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Supporting information
Costunolide specifically binds and inhibits thioredoxin reductase 1 to
induce apoptosis in colon cancer
Weishan Zhuge1,2, Ruijie Chen1, Vladimir Katanaev3, Xidan Dong4, Khan Zia5, Xiangwei Sun6,
Xuanxuan Dai1, Miao Bao1, Xian Shen2,6,*, Guang Liang1,*
1 Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China
2 Department of Gastrointestinal Surgery, The First Affiliated Hospital of Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China
3 School of Biomedicine, Far Eastern Federal University, Sukhanova Street 8, Vladivostok,
690922, Russian Federation
4 Department of Pathology, The First Affiliated Hospital of Wenzhou Medical University,
Wenzhou, Zhejiang 325035, China
5 Department of Pathology and Laboratory Medicine, Western University, London, ON N6A5C1,
Canada
6 Department of Gastrointestinal Surgery, The Second Affiliated Hospital of Wenzhou Medical
University, Wenzhou, Zhejiang 325000, China
Author information*Corresponding author:Guang Liang, Ph.D, ProfessorAddress: Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, ChinaTel: +86-577-86699892; Fax: +86-577-86689982; E-mail: [email protected]
* Co-Corresponding authors: Xian Shen, ProfessorAddress: Department of Gastrointestinal Surgery, Department of Gastrointestinal Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, ChinaE-mail:[email protected]
ContentsSupplemental information includes detailed methodology, 4 figures, and 1 table.
Detailed Methodology
Cell viability, cell cycle, and cell apoptosis assays
Cells were seeded onto 96-well plates at a density of 8 x103 per well and allowed to
attach overnight in complete growth media. The next day, cells were exposed to CTD
which was dissolved in DMSO and diluted using McCoy’s 5A media. Concentrations
tested for viability included 1, 2.5, 5,10 ,25, 50, 75, 100, and 200 μM. Tumor cells
were incubated with CTD for 24 h or 48 h before performing the MTT assay.
To assess cell cycle distribution and apoptosis, cells were plated on 60-mm dishes
for 24 h, and then treated with 10, 20 or 30 μM CTD. NAC pre-treatment, where
indicated, was carried out at 5 mM for 1 h. Following CTD exposure (15 h for cell
cycle analysis and 20 h for apoptosis detection), cells were harvested, washed and
fixed, and stained. PI staining was used for cell cycle analysis and Annexin V/PI for
apoptosis detection. Samples were analyzed using FACS Calibur flow cytometer (BD
Biosciences, CA). Data for apoptosis and cell cycle distribution was analyzed using
FlowJo7.6 software. For apoptotic statistics, the apoptotic cells contain Annexin V/PI double-
positive cells and Annexin V-positive/PI-negative cells.
Colony formation assay
HCT-116 cells were plated at 500 cells per well in 6-well plates and cultured
complete growth media for 24 h. Cell were then exposed to 30 µM CTD for 5 hours,
with or without 5 mM NAC pretreatment for 1 h. Cells were allowed to grow for 9
days and colonies emerging were stained with crystal violet solution. A colony was
defined as a cluster of at least 50 cells that can often only be determined
microscopically.
Western blotting analysis
Total proteins from cultured cells or tumor tissues were isolated and the
concentrations were measured by the Bradford protein assay kit (Bio-Rad, Hercules,
CA). Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred onto poly-vinylidene difluoride
membranes. After blocking with tris-buffered saline containing 0.05% Tween 20
(TBST) and freshly prepared 5% nonfat milk for 1.5 h at room temperature,
membranes were incubated with different primary antibodies overnight at 4 °C.
Membranes were then washed in TBST and incubated with HRP-conjugated
secondary antibodies for 1 h at room temperature. Immunoreactivity was visualized
using an ECL substrate (Bio-Rad, Hercules, CA). Densitometric measurements were
performed using Image J (National Institute of Health, MD).
H&E staining, and DHE/DCFH-DA immunofluorescence staining for ROS
Harvested tumor tissues, as well as heart, liver and kidney tissues were fixed in 10%
formalin at room temperature. Samples were dehydrated using ethanol gradient, and
processed and embedded in paraffin. Parraffin-embedded tissues were sectioned at 5
μm thickness. Tumor tissue sections were stained for Ki-67 and cleaved-caspase 3
using primary antibodies at 1:200 and 1:50 dilution, respectively. HRP-conjugated
secondary antibodies and diaminobenzidine (DAB) were used for detection. Heart,
liver and kidney tissues from mice were also stained with hematoxylin and eosin
(H&E) using routine procedures.
Frozen tissue sections were used for immunofluorescence staining for ROS.
Sections were washed and incubated with DHE (Sigma) or DCFH-DA (Beyotime
Biotech). Slides were then counterstained with DAPI for 10 minutes at 37℃.
Surface plasmon resonance analysis for CTD-TrxR1 interaction
The binding affinity of CTD with recombinant human TrxR1 protein (Sigma) was
determined using a ProteOn XPR36 Protein Interaction Array system (Bio-Rad) with
a GLH sensor chip (ProteOn). Briefly, recombinant human TrxR1 protein (Sigma)
was loaded to the sensor, which was activated with 10 mM NiSO4, and the CTD
samples were prepared with running buffer (PBS, 0.1% SDS, 5% DMSO). Sensor and
sample plates were placed on the instrument. The CTD samples were then captured in
the first flow cells, and the second flow cell was left as a blank. Five concentrations
were simultaneously injected at a flow rate of 30 mM min−1 for 120 seconds of
association phase, followed with 120 seconds of dissociation phase at 25℃. The final
graphs represent the difference between the duplex or quadruplex sensorgrams and the
blank sensorgrams. Data analysis was done using the ProteOn Manager Software, and
the KD was calculated by aligning the kinetic data from various concentrations of
CTD to the 1:1 langmuir binding model.
Molecular docking of CTD to the TrxR1 structural model
To probe the interaction mode between CTD and TrxR1, molecular docking study
was carried out by using AutoDock (version 4.2.6) [1]. AutoDock package is a
flexible docking program which is based on Lamarkian genetic algorithm to search
the optimal conformation of ligand in a macromolecule. The crystal structure of the
human TRXR1 (PDB code: 2ZZ0) was derived from Protein Data Bank as the
receptor [2]. AutoDockTools (version 1.5.6) was employed to generate the docking
input files [1]. A grid box size of 60 × 60 × 60 points with a spacing of 0.375 Å
between the grid points was implemented. The affinity maps of TRXR1 were
calculated using AutoGrid. The Lamarckian genetic algorithm (LGA) was applied to
deal with the TrxR1 and CTD interaction. The docking parameters are as follows:
trials of 200 dockings, the number of individuals in population was set to 300 and 25
million energy evaluations. Other settings were set to default. AutoDockTools version
1.5.6 and PyMol was used to analyze the docking results [1, 3].
[1] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility, J Comput Chem, 30 (2009) 2785-2791.[2] Y.C. Lo, T.P. Ko, W.C. Su, T.L. Su, A.H. Wang, Terpyridine-platinum(II) complexes are effective inhibitors of mammalian topoisomerases and human thioredoxin reductase 1, J Inorg Biochem, 103 (2009) 1082-1092.[3] W.L. DeLano, The PyMOL molecular graphics system., DeLano Scientific, San Carlos, CA, 2002.
Supplementary Figure S1. CTD induced growth arrest and apoptosis is dependent on ROS generation.(S1A, S1B) Quantification of DFC fluorescence (mean fluorescence intensity) in cells exposed to CTD at different concentrations (S1A) or exposed to 30 µM CTD, with or without pretreatment with 5 mM NAC (S1B). (S1C) Flow cytometric determination of cell cycle phase in cells exposed to 30 µM CTD for 15 h. NAC pretreatment, where indicated, was performed at 5 mM for 1 h. (S1D) Apoptosis in colon cancer cells as assessed by Annexin V/PI staining and flow cytometric measurements. Cells were exposed to 30 μM CTD for 20 h, with or with NAC pretreatment (5 mM, 1 h).
Supplementary Figure S2. Morphological alterations in mitochondria following exposure of cells to CTD.Electron microscopy images showing the effect of CTD on mitochondria. HCT-116 cells were exposed to 30 µM CTD for 5 h. NAC pretreatment was carried out at 5 mM for 1 h. Images showing 8,000x and 25,000x mag.
Supplementary Figure S2. The No.2 siRNA sequence silenced ATF-4 expression and block
CTD-induced cell death in HCT116 cells. (A) Western blot analysis of ATF4 protein following
No.2 siRNA transfection in HTC-116 cells [Negative Control = negative control siRNA
transfected cells treated with vehicle, Veh+CTD = negative control siRNA transfected cells treated
with CTD, siRNA = ATF4 siRNA transfected cells treated with vehicle, siRNA+CTD = ATF4
siRNA transfected cells treated with CTD]. (B) Densitometric quantification for panel A [3
independent experiments; *p<0.05]. (C) Assessment of cell viability in HTC-116 cells following
knockdown of ATF4 and exposure to 30μM CTD. Viability was assessed by MTT assay
[***p<0.001].
Supplementary Figure S3. Histological analysis of liver, kidney, and heart tissues did not reveal any pathological alterations upon CTD administration
Supplementary Table S1. The Clinical Characteristics of Patients According to the expression of TrxR1The chart is based on TNM staging of UICC (International Union Against Cancer) eighth edition of Malignant Tumors.MAC Stage is improved Astler-Coller staging.