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: wzmcliangguang@163.com

* 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:13968888873@163.com

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.