Total Synthetic Protoapigenone WYC02 Inhibits Cervical Cancer
Cell Proliferation and Tumour Growth through PIK3 Signalling
Pathway
Yun-Ju Chen1,2,3, Nari Kay2*, Jinn-Moon Yang4,5, Chih-Ta Lin4, Hsueh-Ling Chang1,3, Yang-Chang Wu6,7,8, Chi-Feng Fu2, Yu Chang9, Steven Lo10, Ming-Feng Hou11, Yi-Chen Lee12, Ya-Ching Hsieh3and Shyng-Shiou Yuan1,2,3,13
1
Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan,2Department of Obstetrics and Gynecology, E-DA Hospital, Kaohsiung, Taiwan,3Department of Medical Research, E-DA Hospital, I-Shou University, Kaohsiung, Taiwan,4Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu, Taiwan,5Department of Biological Science and Technology,
National Chiao Tung University, Hsinchu, Taiwan,6Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical
University, Taichung, Taiwan,7Natural Medicinal Products Research Center, China Medical University Hospital, Taichung, Taiwan,8Graduate
Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan,9Department of Obstetrics and
Gynecology, Kaohsiung Medical University Hospital, Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan,10Department of Plastic and Reconstructive Surgery, E-DA Hospital, Kaohsiung, Taiwan,11Cancer Center, Kaohsiung Medical
University Hospital, Kaohsiung, Taiwan,12Graduate Institute of Medicine, College of Medicine and Department of Anatomy, Kaohsiung Medical
University, Kaohsiung, Taiwan and13Translational Research Center, Cancer Center and Department of Obstetrics & Gynecology, Kaohsiung
Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan (Received 16 November 2012; Accepted 28 January 2013)
Abstract: Flavonoids have been intensively explored for their anticancer activity. In this study, a total synthetic flavonoid pro-toapigenone, known as WYC02, was analysed for its potential anticancer activity on human cervical cancer cells as well as the underlying mechanisms for these effects. The site-moiety maps are used to explore the binding site similarity, pharmacophore and docking pose similarity. The effect of WYC02 on cell viability, migration, invasion and apoptosis as well as the underlying mechanisms was analysed in vitro using human cervical cancer cells. The effect of WYC02 on in vivo tumour growth was assessed in a tumour xenograft study. WYC02 inhibited cell proliferation, MMPs activity, migration and invasion in cervical can-cer cells. We speculated that WYC02 might inhibit the activities of PIK3 family proteins, including PIK3CA, PIK3CB, PIK3CD and PIK3CG. Indeed, WYC02 decreased the expression of PIK3 family proteins, especially PIK3CG, through ubiquitination and inhibited the activities of PIK3CG and PIK3 downstream molecules AKT1 and MTOR in cervical cancer cells. Furthermore, PIK3 signalling pathway was involved in the inhibitory effect of WYC02 on cervical cancer cell proliferation and tumour growth in vitro and in vivo. WYC02 inhibits cervical cancer cell proliferation and tumourigenesis via PIK3 signalling pathway and has the potential to be developed as a chemotherapeutic agent in cervical cancer.
Cervical cancer is one of the major gynaecological
malignan-cies among women throughout the world, especially in
devel-oping countries [1]. Conventional therapies for cervical cancer
include surgery, radiotherapy and chemotherapy [2]. For
locally advanced cervical cancer, neoadjuvant chemotherapy
(NACT) before surgery or during radiotherapy is an accepted
primary treatment, with the ability of NACT to reduce tumour
size, radiosensitize tumours and to improve disease control by
decreasing the repair of cancer cell damage caused by
radia-tion [3,4]. The most commonly used chemotherapy regimen in
cervical cancer is platinum-based chemotherapy that can
sig-nificantly reduce local treatment failure and improve overall
disease-free survival [5,6]. However, resistance to
platinum-based chemotherapy is relatively common [7], and therefore,
development of new chemotherapeutic agents is required.
The application of naturally existing dietary regimens in
cancer prevention has been well reported [8,9] and among
these natural diet regimens, flavonoids have been intensely
studied in recent years. Flavonoids are polyphenolic,
second-ary metabolites with broad-spectrum pharmacological activities
and have various biological effects, including induction of
cytotoxicity, apoptosis and antiproliferation [2,10]. Some
flavonoids, for example, LYG-202, N101-2, nobiletin and
his-pidulin, have been shown to inhibit angiogenesis and cell
growth of cervical, gastric and pancreatic cancers through
phosphatidylinositol 3-kinase (PIK3)/AKT1 signalling [11–
14]. Activation of class I PIK3s is one of the most important
signal transduction pathways used by cell-surface receptors to
control intracellular events, known to be involved in the
regu-lation of cell growth, survival, proliferation, movement and
inflammation [15–18]. There are four isoforms of the catalytic
subunit of class I PIK3s: PIK3CA, PIK3CB, PIK3CD and
Authors for correspondence: Shyng-Shiou Yuan, Department of Obstetrics and Gynecology, E-DA Hospital, No.1, E-DA Road, Yan-Chau District, Kaohsiung 824, Taiwan (fax +886 7 6155352, e-mail yuanssf@ms33.hinet.net).
Ya-Ching Hsieh, Department of Medical Research, E-Da Hospital, I-Shou University, No. 6, Yi-Da Road, Yan-Chau District, Kaohsiung 824, Taiwan (fax +886 7 6150945, e-mail: yaching.hsieh@gmail.com). *Co-first author.
PIK3CG [15,16]. Presently, class I PIK3 signalling pathway is
emerging as an exciting new area for the development of
novel therapeutic strategies.
Recently developed drug design models, based on structure–
activity relationship and pharmacological interaction, have
been used to explore the ligand-binding possibility of a
thera-peutic target. Most current virtual screening (SV) methods
employ flexible docking tools, such as incremental and
frag-ment-based approaches (DOCK and FlexX) and evolutionary
algorithms (GOLD, AutoDock and GEMDOCK), to identify
lead compounds for the target proteins [19–21]. These
meth-ods apply the pharmacological interaction preferences to select
the ligands that form pharmacological interactions with target
proteins and use the ligand preferences to eliminate the ligands
that violate electrostatic or hydrophilic constraints. Recently,
an innovative technology iGEMDOCK has been developed to
facilitate steps from preparation of target proteins and ligand
libraries towards post-screening analysis [22]. iGEMDOCK is
especially useful for post-screening analysis and inferring
pharmacological
interactions
from
screening
compounds.
When the structure of the target protein is known,
receptor-based computational methods can be employed. In a previous
study, we applied virtual molecule docking to discover the
pharmacological interactions on three therapeutic protein
targets, including oestrogen receptor
a for antagonists and
agonists [23]. Our results also revealed that the derived
phar-macological interactions are often essential for ligand binding
or maintaining biological functions of these targets.
In our initial screening, the total synthetic protoapigenone
WYC02 contains cytotoxic activity against human cancer cells
in vitro [24]. In this study, the virtual screening (SV) method
that employed flexible docking tools was first applied to
iso-late candidate cellular targets of WYC02, followed by in vitro
and in vivo studies to further clarify potential anticancer
activ-ity and the underlying mechanisms against cervical cancer
cells.
Materials and Methods
Origins of total synthetic protoapigenone WYC02. The plant-derived natural flavonoid protoapigenone was first isolated from Thelypteris torresiana (Gaud.), followed by total synthesis and renamed WYC02 [24].
Cell culture. HeLa cervical adenocarcinoma and SiHa cervical sarcoma cell lines used in this study were cultured according to the instructions from American Type Culture Collection (ATCC, Manassas, VA, USA). The genotypes and phenotypes of the cell lines were authenticated by Bioresource Collection and Research Centre (Hsinchu, Taiwan). Cells were grown in DMEM medium (Invitrogen, Carlsband, CA, USA), supplemented with 10% foetal bovine serum (Hyclone, Logan, UT) and antibiotics (100 units/mL penicillin, 100lg/mL streptomycin and 2.5 lg/mL amphotericin B) (Biological Industries, Haemek, Israel).
Colony formation assay. To determine long-term effects of WYC02 on cell proliferation, HeLa cells were treated with WYC02 for 3 hr. The detailed colony formation assay procedure followed the previous report [25].
Cell cycle analysis. Fluorescence-activated cell sorting (FACS) analysis was applied to analyse the cell cycle distribution. In brief, HeLa cells were treated with WYC02 for 24 hr and FACS analysis was performed according to a previous article [26].
Immunoblotting. Immunoblotting was performed according to a previous article [26]. Antibodies against CDC25A and P-RB1(Thr356) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). RB1, ACTB, PIK3CA, PIK3CB, PIK3CD, PIK3CG, P-AKT1 (Thr308and Ser473), AKT1, ubiquitin and Flag were obtained from Genetex (Irvin, CA, USA). P-CDC25C(Ser216), P-CDC2(Thr161), cleaved CASP8, cleaved CASP9, cleaved CASP3, cleaved PARP1, P-MTOR(Ser2448) and MTOR were obtained from Cell signalling Technology (Beverly, MA, USA). P-PIK3CG(Ser1100) was obtained from Abgent (San Diego, CA, USA).
Annexin V apoptosis assay. Annexin V-FITC fluorescence microscopy kit (BD Biosciences, San Jose, CA, USA) was used to detect early apoptotic cells during apoptotic progression. HeLa cells on chamber slides were treated with 10lM WYC02 for 3 hr, annexin V apoptosis assay was performed according to a previous article [27].
Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labelling Assay. HeLa cells were treated with 10lM WYC02 for 24 hr and then stained for determination of apoptotic cells using the DeadEnd Colorimetric TUNEL system (Promega, Madison, WI, USA). Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labelling (TUNEL) assay was performed according to a previous article [27].
Wound-healing assay. About 19 105HeLa cells were seeded in 12-well plates and allowed to reach 100% confluence. Cell monolayer was scratched with 200-lL pipette tip of constant width. Cells were then treated with WYC02 for 48 hr. and wound-healing assay was performed according to a previous article [28].
Transwell invasion assay. About 79 103HeLa cells were seeded on 8-lm-pore ECM-coated insert chamber (Corning, NY, USA) and allowed to reach 100% confluence. Cells were treated with WYC02 for 48 hr, and invasion assay was performed according to a previous article [29].
Gelatin zymography analysis. Gelatin zymography is mainly used for the detection of gelatinase activity. 59 104HeLa cells were plated in 24-well plates and allowed to reach 100% confluence. Cells were treated with WYC02 for 48 hr, and gelatin zymography analysis was performed according to a previous article [30].
Protein sequence analysis and molecular modelling. We obtained protein sequences of PIK3 catalytic subunits from GenBank and aligned them using the default settings with ClustalW2. The docking of WYC02 into the binding site of the PIK3 catalytic subunits was explored using iGEMDOCK software [24]. The 3D structure of WYC02
was prepared by DS VIEWERPRO6.0 from Accelrys, and the structures
of the quercetin and ATP were extracted from the PIK3CG crystal structures (PDB code 1E8W and 1E8X, respectively) in the Protein Data Bank (PDB). Homology modelling of HsPIK3CB and HsPIK3CD was done using Swiss-Model with 2Y3A and 2WXJ of crystal structures in the PDB as templates. The binding pockets of the HsPIK3CA (PDB code 3HHM), HsPIK3CB (PDB code 2Y3A), HsPIK3CD (PDB code 2WXP) and HsPIK3CG (PDB code 3DBS) were defined to include the residues within an 8A radius sphere centred around the binding site of their ligands. The coordinates of the atoms in the binding pockets were obtained from the PDB. The interaction profile was performed with dChip, and the algorithm of
hierarchical clustering centroid linkage was employed. The site-moiety map analyses of PIK3 catalytic subunits were performed with SiMMap.
Reverse transcription polymerase chain reaction. Reverse transcription polymerase chain reaction (RT-PCR) was analysed with One-s RT-PCR kit (QIAGEN, Foster, CA, USA). Specific cDNA for the PIK3CG and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified with primer pairs (PIK3CG: 5′-GCTTGA AAACCTGCAGAATTCTCAAC-3′ and 5′-CGTCTTTCACAATCTC GATCATTCC-3′; GAPDH: 5′-TGATGACATCAAGAAGGTGGTG AAG-3′ and 5′-TCCTTGGAGGCCATGTGGGCCAT-3′) by PCR, which were performed according to a previous article [31].
Immunoprecipitation. Cells were re-suspended in lysis buffer (Millipore Corporation, Billerica, MA, USA). 50lg cell lysates served as input control and 1.2 mg cell lysates were incubated with 3lg of PIK3CG antibody at 4°C overnight. To study the ubiquitination of PIK3CG, immunoprecipitation was performed by Catch and Release v2.0 reversible immunoprecipitation system (Millipore Corporation).
Ex vivo tumour xenograft study. All experiments using mice were performed according to the guidelines of the Animal Committee and with ethics approval from the institutional review board of E-Da Hospital/I-Shou University (Approval No.: IACUC-ISU-96024). Six-week-old female immunodeficient (Foxnlnu/Foxnlnu) mice were injected subcutaneously with 59 106 HeLa cells at the right flank.
When tumours became visible (approximately an average diameter of 3 mm), mice were treated intraperitoneally with WYC02 at 1.09lg/g (a dose equals to the IC50) body-weight or vehicle (PBS) every 2 days.
Tumour volumes were calculated according to a standard formula: width29 length/2 and performed according to a previous article [27].
Immunohistochemistry. Immunohistochemistry protocol was followed accordingly to a previous report [32]. Tissue sections were incubated overnight at 4°C with 100X diluted of PIK3CA, PIK3CB, PIK3CG or PIK3CD antibodies, which were obtained from Genetex.
Transfection of PIK3CG plasmid DNA. HeLa cells were transfected for 16 hr with human PIK3CG expression plasmid or empty vector, purchased from Addgene (Cambridge, MA, USA; Cat#20574) using Lipofectamine 2000 according to Invitrogen’s respective protocol (Invitrogen). After removal of transfection medium, the cells were incubated with fresh medium for 48 hr and then selected for neomycin-resistant cells using 500lg/mL neomycin (A.G. Scientific, San Diego, CA, USA) for 7 days.
Transfection of PIK3CG siRNA. HeLa cells were seeded at 59 103 cells per well in a 96-well dish. At 20 hr after seeding, the cells were transfected with human PIK3CG siRNA-SMARTpool (Dharmacon, Lafayette, CO, USA, Cat# DAMD-005274-02) (Target sequences: CUACAGCCCUAUCAAAUGA, GGUCCAGGCUGUGAAAUUU, AGAAAUCUCUGAUGGAUAU, GACGUCAGUUCCCAAGUUA) or non-target siRNA Pool (Dharmacon, Cat# DAMD-001206-13) using DharmaFECT1 transfection reagent (Dharmacon Cat# DAMD-2001-02). Briefly, 4lL of DharmaFECT1 was diluted in 196 ll of serum-free medium and was incubated at room temperature for 5 min. In a separate sterile tube, 10lL of siRNA oligos (5 lM stock) was mixed with 190lL of serum-free medium and incubated at room temperature for 5 min. The diluted DharmaFECT1 and diluted siRNA oligos were then mixed together and incubated at room temperature for another 20 min. At the end of the incubation period, 1.6 mL of complete growth medium was added to the mixture, and 100lL of this final mixture was dispensed to each of the 96 wells for 16 hr.
After removal of transfection medium, the cells were incubated with fresh medium for 48 hr and then treated WYC02 for 48 hr.
Statistical analysis. Quantitative data are presented as mean S.E.M. The statistical significance among three or more groups was analysed by one-way analysis of variance (ANOVA) and Duncan’s test.
Two-sided Student’s t-test was used to determine the significance between two groups. p< 0.05 was considered statistically significant.
Results
Total synthetic protoapigenone WYC02 inhibited cervical
cancer cell viability.
The cytotoxicity of WYC02 on human cervical cancer cells
was first analysed in this study. WYC02 was toxic to the three
tested cell lines HeLa, C33A and SiHa [inhibitory
concentra-tion (IC
50)
= 4.23 lM, 5.37 lM and 8.12 lM, respectively].
WYC02 had higher cytotoxic activity against HeLa cells than
the clinically used drug cisplatin at 48 hr of treatment
(IC
50= 4.23 lM and 11.80 lM, respectively). The effect of
WYC02 on cell colony formation and cell cycle distribution
was further analysed. WYC02 treatment significantly inhibited
HeLa cell clonogenicity (fig. 1A). WYC02 treatment
accumu-lated HeLa cells at S and G2/M phases in a dose-dependent
manner (fig. 1B). In agreement with cell cycle distribution, an
increased phosphorylation of G1-S regulator RB1 and the
decreased levels of G2/M regulators CDC25A and P-CDC2
were observed in HeLa cells after WYC02 treatment (fig. 1C).
WYC02-induced apoptotic cell death in cervical cancer cells.
To study the effects of WYC02 on cell apoptosis, annexin V
(marker of early apoptosis) assay and TUNEL (marker of late
apoptosis) assay were applied. A significant increase in
annex-in V and TUNEL positive cells were observed annex-in HeLa cells
after 10
lM WYC02 treatment (figs 1D,E). Further study by
immunoblotting analysis demonstrated that WYC02 treatment
induced a dose-dependent cleavage CASP8, CASP9, CASP3
and PARP1 in HeLa cells (fig. 1F).
WYC02 decreased MMPs activities and inhibited migration/
invasion in cervical cancer cells.
A critical event in cancer cell migration and invasion is the
degradation of extracellular matrix (ECM), while the
expres-sion of matrix metalloproteinases (MMPs) is necessary for
ECM degradation [33]. MMP-2 (gelatinase A) and MMP-9
(gelatinase B) are able to degrade most of the ECM
compo-nents and are the major MMPs secreted from HeLa cells [34].
In this study, we found that WYC02 decreased the efficiency
of cell migration, invasion, activities of MMP-2 and MMP-9
in HeLa cells in a dose-dependent manner (fig. 2). However,
no significant cytotoxicity was observed in 100% confluent
HeLa cells when treated with 2
lM WYC02 for 48 hr
[(IC
50)
= 10.79 lM for HeLa cells] (fig. S1).
WYC02 has inhibitory potential on PI3K catalytic subunits.
To explore the target proteins, especially kinases, for WYC02
activity, ligand similarity was applied. We speculated that
WYC02 had inhibitory effects on PIK3 catalytic subunits,
based on the results of sequence conservation of key
interact-ing residues among PIK3 catalytic subunits, WYC02 docked
conformations of PIK3 catalytic subunits, interaction profile of
known PIK3 catalytic subunit general inhibitors and
site-moi-ety map analysis of PIK3 catalytic subunits. To further analyse
the inhibition mechanisms of PIK3 catalytic subunit general
inhibitors on PIK3 catalytic subunits, key interactions were
extracted from the cocrystal structures of ATP and PIK3
cata-lytic subunit general inhibitors to PIK3 catacata-lytic subunits, and
the binding sites of PIK3 catalytic subunits were divided into
several motifs and regions, including P-loop, hinge, catalytic
loop (C-loop), activation loop (A-loop), adenine pocket (AP),
hydrophobic region I and II (HI and HII), phosphate-binding
region (PB), specific pocket (SP), ribose-binding region (RB)
(fig. 3A). All PIK3 catalytic subunit general inhibitors form
hydrogen bonds with hinge, hydrophobic region I and
phos-phate-binding region, and hydrophobic interactions with
ade-nine pocket, hydrophobic region I, phosphate-binding region
and specific pocket among PIK3 catalytic subunits and
com-pete with ATP by targeting ATP-binding site (fig. 3B).
According to the hierarchical cluster (C2) of interaction
pro-file, similar inhibitors of PIK3 catalytic subunits have a similar
interaction profile. In addition, WYC02 docked conformations
of PIK3 catalytic subunits (C1) showed a similar interaction
profile to PIK3CG inhibitor (IC
50= 3.8 lM, K
d= 0.28 lM),
QUE, quercetin, a similar flavonoid to WYC02 (fig. 3B).
The sequences of PIK3 catalytic subunits are highly
con-served, especially in interacting residues of ATP and PIK3
catalytic subunit general inhibitors to PIK3 catalytic subunits,
catalytic residues of PIK3 catalytic subunits (fig. 3C). The
similarities of PIK3 catalytic subunits are not only shown in
sequences, but also presented in binding environments. The
site-moiety map analysis showed that PIK3 catalytic subunits
are highly similar in anchors, interacting residue compositions
and moiety preferences of each anchor (fig. 3D). All PIK3
catalytic subunits have five consensus anchors, three H-bond
interacting anchors and two van der Waals interacting anchors.
The consensus anchors also consist of conserved interacting
residues. In moiety preference, all H-bond interacting anchors
of PIK3 catalytic subunits prefer to form H-bond with oxygen
atoms, including hydroxyl moiety and carbonyl moiety. All
Cell cycle distribution (%)
0 20 40 60 80 WYC02 (µM) 0 1 2.5 5 10 0 1 2.5 5 10 0 1 2.5 5 10 G0/G1 S G2/M B
Colony formation (% of control) 0 20 40 60 80 100 120 WYC02 (µM) 0 1 2.5 5 10 b b c d A 0 1 2.5 5 10 WYC02 (µM) WYC02 (µM) 0 1 2.5 5 10 a b b c d a a a b b b b c d a a P-CDC2(T161) Rb1 ACTB P-Rb1(T356) CDC25A WYC02 (µM) 0 1 2.5 5 10 Rb1 P-Rb1 C
Annexin V positive cells (%) 0 10 20 30 40 WYC02 (µM) 0 10 *
TUNEL positive cells (%)
0 20 40 60 80 WYC02 (µM) 0 10 * D E WYC02 (µM) 0 10 DAPI Annexin V WYC02 (µM) 0 10 TUNEL Cleaved CASP8 Cleaved CASP9 Cleaved CASP3 Cleaved PARP1 ACTB F WYC02 (µM) 0 1 2.5 5 10
Fig. 1. WYC02 decreased cell viability and induced apoptosis in HeLa cervical cancer cells. (A) and (B) HeLa cells after treatment with vehicle control or WYC02 were analysed for colony formation and cell cycle distribution by flow cytometry. (C) Immunoblotting analysis of the expres-sion of cell cycle regulatory proteins in HeLa cells at 24 hr after WYC02 treatment. (D and E) HeLa cells treated with 10lM WYC02 were analy-sed by annexin V and TUNEL assay. (F) Immunoblotting analysis of the expression of apoptosis-related proteins at 24 hr after WYC02 treatment. Each bar represents the mean S.E.M. *Indicates a significant difference (p < 0.05) when compared with the vehicle control without WYC02.
van der Waals interacting anchors of PIK3 catalytic subunits
prefer to form hydrophobic interactions with aromatic moiety.
Most PIK3 catalytic subunit general inhibitors agree with these
five anchors. In addition, WYC02 docked conformations of
PIK3 catalytic subunits have common anchors, H1
(corre-sponding to hinge), V1 (corre(corre-sponding to adenine pocket) and
V2 (corresponding to hydrophobic region I). Furthermore,
WYC02 docked conformations of PIK3CA, PIK3CB and
PIK3CG target H2 anchor (corresponding to
phosphate-bind-ing region); WYC02 docked conformations of PIK3CB and
PIK3CD target H3 anchor (corresponding to hydrophobic
region I). All WYC02 docked conformations of PIK3 catalytic
subunits target ATP-binding site, consistent with other PIK3
catalytic subunit general inhibitors, especially in a similar
inhibitor, quercetin. Therefore, according to the WYC02
docked conformations, the interaction profile of PIK3 catalytic
subunit general inhibitors, key interacting residue/motif
con-servation of PIK3 catalytic subunits and binding environments
of PIK3 catalytic subunits, we believe that WYC02 has
inhibi-tory potential effects on PIK3 catalytic subunits.
WYC02-inactivated PIK3/AKT1 signalling pathway in cervical
cancer cells.
From the results of site-moiety maps for exploration of the
binding site similarity, pharmacophore and docking pose
simi-larity, we speculated that WYC02 might inhibit the activities
of PIK3 family proteins including PIK3CA, PIK3CB, PIK3CG
and PIK3CD. Further literature search also revealed that
flavo-noids suppressed cell proliferation in leukaemia cells, gastric
wound closure (%) 0 20 40 60 80 100 120
Relative invasion efficiency 0
20 40 60 80 100 MMP-2 secretion 0 20 40 60 80 100 120 MMP-9 secretion 0 20 40 60 80 100 120 0 0.25 0.5 1 2 0 0.25 0.5 1 2 WYC02 (µM) WYC02 (µM) 0 0.25 0.5 1 2 WYC02 (µM) 0 0.25 0.5 1 2 WYC02 (µM) Wound healing Invasion 0 0.25 0.5 1 2 WYC02 (µM) b c d a a b c d a a c c c a b c c d a b
Fig. 2. WYC02 decreased HeLa cell migration, invasion and MMPs activities. HeLa cells were treated with WYC02 at various doses and the migration and invasion efficiencies were determined by wound-healing assay and ECM-coated transwell system. MMP-2 and MMP-9 activities were measured using gelatin zymography analysis. All data are shown as mean S.E.M.
Fig. 3. Inhibitory potential effects of WYC02 on PI3K catalytic subunits. (A) Schematic representation of a PIK3 protein kinase ATP-binding pocket. (B) Interaction profile of PIK3 inhibitor complexes and docked conformations of WYC02 in PIK3 catalytic subunits. (C) The sequence conservation of key interacting residues among four PIK3 catalytic subunits. The catalytic residues are coloured in yellow. The key interacting resi-dues of ATP and general inhibitors of PIK3 catalytic subunit are circled and coloured in grey, respectively. (D) Chemical structure of WYC02 and site-moiety map analysis of PIK3 catalytic subunits. The key interacting residues of ATP and general inhibitors of PIK3 catalytic subunit in the table are shown in bold and coloured in grey, respectively. The interacting residues of each anchor are labelled and the hydrogen bonds (dash with green line) between WYC02 (blue) and the PIK3 catalytic subunits (grey) are indicated. The ATP (orange) and QUE (pink) were extracted from the PIK3CG crystal structures (PDB code 1E8W and 1E8X, respectively) as the reference. The interacting anchors of H-bond and van der Waals are shown in green and grey, respectively. The figures were drawn using PyMOL software.
adenocarcinoma cells and lung cancer cells, through inhibition
of PIK3/AKT1 signalling cascades [14,35,36]. Therefore,
immunoblotting analysis was applied to confirm the
suppres-sive effect of WYC02 on PIK3 family proteins. The results
were consistent with the data of site-moiety maps showing that
WYC02 inhibited the expression of PIK3CA, PIK3CB,
D841 D836 D810 D933 K776 D964 I879 V2 V2 V1 V1 I963 I831 M953 W780 V850 I932 I800 I848 Y836 V881 K833 ATP ATP QUE QUE WYCO2 WYCO2 ATP QUE WYCO2 H1 H1 H3 H3 H2 H2 Y867 E880 V882 S806 E849 V851 S854 K802 D813 D787 D782 K779 Y813 K805 D937 Y839 I936 D937 E826 D911 V828 S831 E852 V854 I851 V853 M926 V2 V1 1825 I910 M900 I777 V2 V1 H3 H2 H1 H1 H3 H2 ATP QUE WYCO2 ATP QUE WYCO2 A C D B
PIK3CG and PIK3CD, with the most significant suppressive
effect on PIK3CG (fig. 4A). WYC02 also showed inhibitory
effects on phosphorylation and activity of PIK3CG (fig. 4B).
We further examined the effect of WYC02 treatment on the
major downstream signalling mediators of PIK3, namely
AKT1 and MTOR, in HeLa cells. Both AKT1 and MTOR
kinases play important roles in cell survival [37]. Upon
WYC02 treatment, the phosphorylation of AKT1 and MTOR,
but not the total expression levels of AKT1 and MTOR, was
suppressed (fig. 4B). These results confirmed that WYC02
decreased PIK3 expression and inactivated PIK3 signalling
cascades in human cervical cancer cells.
Several mechanisms are well known to negatively regulate
kinase activity, including reduction in mRNA level and
induc-tion of protein degradainduc-tion [38], and PIK3 can be degraded
through ubiquitination/proteasome pathway [39,40]. In this
study, the RNA levels of PI3KCG were not changed in HeLa
cells
upon
WYC02
treatment,
determined
by
RT-PCR
(fig. 4C). On the other hand, we observed that MG132, an
inhibitor of proteasomal protease activity, reversed
WYC02-induced decrease in PI3KCG protein level, suggesting the
decrease in PI3KCA expression upon WYC02 treatment was
caused by protein degradation (fig. 4D). We further tested the
possibility that WYC02-induced PIK3CA degradation in HeLa
cells was caused by protein ubiquitination. Upon WYC02
treatment in HeLa cells, PIK3CG coimmunoprecipitated with
ubiquitin, indicating a physical interaction between the two
proteins. Ubiquitination of PIK3CG was increased in HeLa
cells upon WYC02 treatment (fig. 4E) and resulted in a
decreased amount of PIK3CG protein (figs. 4A,D). To further
analyse the involvement of PIK3CG in WYC02-induced
cytotoxicity, PIK3CG-Flag was over-expressed in HeLa cells
followed by cell viability analysis upon WYC02 treatment. A
statistically significant reverse in cell viability was observed in
PIK3CG-Flag-overexpressing HeLa cells compared with
vec-tor control cells (fig. 5A). Furthermore, PIK3CG levels were
significantly reduced by siRNA knockdown, and the reduction
in PI3KCG expression resulted in a significant decrease in
HeLa cell viability but blocked the cytotoxicity caused by
WYC02 treatment (fig. 5B).
WYC02 decreased the expression of PI3K subunits and
suppressed xenograft tumour growth in nude mice.
To determine the suppressive effect of WYC02 on cervical
cancer cell growth in vivo, nude mice xenograft model was
applied. The tumour growth was significantly suppressed in
the WYC02-treated group (fig. 6A). There was also no
signifi-cant alteration of body-weight, haematopoiesis, liver function,
renal function and organ histology in the WYC02-treated
group (fig. 6B, fig. S1 and table S1). Moreover, the
expres-sion of PIK3 catalytic subunits, PIK3CA, PIK3CB, PIK3CD
and PIK3CG, in xenograft tumours was decreased in
WYC02-treated tumour tissues. This result confirmed that WYC02
decreased the expression of PIK3CA, PIK3CB, PIK3CD and
PIK3CG in cervical cancer cells both in vitro and in vivo
(fig. 4A and fig. S2).
Discussion
Virtual screening of the candidate anticancer compound
WYC02.
Exploring the potential antiproliferative effects of
phytochemi-cals, such as vinblastine and adriamycin, may open new
ave-nues in anticancer drug design [41]. One such phytochemical,
flavonoids (a subclass of polyphenols), has been previously
explored in cancer therapy in their ability to suppress cancer
cell proliferation, induce cell cycle arrest and promote apoptosis
[10,35,42]. The present study investigated the inhibitory effect
of the candidate synthetic flavonoid protoapigenone WYC02,
on HeLa cell proliferation, cell cycle progression, migration,
invasion, as well as an apoptosis-promoting effect. Nonetheless,
thousands of mechanisms may underlie these therapeutic
effects, and virtual screening was therefore employed as an
effi-cient route to reduce the complexity of identifying potential
therapeutic targets and underlying mechanisms of action. Using
such an approach, with site-moiety mapping, we identified that
the candidate synthetic flavonoid protoapigenone WYC02
potentially interacts with PIK3 catalytic subunits. This allowed
the present study to focus on the PIK3 signalling pathway.
Cell-based assays and an in vivo mouse xenograft model indeed
confirmed that WYC02 inhibited tumour progression through
inhibition of PIK3/AKT1/mTOR signalling and suppressed cell
invasion/migration through inhibition of MMP-2/MMP-9.
WYC02-inhibited cervical cancer cell growth and migration/
invasion.
The synthetic protoapigenone WYC02 exhibited a number of
inhibitory effects on cervical cancer (HeLa) cells. WYC02
inhibition of colony formation, induction of S-G2/M cell cycle
arrest (fig 1A–C) and promotion of Hela cell apoptosis
(fig 1D
–F) were matched by a corresponding suppression of
xenograft tumour growth in nude mice (fig. 6A). WYC02 also
demonstrated effects on tumour migration and invasion,
medi-ated by suppression of MMP-2/-9-dependent cell invasion/
migration (fig. 2). WYC02 may therefore have merit as a
potential therapeutic agent in both cervical tumour growth
suppression and in inhibition of invasion.
WYC02 increases cervical cancer cell apoptosis via inhibition
of the PIK3/AKT1/mTOR pathway.
The
PIK3/AKT1
signalling
pathway
regulates
cellular
responses and plays a critical role in maintaining the balance
between cell survival and apoptosis [15]. Recent studies
indi-cate that activation of the PIK3/AKT pathway by
amplifica-tion, mutation and translocation occurs on a more frequent
basis than in other pathways in patients with cancer [43].
Inhi-bition of the PIK3 pathway may therefore provide an
appropri-ate target for cancer therapeutic options. Using site-moiety
maps to explore binding site, pharmacophore and docking
pose similarity, we observed that WYC02 may inhibit the
activities of PIK3s (PIK3CA, PIK3CB, PIK3CD and PIK3CG)
(fig. 3). Over-expression of PIK3 has been associated with
PIK3CA/ACTB ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 PIK3CG/ACTB ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 PIK3CD/ACTB ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 PIK3CB/ACTB ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 PIK3CG ACTB PIK3CA ACTB PIK3CD ACTB 0 0.5 1 2 4 6 24 (H) 5 µM WYC02 PIK3CB ACTB 0 0.5 1 2 4 6 24 (H) 5 µM WYC02 P-PIK3CG(S1100) MTOR P-MTOR(S2448) P-AKT1(T308) AKT1 P-AKT1(S473) ACTB WYC02 (µM) 0 1 2.5 5 10 A B a a a a a b b a a b a a b b c c c a b d e b c c a a c c
MG132 (10
µM) – + – +
5 µM WYC02
Control
PIK3CG
GAPDH
34 kDa 26 kDa 17 kDa 10 kDa 43 kDaWYC02 (5
µM) – +
IP: PIK3CG
Input
IB: ACTB
IB: ubiquitin
IB: PIK3CG
110 kDa5
µM WYC02
Control
PIK3CG
ACTB
43 kDa
C D E ▲▲ ▲ ▲ ▲Fig. 4. WYC02 decreased PIK3CG expression and activity through ubiquitination. (A) HeLa cells were treated with 5lM WYC02 for different time periods and the cell lysates were analysed by immunoblotting for PIK3 catalytic subunits, including PIK3CA, PIK3CB, PIK3CG and PIK3CD. (B) HeLa cells were treated with 1–10 lM WYC02 for 4 hr and cell lysates were analysed by immunoblotting for the activities of PIK3CG and PIK3 downstream molecules AKT1 and MTOR. (C) HeLa cells were treated with WYC02 for 6 hr and PIK3CG RNA level was determined by RT-PCR. (D) HeLa cells were treated with WYC02 for 24 hr and the effect of MG132, an inhibitor of proteasomal protease activity, on the expression of PIK3CG was determined by immunoblotting. (E) HeLa cells were treated with WYC02 for 8 hr and PIK3CG ubiquitination was determined by immunoprecipitation with anti-PI3KCG followed by immunoblotting for PIK3CG and ubiquitin antibodies, respectively. Input ACTB served as an internal control. Arrowheads mark the position of discrete bands, consistent with addition of a different number of ubiquitin moieties (approximately 8.5 kDa per ubiquitin).
tumour stage, grade, lymph node metastasis and poor
progno-sis in cervical cancer [44,45]. Using site-moiety maps and
detection of cellular biological activity in cervical cancer cells,
we found that WYC02 inhibited the expression of PIK3CA,
PIK3CB, PIK3CD and PIK3CG in a dose-related manner,
with the most marked inhibitory effect on PIK3CG (fig. 4A).
Decreased PIK3CG activity was accompanied by a decrease in
levels of downstream effectors P-AKT1 and mTOR (fig. 4B).
The inhibitory effect of WYC02 on cell viability was also
reversed by PIK3CG over-expression (fig. 5A). However, this
reverse effect was not seen in cervical cancer cells when
PI3KCG expression was knockdowned (fig. 5B). It suggests
that WYC02 may act by inducing apoptosis in cervical cancer
cells, with inhibition of PIK3CG/AKT1/mTOR contributing to
the activation of caspases 3, 8 and 9, and PARP cleavage. The
precise mechanism by which this occurs is discussed below.
WYC02 targets the PIK3 pathway by promoting PIK3CG
ubiquitination.
Distinct from other members in the PIK3 family, PIK3CG is
acti-vated by G-protein coupled-receptors and is involved in other
processes including inflammation, allergy and thrombosis
[46,47]. PIK3CA, PIK3CB and PIK3CD have been implicated as
possible oncogenes in human cancers including brain, colon and
bladder [48
–51]. Emerging data suggest that PIK3CG may also
have a role in cancer growth, invasion and metastasis [52,53],
suggesting its potential as an oncological therapeutic target.
Proteasome degradation of ubiquitin-targeted proteins is an
important mechanism that negatively controls activated
signal-ling pathways [54]. PIK3 degradation via the ubiquitination/
Tumor volume (mm 3) 0 100 200 300 400 500 600 Control WYC02 Week 0 1 2 3 4 5
*
*
*
*
*
*
*
Body weight (g) 0 5 10 15 20 25 30 ● Control ○ WYC02 Week 0 1 2 3 4 5 6 7 6 7 A BFig. 6. WYC02 suppressed xenograft tumour growth in nude mice. Nude mice bearing HeLa tumours were treated with vehicle control or 1.09lg/g (a dose equals to the IC50) WYC02 every 2 days. (A)
Tumour volumes were measured per week and data presented as means S.E.M. (B) Body-weight was measured per week and data presented as means S.E.M. *Indicates a significant difference (p< 0.05) when compared with the vehicle control without WYC02 treatment. Cell viability 0 20 40 60 80 100 120 Parentral Vector Control PIK3CG PIK3CG-Flag PIK3CG ACTB Vector control + + + + PIK3CG + + + + 0 1 2.5 5 A B * * 113 kDa 43 kDa 110 kDa * Cell viability 0 20 40 60 80 100 120 Control siRNA + + + + PIK3CG siRNA + + + + WYC02 (µM) WYC02 (µM) 0 1 2.5 5 * *
Parentral Control PIK3CG PIK3CG ACTB 43 kDa 110 kDa siRNA a b c d a b c d
Fig. 5. PIK3CG was involved in the cytotoxic activity of WYC02 on HeLa cells. (A) Immunoblotting analysis of the expression of PIK3CG-Flag in parental, empty vector and PIK3CG-overexpressing HeLa cells. HeLa cells were transfected with PIK3CG-Flag plasmid and then treated with WYC02. (B) Immunoblotting analysis of the expression of PIK3CG in parental, control siRNA- or PIK3CG siRNA-transfected HeLa cells and then treated with WYC02. Cytotox-icity was determined by XTT assay. Each bar represents mean S.E.M. (n = 6). *Indicates a significant difference (p < 0.05) compared with their respective controls after different doses of WYC02 treatments.
proteasome pathway has previously been shown [39,40]. In
this study, we determined whether WYC02 negatively controls
PIK3CG signalling via ubiquitination degradation. WYC02
promoted PIK3CG ubiquitination in HeLa cells (fig. 4E), while
addition of the proteasome inhibitor MG132 reversed this
effect (fig. 4D). We also confirmed that WYC02 had no effect
on the mRNA levels of PIK3CG (fig. 4C), further supporting
the direct inhibitory effect of WYC02 on protein degradation.
Therefore, it was demonstrated that the WYC02-induced
decrease in PI3KCG activity was mediated by ubiquination.
Conclusions
In conclusion, using site-moiety maps as an initial approach,
we demonstrated that the total synthetic protoapigenone
WYC02 suppressed cervical cancer cells in vitro and in vivo
through inhibition of PIK3 signalling pathway. This is the first
study to demonstrate the involvement of PIK3CG molecule
in cervical tumour progression and that ubiquitination
degrada-tion is responsible for the WYC02 inhibitory effect on cancer
cell proliferation. Ubiquitination of PIK3CG results in
inhibi-tion of AKT1/MTOR activity, leading to activainhibi-tion of caspases
3, 8 and 9, and PARP cleavage, and promotion of apoptosis in
cervical cancer cells. WYC02 therefore merits further
investi-gation as a potential therapeutic target in cervical cancer.
Acknowledgements
This manuscript was supported by grants from National
Health
Research
Institutes,
Taiwan,
ROC
(NHRI-EX98,
99,100-9829BI,
NHRI-EX102-10212BI,
EDPJ99007
and
EDPJ100003) to SSY, Department of Health, Taiwan, ROC
(DOH101-TD-C-111-002) to MFH and E-DA hospital,
Tai-wan, ROC (EDAHP99040 and EDAHP101023) to NK.
Conflict of i nte re st
The authors declare no conflicts of interest.
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