancer Center, Sichuan University, China. EJ and
T24 cells were cultured
in RPMI-1640
medium containing 1%
penicillin-streptomycin and 10% (v/v) FBS in moist air with 5% CO2 at 37°C.
2
.4 Cell proliferation assay
Cell proliferation was examined through MTT assays. Briefly, EJ and T24 cells
4
were seeded into 24-well plates at 2 × 10 cells per well and incubated for 24 h. After
treatment, the cells were incubated for 48 h, then 0.5 mL of MTT solution (final
concentration of 1 mg/mL) was added to each well and incubated for 4 h. The MTT
solution was then removed, and 0.5 mL of acidified isopropanol (with 0.4% HCl) was
added to solubilize the formazan crystals. Three replicates were performed, and the
OD570 was analysed using a microplate reader (BD Biosciences, Mountain View, CA,
USA). The ratio of viability (%) was calculated as ODsample/ODcontrol × 100%.
2
.5 Cell cycle synchronization analysis
The cell cycle synchronization of EJ and T24 cells was achieved by serum
starvation. The cells were treated with serum-free medium for 24 h, pretreated with
Z-VAD-FMK (20 µM) and SP600125 (10 µM) for 2 h, and then treated with Erianin
for 24 h. The cells were harvested, fixed with 70% (v/v) ethanol overnight at -20°C
and washed with PBS and then labelled with propidium iodide (50 µg/mL) and
incubated at RT in the dark for 30 min. Approximately 10000 cells in each sample
were analysed with a FACScan flow cytometer (BD Biosciences, Mountain View, CA,

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USA), and the percentages of cells in the G1, S, and G2/M cell cycle phases were
investigated using ModFIT software as described previously (Tang et al., 2008).
2
.6 Cell morphology observation and DAPI staining
EJ and T24 cells in the logarithmic phase were plated in six-well culture plates.
after the cells adhered to the plate, After Erianin treatments for 24 h, the medium was
removed, and the cells were washed three times with PBS. The cells were then
observed and photographed using an inverted microscope (Olympus, Tokyo, Japan).
The cells were inoculated on a cover slip treated with Erianin. After the treatments,
the cells were fixed with 4% paraformaldehyde for 30 min and stained with 1 µg/mL
DAPI for 5 min. Finally, the cells were observed with a fluorescence microscope
(Olympus, Tokyo, Japan) using emission and excitation wavelengths of 460 nm and
3
50 nm, respectively.
2
.7 Annexin V/PI analysis
To quantify the cell death modality, EJ and T24 cells were stained using an
Annexin V-FITC detection kit. Cells in the exponential growth phase were seeded in
2
6
0-mm dishes, incubated at 37 ℃ for 24 h, pretreated with Z-VAD-FMK (20 µM)
and SP600125 (10 µM) for 2 h, and then treated with Erianin for 24 h. The cells were
then collected, washed twice with PBS, resuspended in 400 µL of Annexin binding
buffer, stained with 5 µL of Annexin V-FITC and 10 µL of PI, and incubated for 15
min at RT in the dark. The stained cells were analysed with a FACScan flow
cytometer (BD Biosciences, Mountain View, CA, USA), and the percentages of

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apoptotic cells were investigated using FlowJo software.
2
.8 Analysis of mitochondrial membrane potential
To investigate the mitochondrial membrane potential, a mitochondrial membrane
potential kit (JC-1) was used. EJ cells were seeded into six-well culture plates,
incubated overnight and treated with various concentrations Erianin for 24 h. The
medium was then removed, 1 mL of JC-1 dye working solution was added (according
to the manufacturer’s recommended protocol), and the cells were incubated for 20
min at 37°C. After incubation, the cells were washed twice with JC-1 dyeing buffer,
and then, 2 mL of cell culture medium was added. Cell staining was observed with a
fluorescence microscope or analysed with a flow cytometer.
2
.9 Measurement of cytochrome c release from mitochondria
Cells were treated with Erianin for 24 h. The cytosol and mitochondria were
separated using a cytochrome c-releasing apoptosis assay kit. The cells were
suspended in cytosol extraction buffer, mixed in a Dounce homogenizer, and
centrifuged (700 × g) for 10 min at 4°C. The supernatant was collected and
recentrifuged (10,000 × g, 30 min) at 4°C. The pellet (mitochondrial fraction) and
supernatant (cytosolic fraction) were used for western blot analysis.
2
.10 Western blot analysis
EJ and T24 cells were lysed in ice-cold radioimmunoprecipitation (RIPA) buffer
(50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate,
0
.1% sodium dodecyl sulphate (SDS), 1 mmol/L ethylenediaminetetraacetic acid

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(
EDTA), and 1% protease inhibitor cocktail (v/v)). Total protein (40–80 µg) was
separated by SDS–PAGE and then transferred to nitrocellulose membranes. The
membranes were blocked with 5% non-fat milk for 1 h, labelled with primary
antibodies overnight at 4°C, subjected to four 10-min washed with PBS with Tween
(PBST), and incubated with horseradish peroxidase-conjugated secondary antibody
for 1–2 h at RT. According to the manufacturer’s recommended protocol, the target
proteins were then detected via enhanced chemiluminescence.
2
.11 Plasmid transfection
The pcDNA3.1, pcDNA3.1-Bcl-2-V5-tag and pCMV-TAM67 plasmids were
generous gifts from Dr. Xiaolin Zi (University of California Irvine, Orange, CA,
USA). EJ and T24 cells were plated in 24-well plates and transiently transfected with
pcDNA3.1, pcDNA3.1-Bcl-2-V5-tag or pCMV-TAM67 using Lipofectamine 2000,
and 48 h after transfection, the cells were treated with Erianin 48 h after transfection.
2
.12 RNA interference
The Bim-targeting sequences were designed using the RNAi Designer
programme, and the RNAi cassettes were cloned into a PENTR™/U6 Entry vector
and subsequently transferred into a pBLOCK-iT™3-DEST vector using Clonase™ to
catalyse a recombination reaction (Invitrogen, Carlsbad, CA, USA). The following
Bim-targeting
’-GGAGACGAGTTTAACGCTTAC-3’. Prior to the experiment, 4 × 10 EJ and
T24 cells were seeded in each well of a 24-well plate. When the cells reached
sequence
was
selected
for
further
studies:
4
5

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approximately 60% confluence, they were transfected with shRNA using
Lipofectamine 2000.
2
.13 In vivo tumour model
NCR-nu/nu (nude) mice were purchased from Taconic, and the animal
experiments were approved by the Committee of Animal Ethics of Chengdu Institute
of Biology, Chinese Academy of Sciences, China (#CIBCAE20160814), and were
performed in strict accordance with the Guide for the Care and Use of Laboratory
Animals of the Ministry of Health, China. At the end of the animal experiment, the
animals were sacrificed via cervical vertebral dislocation to reduce the pain of the
animals. Erianin was dissolved in 10% grain alcohol in 0.9% saline and administered
6
via gavage to the mice. Then, 2 × 10 EJ cells per 100 µL of PBS were injected s.c.
3
into the right flank of the nude mice. The tumours grew to approximately 120 mm
after seven days, and the mice bearing EJ tumours were randomly divided into the
control and treatment groups, with 10 mice in each group, and dosed daily with
vehicle or 50 mg/kg Erianin. Because no in vivo data regarding s.c. Erianin
administration are mentioned in the literature (only i.p. injections are described)
(Wang et al., 2016; Yu et al., 2016), the Erianin dose (50 mg/kg) used in this study
was based on the pharmacologically effective and non-toxic doses of other
compounds determined in our laboratory. The water consumption, diet and body
weight were recorded thrice weekly. Once xenografts started growing, the tumour size
was measured every three days, and the volume was calculated using the formula
2
0
.5236 L1 (L2) , where L1 is the long axis and L2 is the short axis of the tumour. At the

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end of the experiment, the tumours were removed, weighed, and stored at −80°C.
2
.14 TUNEL assay
For detection of the apoptotic induction effect, we used a One-Step TUNEL
Apoptosis Detection Kit according to the manufacturer’s recommended protocol.
Briefly, the samples were fixed with 4% formaldehyde solution for 40 min, washed
twice in PBS treated with 0.1% Triton X-100 in PBS and incubated on ice for 2 min.
The reaction mix was prepared according to the manufacturer’s recommended
protocol. After two washes in PBS, staining was performed at 37°C for 60 min away
from light, and the resulting staining was observed with a fluorescence microscope
(Olympus, Tokyo, Japan).
2
.15 H&E staining
At the end of the study, tumour tissues were harvested from each mouse and
fixed in 10% phosphate-buffered formalin for 24 h. The formalin-fixed tissues were
paraffin-embedded, sectioned, and stained with H&E. The sections were evaluated for
the degree of tumour cell apoptosis, and the apoptotic cells were counted under a
microscope.
3
. Results
3
.1 Erianin inhibited human bladder cancer cell proliferation but had minimal
effects on normal cells

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Erianin is a natural compound primarily extracted from the precious traditional
Chinese herb Dendrobium; however, considering the low extract yield, Erianin was
synthesized in this study from 3,4,5-trimethylbenzaldehyde using a series of reactions,
including hydroboration, Michaelis-Arbuzov, and Witting reactions (Fig. 1A)
(
Cushman M, et al., 1991) with an excellent yield (95%). The structure of Erianin
was confirmed by nuclear magnetic resonance spectroscopy, and the final product was
of analytical grade (Supporting Information Figs. A and B in S1 File).
To investigate the inhibitory effects and cytotoxicity of Erianin in bladder cancer
cells, EJ and T24 cells were treated with various Erianin concentrations for 48 h and
then subjected to MTT assays. As shown in Fig. 1B, Erianin inhibited EJ and T24 cell
growth in a dose-dependent manner. The IC50 values of Erianin in the EJ and T24 cell
lines were estimated to equal 65.04 nM and 45.9 nM. However, Erianin had a
minimal effect on normal liver cell growth. The selective killing effect of Erianin on
bladder cancer cells over normal cells suggest its potential value for bladder cancer
treatment.
3
.2 Erianin induced G2/M arrest in EJ and T24 cells
To explore whether the proliferation inhibition effect of Erianin was induced
through disruption of cell cycle progression, a flow cytometry analysis was performed
to examine the cell cycle distribution. The results indicated that the relative
percentage of cells in the G2/M phase increased from 3.00% in the control population
of EJ cells to 55.0% in the population of EJ cells treated with different Erianin

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concentrations (40, 80, or 160 nM) (P < 0.05) and from 8.29% in the control
population of T24 cells to 51.9% in the population of T24 cells treated with various
Erianin concentrations (25, 50, 100 nM) (Fig. 2A-2B), and these increases were
accompanied by decreases in the numbers of cells in the G0/G1 phases. However,
Erianin couldn’t induce the cell cycle arrest in the normal liver LO2. Furthermore, a
western blotting analysis was performed to examine the protein profile of cell cycle
markers and DNA damage response proteins. The results showed that phospho-Chk2,
phospho-cdc2 and phospho-H2A.X expression was up-regulated and the level of
phospho-Cdc25c was down-regulated (Fig. 2C), but Erianin not induced the
expression change of phospho-Chk2, phospho-cdc2, phospho-H2A.X and
phospho-Cdc25c in LO2 cells, indicating that Erianin induced G2/M cell cycle arrest
by modulating cell cycle-related protein expression in EJ and T24 cells but not in the
normal liver LO2 cells. In addition, we used the different dose range for EJ cells and
T24 cells because of the different IC50 values of Erianin in both cells.
3
.3 Erianin inhibited EJ and T24 cell proliferation by inducing apoptosis
Apoptosis is a crucial event in tumour cell fate decisions. To explore whether the
proliferation inhibition effect of Erianin was related to apoptosis induction, DAPI
staining was performed to investigate morphological changes in bladder cancer cells
treated with Erianin. The Erianin-treated cells exhibited a typical apoptotic
morphology, including cell shrinkage, nuclear changes, and chromatin fragmentation,
whereas these characteristics were not observed in control cells (Fig. 3A) and LO2
cells(Fig. S4). To further verify a causal relationship between cell growth inhibition

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and apoptosis, the effects of Erianin on apoptosis and cell death in bladder cancer
cells were examined by Annexin V-FITC/PI staining followed by flow cytometric
analysis. Erianin treatment significantly increased the number of apoptotic cells
compared with the control treatment (Fig. 3B). Furthermore, treatment of the cells
with the combination of Erianin and Z-VAD-FMK (20 µM) conspicuously decreased
the number of apoptotic cells and the reversed induced G2/M cell cycle arrest (Fig.
3
C). Nevertheless, Erianin treatment not increased the number of apoptotic cells
compared with the control treatment(Fig. S6) in LO2. The activation of caspase
cascades plays an executive role in the apoptosis process. As shown in Fig. 3D,
Erianin induced activation of caspase-9/7/3 cleavage, thus inducing caspase cascades
and eventually leading to PARP cleavage in both EJ and T24 cells, but Erianin
couldn’t induce PARP cleavage in LO2 cells(Fig.S6). Furthermore, the caspase
inhibitor Z-VAD-FMK (20 µM; the Z-VAD-FMK dose response curve in EJ and T24
cells is shown in Supporting Information Fig. 2A in S1 File) was used to intervene
with Erianin-induced apoptosis in bladder cancer cells. Consistently, the treatment of
bladder cancer cells with the combination of Erianin and Z-VAD-FMK significantly
decreased the number of cells (Fig. 3E). Hence, the proliferation inhibition effect of
Erianin is primarily associated with apoptosis induction through activation of caspase
cascades.
3
.4 Erianin induced cytochrome c release and related changes in the mitochondrial
membrane potential in bladder cancer cells
Apoptosis can be induced by the extrinsic pathway, which is associated with

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death receptor stimulation on the cell surface, or by the intrinsic pathway, which is
characterized by mitochondrial dysfunction. To explore whether apoptosis induced by
Erianin was related to the intrinsic pathway characterized by mitochondrial
involvement, the effect of Erianin on mitochondrial membrane potential was
determined using the mitochondrial probe JC-1. A fluorescence microscopy analysis
indicated that Erianin markedly enhanced green fluorescence compared with that
observed in the control group, whereas a flow cytometry analysis showed that 55% of
EJ cells presented an altered membrane potential compared with the control cells after
treatment with 160 nM Erianin (Fig. 4A-4B).
Furthermore, considering the crucial role of cytochrome c release in the
mitochondrial-related intrinsic pathway, cytochrome c expression in the mitochondria
and cytoplasm was examined by western blotting. The results showed that in both EJ
and T24 cells, Erianin treatment induced cytochrome c release from the mitochondria
into the cytosol (Fig. 4C), indicating that the mitochondrial-mediated apoptotic
pathway was activated.
3
.5 Erianin activated the mitochondrial-mediated apoptotic pathway by regulating
the protein expression of Bcl-2 family members
Bcl-2 family members are important in the apoptosis process and include
anti-apoptotic and pro-apoptotic proteins, and regulation of the delicate balance
among these members determines cell fate. The expression of the pro-apoptotic
proteins Bik, Bax, Bim, BID and Bad and the anti-apoptotic proteins Mcl-1 and Bcl-2

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in EJ, T24 and LO2 cells treated with 0.1% DMSO or Erianin was investigated by
western blotting. The results indicated that Erianin up-regulated Bim, Bik, BID and
Bad and clearly down-regulated Bcl-2 and Mcl-1 but had no effect on Bax in EJ cells.
In T24 cells, Erianin up-regulated Bim and Bad and down-regulated Bcl-2 (Fig. 5A).
However, the expression of pro-apoptotic proteins Bik, Bax, Bim, BID and Bad and
the anti-apoptotic proteins Mcl-1 and Bcl-2 were unchanged in LO2 compared with
control. Taken together, these results indicate that Erianin activated the
mitochondria-mediated apoptotic pathway by regulating the balance between
pro-apoptotic and anti-apoptotic protein expression.
In addition, given that Bim up-regulation and Bcl-2 down-regulation were
confirmed in both EJ cells and T24 cells, we further examined their roles in the
growth inhibitory effect of Erianin. EJ and T24 cells were subjected to the knockdown
of Bim via short hairpin RNAs and the overexpression of Bcl-2 followed by Erianin
treatment (40 nM, 80 nM, or 160 nM (EJ cells) and 25 nM, 50 nM, or 100 nM (T24
cells)) for 48 h, and cell proliferation was then measured through MTT assays.
Consistent with the above-described results, the suppression of Bim expression and
the overexpression of Bcl-2 attenuated the growth inhibition effect of Erianin on
bladder cancer cells (Fig. 5B-5C). These results suggest that Bim and Bcl-2 are, at
least in part, required for the cell growth inhibition effect of Erianin.
3
.6 Erianin induced apoptosis via JNK pathway activation
The JNK family plays a core role in the signalling pathways associated with cell

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differentiation, proliferation and apoptosis. Therefore, to examine whether Erianin
induced apoptosis through the JNK pathway, the cells were treated with Erianin only
or pretreated with the JNK inhibitor SP600125 (10 µM; the SP600125 dose response
curve for EJ and T24 cells is shown in Supporting Information Fig. B in S2 File) and
then treated with Erianin. Western blotting results showed that treatment with Erianin
activated JNK signalling through phospho-JNK, phospho-c-Jun and phospho-Bcl-2
(Ser70) up-regulation. However, the JNK inhibitor SP600125 potently inhibited the
JNK pathway activation induced by Erianin (Fig. 6A) in EJ cells. Next, to verify the
effect of JNK pathway activation on Erianin-induced growth inhibition, the cells were
first pretreated with SP600125 and then treated with Erianin for an additional 24 h. As
shown in Fig. 6B, pretreatment with SP600125 clearly attenuated the inhibition of
proliferation induced by Erianin. Furthermore, the transient transfection of EJ cells
with plasmid encoding dominant negative c-Jun (pCMV-TAM67) to inhibit c-Jun
activation, the growth inhibition induced by Erianin was also attenuated (Fig. 6C). A
flow cytometric analysis also indicated that SP600125 attenuated the Erianin-induced
G2/M arrest (Fig. 6D), and Annexin V-FITC/PI staining showed that SP600125
reduced the apoptotic effect of Erianin (Fig. 6E). Taken together, these results
confirmed that Erianin-induced activation of the JNK pathway plays a crucial role in
the induction of both cell proliferation and apoptosis in EJ and T24 cells.
3
.7 Erianin inhibited tumour growth in vivo
To further examine the anti-proliferation effects of Erianin in vivo, a xenograft
mouse model was constructed through the subcutaneous transplantation of EJ cells.

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After two weeks, the mice were orally administered 50 mg/kg Erianin daily for 24
days. The growth rate of EJ cell-derived tumours treated with Erianin significantly
decreased compared with that of the control group (P < 0.05) (Fig. 7A). The wet
tumour weights were measured, and the mean results for the control and
Erianin-treated groups were 1479 ± 26 and 516 ± 2.3 mg, respectively. Erianin
treatment reduced tumour growth by approximately 65% (Fig. 7B). Simultaneously,
to examine the potential apoptosis-inducing effect of Erianin in tumour tissues, a
TUNEL staining analysis was performed, and the results demonstrated a notable
increase in DNA breakage in the Erianin-treated tumour tissues in comparison with
the control group (Fig. 7C). Haematoxylin and eosin (H&E) staining of tumour tissues
obtained at the end of the study showed that the untreated tumour cells and their
nuclei exhibited an uneven staining intensity, and the morphology and eosin staining
of the cytoplasm of the untreated tumour cells was lighter than that of Erianin-treated
tumour cells; in addition, tissue from Erianin-treated mice exhibited intense
eosinophilic staining, which indicated massive apoptosis of tumour cells (Fig. 7D).
These data suggest that Erianin exhibited potent anti-tumour activity by inducing
apoptosis in vivo.
4
. Discussion
Natural medicinal plants are important sources for the research and development

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of novel drugs due to the diversity of their biological sources and chemical structures.
Dendrobium is commonly known as one of the largest and most important
(
medicinally and ornamentally) herbs in the Orchidaceae family (Teixeira et al., 2014),
which includes various species that are extensively used as Chinese medicinal
materials, such as D. aphyllum, D. candidum, D. chrysanthum, and D. densiflorum (Li
et al., 2011; Ng et al., 2012). Dendrobium species have been reported to exert various
bioactivities,
including
antioxidant,
immunomodulatory,
neuroprotective,
anti-inflammatory, and strong anti-tumour activities, in hepatic carcinoma, lymphoma,
and lung cancer (Gong et al.,2004; Prasad and Koch, 2014; Charoenrungruang et al.,
2
014). Erianin is the most abundant active component extracted from Dendrobium
and shows significant anti-tumour activity in some human cancers, such as human
osteosarcoma cells (Wang et al., 2016), but its effect on bladder cancer has not been
previously examined. The cytotoxicity observed in this study indicated that Erianin
clearly inhibited EJ and T24 cell proliferation but exhibited little cytotoxicity toward
normal liver LO2 cells in vitro. In addition, Erianin inhibited in vivo tumour growth
without obvious toxicity. Furthermore, our results showed that Erianin caused G2/M
cell arrest and DNA damage, because of phospho-H2A.X in the sign of DNA damage.
Cells instantaneously block cell cycle progression, to allow time for repair when DNA
damage( Shaltiel IA, et al., 2015). Erianin could up-regulated the expression of
phospho-Chk2, phospho-cdc2, phospho-H2A.X and down-regulated the expression of
phospho-Cdc25c at 160 nM, indicating Erianin induced the G2/M arrest by DNA
damage and induced apoptosis through the mitochondria-related intrinsic pathway and

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that the JNK signalling pathway played a critical role. Thus, Erianin might have
potential value for bladder cancer treatment due to its low toxicity and high apoptosis
induction efficiency.
Cell death is commonly induced by apoptosis, necrosis or autophagy, but among
these, apoptosis plays more important role in deciding cell fate, particularly in cancer
cells. Apoptosis leads to substantial changes in cell morphology, such as membrane
blebbing and cell shrinkage, and to DNA fragmentation, chromatin condensation and
the formation of apoptotic bodies (Li et al., 2007). These changes were induced in the
cells through activation of the caspase signalling pathway. Caspase family proteins are
the critical executors of the apoptosis pathway. All caspases are initially present in
inactivate zymogen form, and when cells receive apoptosis signals, the mitochondrial
membrane potential decreases, cytochrome c is released from the mitochondria, and
apoptotic bodies are formed. The apoptotic bodies activate the initiator caspase-9,
which acts on the downstream target effector caspase-7/3 (Shi, 2002; Ohtsuka et al.,
2
003). Activated caspase-9/7/3 induces PARP cleavage, which is a hallmark of
apoptosis and important in many cellular processes, such as DNA repair, cell death,
chromatin function and genomic stability. PARP cleavage occurred via caspase-9/7/3
activation in Erianin-treated EJ and T24 cells. In addition to the caspase family,
another protein family is known to be very important in the apoptotic process: Bcl-2
family proteins (Wang et al., 2007). The Bcl-2 family proteins include
apoptosis-promoting proteins (e.g., Bim, BID, Bad, Bax, and Bik) and anti-apoptotic
proteins (e.g., Bcl-2 and Mcl-1). Among these members, Bim has been demonstrated

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to be essential and can indirectly induce Bax and Bak oligomerization on
mitochondria, and polymeric Bax forms holes in liposomes through which
cytochrome c is released into the cytoplasm to induce apoptosis. In this study, Erianin
induced Bim up-regulation, and the silencing of Bim expression significantly rescued
the proliferation inhibition effect of Erianin on EJ and T24 cells. Therefore, we
speculate that Bim might be an important element for the growth inhibition and
apoptosis induction effects of Erianin.
SAPK/JNK is a member of the MAPK family and is activated by multifarious
environmental stressors, growth factors, inflammatory cytokines, and
G
protein-coupled receptors. Overwhelming evidence suggests that JNK plays an
important role in cell proliferation, differentiation, apoptosis and other cell regulation
processes. Activation of the JNK signalling pathway by phosphorylated JNK/c-Jun
can promote mitochondrial cytochrome c release and induce mitochondrial-mediated
apoptosis (Lai et al., 2011). In addition, activated JNK is transferred to the nucleus
and trans-activates c-Jun, thereby up-regulating apoptotic precursor genes and
inducing apoptosis (Chen, 2012). Previous studies have shown that Bim is a
significant convergence point in apoptotic signalling regulated by AKT and ERK. The
experimental results showed that erianin increased the p-JNK levels and induced
c-Jun and Bcl-2 phosphorylation. Thus, the JNK pathway is involved in the
Erianin-mediated apoptotic response in bladder cancer.
Compared with traditional chemotherapy and radiation therapy, targeted tumour
therapy has the benefits of high specificity and low toxicity. However, few targeted

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anti-cancer drugs have been developed. Drug discovery typically takes one of two
approaches: phenotypic screening or a target-based approach. Phenotypic screening
involves small molecule libraries that are filtered against live cells with a phenotypic
output. However, this method has challenges, such as poor cell uptake or cell removal
of the compound and non-specific cytotoxicity effects. The target-based approach has
many advantages, including known compound patterns of action and the ability to
design compounds through a guiding structure. Therefore, molecular target screening
has become the main early drug discovery method. Natural products, as the basis of
drug development, have been widely applied in clarifying cellular mechanisms and
drug development. In our study, we demonstrated that Erianin exerts significant
inhibitory effects on bladder cancer cells and low toxicity toward normal liver cells,
which hints at the huge potential of Erianin and its targets for drug development.
Therefore, the identification of Erianin targets is of great significance for revealing the
mechanism of bladder cancer and for the development of more targeted drugs.
In conclusion, Erianin exerts a prominent inhibitory effect on bladder cancer cell
growth by inducing G2-M phase arrest and apoptosis. We demonstrated that
Erianin-induced apoptosis is primarily associated with the mitochondria-mediated
intrinsic apoptosis pathway. Additionally, JNK pathway activation contributes to the
anti-proliferation and apoptosis induction effects of Erianin. These findings provide a
scientific rationale for the clinical use of Erianin for bladder cancer treatment, and the
low toxicity of Erianin indicates that it might be a safe and effective agent for bladder
cancer prevention. Of course, Erianin derivatives or newly identified Erianin targets

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could also provide new therapeutic strategies for bladder cancer.
Conflicts of interest
The authors have declared no conflicts of interest.
Acknowledgements
The authors sincerely thank all the investigators and coordinators who
contributed to this study. This study was financially supported by the National Natural
Science Foundation of China (# 81573662, Yaxiong Tang) and the Sichuan Science
and Technology Program (No. 2018HH0079).
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Fig. 1: Erianin exerts anti-proliferative effects in human bladder cancer EJ and T24
cells. A. Chemical synthetic schedule of Erianin. B. Cytotoxicity effect of various
concentrations of Erianin on EJ, T24, and LO2 cells after 48 h of exposure. MTT
assays were performed to examine cell growth (as % viability). Each point is the
mean ± SE of three independent experiments. Each bar represents the mean ± SE of
three independent experiments. Each sample count was performed in duplicate.
Fig. 2: Effect of Erianin on EJ and T24 cell cycle progression. EJ cells were treated
with 0.1% DMSO or 40, 80, or 160 nM Erianin, and T24 cells were treated with 0.1%

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DMSO or 25, 50, or 100 nM Erianin. After treatment for 24 h, the cell cycle
distribution was analysed by flow cytometry and western blotting. A, B. The cells
were stained with propidium iodide for the analysis of cell cycle distribution. Each bar
represents the mean ± SE of three independent experiments. C. Western blotting
analysis of cell cycle markers in EJ and T24 cells following Erianin treatment. The
cell cycle-related proteins p-Chk2, p-cdc2, p-H2A.X and p-cdc25C were analysed
using specific antibodies. β-Actin served as the loading control.
Fig. 3: Effect of Erianin on the expression of apoptosis-related proteins. A. The live
cell morphology of EJ or T24 cells treated with Erianin at a concentration of 160 nM
or 100 nM for 48 h was observed with a phase-contrast light microscope
(
magnification × 100). DAPI staining was used to visualize the nuclear morphology
using a fluorescence microscope (magnification × 200). Control cells were treated
with 0.1% DMSO. B. Cells were pretreated in the presence or absence of
Z-VAD-FMK (20 µM) for 2 h and treated with Erianin for 24 h. Apoptotic cells were
identified through Annexin V-FITC/PI staining. C. The cell cycle distribution after
treatment with Z-VAD-FMK (20 µM) and Erianin was analysed by flow cytometry. D.
The cells were treated with 0.1% DMSO and Erianin (40, 80, or 160 nM (EJ cells)
and 25, 50, or 100 nM (T24 cells)) for 24 h, and the expression of cleaved caspase-9,
-
7, and -3 and PARP was determined by western blotting. E. The effect of the caspase
inhibitor on Erianin-induced apoptosis was measured. Inhibition of caspase activity
by Z-VAD-FMK (20 µM) decreased the anti-proliferative effect of Erianin in EJ and