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Effect of euphorbia factor L1 on oxidative stress, apoptosis, and autophagy in human gastric epithelial cells
Accepted Manuscript
Effect of Euphorbia factor L1 on oxidative stress, apoptosis and autophagy in human gastric epithelial cells An Zhu , Yuqing Sun , Qianwen Zhong , Jinlan Yang , Tao Zhang , Jingwei Zhao , Qi Wang PII: DOI: Article Number: Reference:
S0944-7113(19)30098-4 https://doi.org/10.1016/j.phymed.2019.152929 152929 PHYMED 152929
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
1 February 2019 11 April 2019 15 April 2019
Please cite this article as: An Zhu , Yuqing Sun , Qianwen Zhong , Jinlan Yang , Tao Zhang , Jingwei Zhao , Qi Wang , Effect of Euphorbia factor L1 on oxidative stress, apoptosis and autophagy in human gastric epithelial cells, Phytomedicine (2019), doi: https://doi.org/10.1016/j.phymed.2019.152929
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ACCEPTED MANUSCRIPT
Effect of Euphorbia factor L1 on oxidative stress, apoptosis and autophagy in human gastric epithelial cells
An Zhua, Yuqing Suna, Qianwen Zhonga, Jinlan Yanga, Tao Zhanga, Jingwei Zhaoa, Qi
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Wanga,b,c,*
a
Department of Toxicology, School of Public Health, Peking University, Beijing 100191, China
b
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Key Laboratory of State Administration of Traditional Chinese Medicine for Compatibility
Toxicology, Beijing 100191, China
c
Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing
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100191, China
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*Correspondence author at: Department of Toxicology, School of Public Health, Peking University,
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82801527
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No. 38 Xueyuan Road, Haidian District, Beijing 100191, People’s Republic of China. Tel.: +86 10
E-mail address: [email protected] (Q. Wang)
ACCEPTED MANUSCRIPT Abstract Background: Euphorbia factor L1 (EFL1), a lathyrane-type diterpenoid from the medicinal herb Euphorbia lathyris L. (Euphorbiaceae), has been reported for many decades to induce gastric irritation, but the underlying mechanisms remain unclear. Purpose: The objective of this study was to investigate EFL1-induced cytotoxicity and the potential
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mechanisms of action on the human normal gastric epithelial cell GES-1.
Methods: GES-1 cells were treated with EFL1 (12.5-200 μM) for different time intervals, and cell survival, LDH release, intracellular reactive oxygen species (ROS), malondialdehyde (MDA)
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content, and superoxide dismutase (SOD) activity were detected. Mitochondrial membrane potential (MMP) assay, DAPI staining, DNA fragment assay, and annexin V-FITC/PI staining were performed. The interaction between EFL1 and Bcl-2, cytochrome c, caspase-9, caspase-3, PI3K,
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AKT, and mTOR proteins was simulated by molecular docking. The mRNA and protein expression of apoptosis and autophagy factors were detected by RT-qPCR and Western blotting.
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Results: EFL1 decreased survival, increased LDH leakage, and induced abnormal production of
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ROS, MDA and SOD in GES-1 cells. Mitochondria-mediated apoptosis was characterized by
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decreased MMP, condensed nuclei, fragmented DNA, and increased apoptosis rate. EFL1 interacted with proteins via hydrogen bonding. The mRNA, total or phosphorylated protein
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expression of Bcl-2, mitochondrial cytochrome c, PI3K, AKT, mTOR and p62 were downregulated; in contrast, those of cytoplasmic cytochrome c, cleaved caspase-9, cleaved caspase-3, LC3-ll and Beclin-1 were upregulated. Conclusion: These findings indicated that EFL1 decreased the survival of GES-1 cells through EFL1-induced oxidative stress, activation of the mitochondria-mediated apoptosis as well as autophagy via inhibition of the PI3K/AKT/mTOR pathway.
ACCEPTED MANUSCRIPT Keywords Euphorbia factor L1; Gastric toxicity; Oxidative stress; Mitochondria; Apoptosis; Autophagy.
Abbreviations EFL1, Euphorbia factor L1
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LDH, lactate dehydrogenase ROS, reactive oxygen species MDA, malondialdehyde
MMP, mitochondrial membrane potential Bcl-2, B-cell lymphoma-2
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PI3K, phosphatidylinositol 3-kinase
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SOD, superoxide dismutase
AKT, protein kinase B
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mTOR, mammalian target of rapamycin
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LC3B, microtubule-associated protein light chain-3
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p62/SQSTM1, sequestosome-1.
ACCEPTED MANUSCRIPT Introduction Euphorbia lathyris L. (Euphorbiaceae) is widely used as a medicinal herb in East Asia, Europe and the USA (Seca and Pinto, 2018). Euphorbia factor L1 (EFL1) is a lathyrane-type diterpenoid mainly found in the seeds of E. lathyris (Adolf et al., 1970). It is one of the main active constituents isolated from E. lathyris and exerts multiple activities, including anticancer, antiadipogenesis,
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antiosteoclastogenesis and multidrug resistance-modulating effects (Hong et al., 2017; Park et al., 2015).
It was recently reported that EFL1 could inhibit the proliferation of tumour cell lines (Wang et al.,
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2018). EFL1 was also reported as a gastrointestinal toxic ingredient, and caused severe toxicity in the gastrointestinal system, manifested by hyperemesis, stomachache, and diarrhoea (Zhu et al., 2018). EFL1 is toxic to tumour cells, but whether it exerts toxicity in normal gastric epithelial cells is
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still unknown, as the mechanisms underlying EFL1-induced gastric toxicity have not been clarified.
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Previous studies suggested that the anticancer mechanisms of lathyrane-type diterpenoids may
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be related to apoptosis (Lin et al., 2017; Zhang et al., 2011). In this study, we hypothesized that EFL1 induced apoptosis in normal gastric cells; consequently, we performed further investigations
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of the interrelated signaling pathways. Cell apoptosis, the process of programmed cell death, plays
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a vital role in maintenance of organism homeostasis (Fuchs and Steller, 2011). Endogenous and exogenous toxicants increase the generation of intracellular reactive oxygen species (ROS), and excessive ROS causes a loss of mitochondrial membrane potential (MMP). During the early stage of mitochondria-mediated apoptosis, the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) was downregulated and therefore unable to inhibit the release of cytochrome c (Lai et al., 2017). After cytochrome c was released from the mitochondria to the cytoplasm, caspase-9 was activated, resulting in the upregulation of cleaved poly ADP-ribose polymerase (PARP) and cleaved
ACCEPTED MANUSCRIPT caspase-3 proteins, involved in the execution of apoptosis (Kuo et al., 2006). The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway is a convergence point of multiple upstream signals; in turn, these regulate various downstream effectors and are therefore involved in the regulation of cell metabolism, survival, proliferation, migration, and protein synthesis, in response to exogenous stimuli (Thorpe et al.,
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2015). Autophagy is a cellular “self-digestion” manner for cell survival in response to extrinsic stress, and PI3K/AKT/mTOR pathway is a negative regulator of autophagy (Shinojima et al., 2014).
The present study used the human normal gastric epithelial cell line GES-1 to investigate the
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potential gastric toxicity of EFL1. The mechanisms underlying the cytotoxicity, including the role of oxidative stress, mitochondria-mediated apoptosis and autophagy via PI3K/AKT/mTOR pathway, were explored by using molecular biology experiments and computational toxicological methods.
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Chemical reagents
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Materials and methods
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EFL1 was purchased from Spring & Autumn Biological Engineering Co., Ltd (Nanjing, China). 13
C and 1H NMR
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The chemical structure (Fig. 1A) of EFL1 was confirmed from the 400 MHz
spectra (Bruker, Rheinstetten, Germany) (Fig. S1). The purity of EFL1 was 98.75% (Fig. 1B) as
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determined by HPLC (Agilent, Santa Clara, CA, USA).
Cell culture and treatments
GES-1 cells derived from the normal epithelium of human gastric mucosa (Cui et al., 2010), were obtained from the Cell Culture Center of Peking Union Medical College (Beijing, China). The cells were cultured in high-glucose Dulbecco's modified Eagle’s medium (DMEM, GE Healthcare HyClone, Logan, UT, USA) supplemented with 10% foetal bovine serum (Gibco, New York, NY
ACCEPTED MANUSCRIPT USA), 100 U/ml penicillin G sodium salt, and 100 μg/ml streptomycin sulphate and maintained in an incubator (Thermo Fisher, Langenselbold, Germany) with an atmosphere of 5% CO 2 at 37°C. EFL1 was dissolved in dimethyl sulfoxide (DMSO), then diluted in DMEM medium to the concentrations 12.5, 25, 50, 100, and 200 μM. The final working concentration of DMSO in cell culture experiments was ≤1%.
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Measurement of cytotoxicity by CCK-8 assay
The cell counting kit (CCK-8) method was used to analyse the cytotoxicity of EFL1 on GES-1 cells. Briefly, the cells were plated into 96-well plates at a density of 1×104 cells/well in a total
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volume of 100 μl, and left to attach and grow for 24 h. The EFL1 concentrations used in this study, 0, 12.5, 25, 50, 100, and 200 μM, were added in a volume of 10 μl to each well and then incubated for 24, 48, and 72 h. Subsequently, 10 μl of CCK-8 solution was added to the cells, incubated 4 h
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at 37°C, and the absorbance at 450 nm was measured by using a microplate reader (Omega,
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Ortenberg, Germany). The EFL1-induced half maximal inhibitory concentration (IC 50) in GES-1
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cells were calculated by probit regression using SPSS 20.0 software (IBM, New York, NY, USA).
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Lactate dehydrogenase release assay
The integrity of the cell membrane was examined by a previously described method (Liu et al.,
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2009) through the measurement of lactate dehydrogenase (LDH) release. After exposure for 24, 48, and 72 h, the 96-well plate was centrifuged at 500 ×g for 5 min; 20 μl of the culture supernatant was transferred to another 96-well culture plate and reacted with solutions of 2,4-dinitrophenylhydrazine and NaOH in succession. After incubation at room temperature for 5 min, LDH activity was measured by using a microplate reader at 450 nm.
ACCEPTED MANUSCRIPT Intracellular ROS detection by flow cytometry
The intracellular ROS levels were detected by using a dichlorofluorescein (DCF) probe. After the addition of 10 μM DCFH-DA and incubation of the GES-1 cells for 1 h at 37°C in the dark, the cells were washed twice with PBS, collected and centrifuged at 500 ×g for 5 min, and washed twice with PBS prior to resuspension to obtain 1×10 6 cells per tube. The cell solutions were then analysed on
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fluorescence channel 1 (FL1) by using flow cytometry (Beckman Coulter, Brea, CA, USA).
Detection of MDA content and SOD activity
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GES-1 cells were treated with various concentrations of EFL1 for 72 h and collected for protein quantification using the bicinchoninic acid (BCA) method (Smith et al., 1985). Malondialdehyde (MDA) was reacted with thiobarbituric acid (TBA) to generate the MDA-TBA adduct, which was
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easily quantifiable at 535 nm. Superoxide dismutase (SOD) catalysed the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, and WST-8 produced
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water-soluble formazan once reacted with a superoxide anion. Formazan was detected at 450 nm.
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MMP detection by flow cytometry
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After the treatment of GES-1 cells with EFL1, JC-10 mitochondrial membrane potential was
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detected by using a previously reported method (Cossarizza et al., 1993). The cells were incubated with JC-10 solution in the dark at 37°C for 20 min, washed twice with PBS, detached by incubation with 0.25% trypsin-0.53 mM EDTA, and collected. The collected cells were centrifuged at 500 ×g for 5 min and then 1×106 cells per tube were resuspended in JC-10 dye buffer prior to analysis on the FL1 channel.
DAPI staining assay by confocal microscope
Nuclear DNA staining was performed to observe EFL1-induced apoptosis through morphological
ACCEPTED MANUSCRIPT nucleolus deformation (Kapuscinski, 1995). GES-1 cells were cultured in glass-bottomed culture dishes and treated with 50, 100 and 200 μM EFL1 for 72 h. After the cells were fixed with 4% formaldehyde for 15 min, the cells were stained with 10 μg/ml DAPI. After 10 min incubation in the dark, the DAPI solution was removed and the cells were washed three times with PBS. The deformation of the nuclei was observed by using laser scanning confocal microscope (Nikon, Tokyo,
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Japan).
DNA fragmentation assay
DNA fragmentation assay was performed to detect the fragmentation of DNA induced by EFL1.
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GES-1 cells were collected, resuspended for lysis, and 200 μl of 100% ethanol was added, centrifuged at 13000 ×g for 2 min, and washed twice with 70% ethanol (v/v). Finally, the DNA was dissolved in 30 μl of eluent. Equal amounts of DNA (1 μg per sample) were migrated in 2%
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horizontal agarose gels at 225 V for 30 min. The gels were premixed with ethidium bromide to
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enable visualization of the fluorescence of the DNA fragments under UV light.
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Annexin V-FITC/PI analysis of apoptosis
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The annexin V-FITC/propidium iodide (PI) probe was used for the detection of apoptosis, as described previously (Schutte et al., 1998). GES-1 cells were seeded in 25-cm2 flasks, pretreated
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with different concentrations of EFL1, and then incubated for 72 h. Thereafter, cells were harvested and washed twice with PBS, and resuspended in 500 μl binding buffer at a density of 5×10 5 cells/ml. Annexin V-FITC (5 μl) and PI (5 μl) were added to the samples and incubated at room temperature for 15 min in the dark, and then samples were processed immediately in flow cytometry. The annexin V-FITC (green) and PI (red) signals were analysed on the FL1 and FL2 channels, respectively.
ACCEPTED MANUSCRIPT Molecular docking
To predict whether EFL1 could interact with proteins involved in apoptosis, molecular docking was performed by SYBYL-X 2.0 software (Tripos, St Louis, MO, USA). The 3D structures of proteins were retrieved from the Protein Data Bank (PDB). The ligand and proteins were prepared prior to docking. The energy of EFL1 was minimized via the addition of Gasteiger-Hückel charges
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in the Tripos force field. To prepare the proteins, we removed the water molecules, metal ions, and solvent molecules, repaired the side chain, fixed the side chain amides, and added the hydrogen atoms. Ligand and proteins were docked in Surflex-Dock Geom mode. Root-mean-square
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deviation (RMSD) was the average distance between the reference structure and the highest ranking docked structure, and RMSD less than 2 Å was deemed to be a proper protein for molecular docking. The total score, a comprehensive evaluation of hydrophobic complementarity,
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RNA isolation and RT-qPCR
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when the value was higher than 5.
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polar complementarity, solvation terms, and entropic terms, was deemed as a stable interaction
RT-qPCR was performed to identify the expression of genes related to the mitochondrial
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apoptotic pathway and the PI3K/AKT/mTOR pathway. Total RNA from GES-1 cells was isolated by
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using TRIzol reagent (Invitrogen, Carlsbad, CA, USA); subsequently, cDNA was synthesized from 10 μl of total RNA by using a reverse transcriptase reaction. To quantify the relative mRNA expression, the primers were synthesized, as shown in Table 1, with β-actin used as the endogenous control. RT-qPCR was performed using a 20 μl reaction volume; relative mRNA expression was calculated by using the 2 -ΔΔCt method (Schmittgen and Livak, 2008) and the fold change was normalized to that observed in the control group.
ACCEPTED MANUSCRIPT Western blotting
Mitochondrial and cytoplasmic proteins contained cytochrome c were extracted using a Mitochondrial Isolation Kit (Applygen, Beijing, China). Total protein was extracted from the GES-1 cells by using the RIPA lysate containing proteinase inhibitors and phosphatase inhibitors and then centrifuged at 14000 ×g for 5 min at 4°C. The protein concentration in the supernatant was
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measured by using the BCA method, and denatured at 100°C for 10 min. Equal amounts of protein (15 μg) were separated on a 4%-15% precast gel and then electrophoretically transferred to polyvinylidene difluoride membranes at 220 mA for 120 min. The membranes were blocked with 5%
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bovine serum albumin in Tris-saline-Tween 20 (TBST) buffer for 120 min at room temperature, and then rinsed three times in TBST for 10 min. The membranes were incubated with primary antibodies (Table 2) in TBST overnight at 4°C and then blots were probed by incubation with a
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secondary antibody for 120 min at room temperature. Finally, the membranes were exposed to ECL
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chemiluminescence solution and imaged by using a Tanon 4500 System (Tanon, Shanghai, China).
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Statistical analysis
The data were analysed by using SPSS software (IBM) and expressed as the mean ± standard
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deviation. One-way analysis of variance (ANOVA) was performed, and the differences between the
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two groups were determined by the least-significant difference test. Values of *p﹤0.05 or **p﹤0.01 were considered statistically significant.
Results Effects of EFL1 on the survival and LDH release of GES-1 cells
The cytotoxic effects of EFL1 on GES-1 cells were evaluated at different time intervals by using the CCK-8 assay and the LDH release assay.
ACCEPTED MANUSCRIPT EFL1 significantly decreased cell viability in a concentration-dependent manner (Fig. 2A). After exposed to 50, 100, and 200 μM EFL1 for 24 h, the cell viability decreased to 90.76%, 86.48% and 82.67% of control, respectively; after exposure for 48 h, the cell viability decreased to 83.39%, 75.89% and 66.95%; and further decreased to 67.89%, 53.90% and 42.15% for 72 h. The IC50 of EFL1 in GES-1 cells following exposure for 72 h was 128.82 ± 14.55 μM, and exceeded 200 μM for
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24 and 48 h.
LDH is a stable cytosolic enzyme that is released into extracellular culture media when the plasma membrane is damaged. The LDH assay was used to confirm the cytotoxicity of EFL1. The
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extracellular LDH activity was 97.36 U/L in control group, after exposure to 50, 100, and 200 μM EFL1 for 72 h, exhibited significant increase (p﹤0.01) in extracellular LDH activity to 321.39, 416.62 and 511.23 U/L, respectively (Fig. 2B). The LDH leakage increased in a concentration- and
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time-dependent manner in response to EFL1 exposure.
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Effects of EFL1 on the oxidative stress in GES-1 cells
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EFL1-induced oxidative injury in GES-1 cells was evaluated based on intracellular ROS, MDA
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content, and SOD activity.
After exposure for 72 h, DCFH-DA fluorescence intensity, representing ROS production,
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significantly increased from 4.93% of the control group to 12.01%, 28.36% and 63.10% of the 50, 100 and 200 μM EFL1 groups (Fig. 3A and B). Excess ROS accumulation in cells was regarded as an adverse molecular event. MDA content significantly increased from 4.43 μM/mg protein of the control group to 7.19, 11.71 and 14.93 μM/mg protein of the 50, 100 and 200 μM EFL1 groups (Fig. 3C), which indicated the oxidative degradation of lipids in GES-1 cells. SOD activities decreased from 17.66 units/mg
ACCEPTED MANUSCRIPT protein in the control group to 16.26, 12.47, and 8.30 units/mg protein in the 50, 100, and 200 μM EFL1 groups, respectively. These results implied that the antioxidant defence system was imbalanced; namely, detoxification capabilities were decreased.
Effects of EFL1 on apoptosis in GES-1 cells
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Several methods were used to identify EFL1-induced apoptosis in GES-1 cells (Fig. 4). The JC-10 probe was used to quantify the MMP of cells; and after exposure for 72 h, green fluorescence intensity of 100 and 200 μM EFL1 groups increased to 1.56 and 2.32 fold of the
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control group (Fig. 4A).
After staining with DAPI, the morphology of the nuclei of GES-1 cells was presented (Fig. 4B). Cells with chromatin condensation and nuclear fragmentation were considered as an indication of
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the induction of apoptosis. In the control group, the nuclei were uniformly distributed with normal morphology, but nuclear fragmentation and chromatin condensation appeared in the EFL1 groups.
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DNA fragmentation assay indicated that the exposure of GES-1 cells to EFL1 for 72 h produced a
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concentration-dependent increase in the degradation of DNA into small fragments (Fig. 4C). DAPI
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staining and DNA fragment analysis provided convincing evidence for EFL1-induced cell apoptosis.
Annexin V-FITC/PI staining was used to estimate the apoptosis rate induced by EFL1 in GES-1
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cells. Early apoptotic cells were determined by counting the percentage of annexin V-FITC+/PIcells, and late apoptotic cells were detected by counting the percentage of annexin V-FITC+/PI+ cells. Annexin V-FITC-/PI+ stained cells were considered to be damaged and the annexin V-FITC-/PI- cells were considered to be surviving cells. After exposure for 72 h, the early apoptosis rate increased from 0.41% of the control group to 6.10%, 25.42% and 12.50% of the 50, 100 and 200 μM EFL1 groups; the late apoptosis rate increased from 0.20% of the control group to 1.60%, 13.72% and 21.54% of the 50, 100 and 200 μM EFL1 groups (Fig. 4D and E).
ACCEPTED MANUSCRIPT Molecular interactions of EFL1 with human proteins in the apoptotic and PI3K/AKT/mTOR pathways
We evaluated the molecular interactions between EFL1 and several key regulatory proteins, including Bcl-2, cytochrome c, caspase-9, caspase-3, PI3K, AKT and mTOR, by using a computational approach. As shown in Fig. 5 and Table 3, all seven proteins had reasonable RMSD
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less than 2 and total score over 5, which indicated potential interactions between EFL1 and these proteins.
Theoretically, EFL1 bound Bcl-2 via the formation of one hydrogen bond at Tyr105, and six
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hydrophobic contacts with Leu198, Tyr199, Gly142, Arg104, Asp100 and Phe101 (Fig. 5A). EFL1 bound cytochrome c via the formation of two hydrogen bonds at Cys17 and Gln16, and nine hydrophobic contacts with Thr28, Met80, Gly29, Lys79, Phe82, His18, Cys14, Lys13 and Il81 (Fig.
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5B). EFL1 bound to caspase-9 via the formation of one hydrogen bond at Gln21, and seven
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hydrophobic contacts at Met84, Glu19, Gln82, Gly81, Asp83, Thr80, and Gln24 (Fig. 5C). EFL1 bound to caspase-3 via the formation of one hydrogen bond at Arg86, and two hydrophobic
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contacts with Leu99 and Lys88 (Fig. 5D).
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EFL1 bound PI3K via the formation of one hydrogen bond at Gln859 and eight hydrophobic
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contacts at Ser774, Ser773, Ile932, Met922, Trp780, Ser854, Thr856, and Met 858 (Fig. 5E). EFL1 bound to AKT via the formation of one hydrogen bond at Lys276 and ten hydrophobic contacts at Glu191, Gly294, His194, Asp292, Glu234, Phe442, Gly157, Gly159, Lys158, Gly162, and Val164 (Fig. 5E). EFL1 bound mTOR via the formation of two hydrogen bonds at Arg2086.HH21 and Arg2086.HE, and four hydrophobic contacts at Cys2085, Phe2048, Glu2052, and Gln2082 (Fig. 5G).
ACCEPTED MANUSCRIPT Effect of EFL1 on the activation of mitochondria-mediated apoptotic pathway in GES-1 cells
The role of oxidative stress in the response of mitochondria related apoptotic molecules in GES-1 cells after EFL1 exposure was further investigated by using RT-qPCR and Western blotting to analyse the mRNA and protein expression of Bcl-2, cytochrome c, caspase-9, and caspase-3 (Fig. 6A and B). EFL1 downregulated the mRNA expression of Bcl-2 to 51.64% and 41.30% of control in
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the 100 and 200 μM groups, but upregulated the mRNA expression of cytochrome c, caspase-9, and caspase-3 in the 50, 100, and 200 μM EFL1 groups. The content of the anti-apoptotic protein Bcl-2 significantly decreased to 43.72% of control after exposed to 200 μM EFL1 (Fig. 6C). The
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elevated protein content of cytoplasmic cytochrome c showed that it was released from the mitochondria to the cytosol, leading to the activation of caspase cascades, namely the upregulation of cleaved caspase-9 and caspase-3. Meanwhile, the proteins of pro-caspase-9 and pro-caspase-3 not
significant
changes.
These
results
indicated
that
EFL1
activated
the
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had
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mitochondria-mediated apoptosis pathway.
Effect of EFL1 on the inhibition of PI3K/AKT/mTOR pathway and activation of autophagy in GES-1
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cells
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The participation of the PI3K/AKT/mTOR pathway in EFL1-induced cytotoxicity was investigated
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by using RT-qPCR and Western blotting. As shown in Fig. 7, the expression of PI3K, AKT, and mTOR mRNA and protein were downregulated in a concentration-dependent manner. The mRNA expression of PI3K, AKT and mTOR decreased to 28.82%, 62.92% and 37.53% of control in the 200 μM EFL1 group. The phosphorylated protein expression of p-AKT and p-mTOR was significantly decreased to 21.53% and 19.33% of control in the 200 μM EFL1 groups, without changes of total AKT and mTOR. The results suggested that EFL1 could inhibit the PI3K/AKT/mTOR pathway, contributing to the suppression of cell survival and proliferation.
ACCEPTED MANUSCRIPT To investigate the effect of the inhibition of PI3K/AKT/mTOR pathway on the autophagy, we detected the protein expression of autophagy markers including microtubule-associated protein light chain-3 (LC3), Beclin-1 and sequestosome-1 (SQSTM1, p62). As shown in Fig. 8, the expression of LC3-ll increased to 2.27 and 2.33 fold of control in 100 and 200 μM EFL1 groups; and Beclin-1 increased to 1.68 and 2.60 fold of control. Whereas the p62 decreased to 34.84% of
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control in the 200 μM EFL1 group. These results indicated that EFL1 activated autophagy in GES-1 cells.
Discussion
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The cytotoxicity of EFL1, EFL2, and EFL3 was recently investigated in A549, MDA-MB-231, KB, KB-VIN, and MCF-7 cell lines (Teng et al., 2018). After exposure for 72 h, it was shown that EFL1 had the highest LC50 (>40 μM) in several tumour cells. In K562 human leukaemia cells, EFL1 was
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cytotoxic with an IC50 value of 33.86 ± 2.51 μM after 96 h (Zhang et al., 2013). In this study, we
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evaluated the cytotoxicity of EFL1 in GES-1 cells at various concentrations and different time intervals. The cell viability and LDH release assays showed that EFL1 was cytotoxic at 50, 100, and
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200 μM after exposure for 72 h. The IC50 of EFL1 in GES-1 cells at 72 h was 128.82 ± 14.55 μM,
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which was much higher than that in tumour cells, suggesting that EFL1 may exert anticancer effects at low concentrations, but may potentially lead to gastric cytotoxicity at relatively high
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concentrations.
The detailed responses of EFL1-induced cytotoxicity in GES-1 cells were evaluated through the analysis of ROS generation, MDA content, and SOD activity. ROS are highly reactive free radicals owing to the presence of unpaired valence shell electrons, and excessive ROS generation is known to disrupt the intracellular redox balance to induce the oxidative reaction of cellular lipids, proteins and nucleic acids, and cell death (Sinha et al., 2013). MDA was generated and selected as a typical
ACCEPTED MANUSCRIPT oxidative marker of the peroxidation of polyunsaturated fatty acids (Gong and Li, 2011). SOD is known as one of the crucial enzymatic antioxidant defences against superoxide radicals; its activity is decreased in disorders of resistance to oxidative stress (Huang et al., 2000). In the present study, EFL1 induced excessive ROS accumulation in GES-1 cells. As an adverse initiation event, ROS can oxidize lipids of cellular membrane and organelle membranes into MDA. In addition, the
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antioxidant system, as represented by SOD, was also disrupted. These findings indicated that EFL1 induced oxidative injury in GES-1 cells.
The relationship between oxidative stress and mitochondria-mediated apoptosis has been
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previously described (Ho et al., 2013). During the process of cell metabolism, the mitochondrial electron transport chain continuously produces ROS, and it is known that excessive ROS can induce mitochondrial dysfunction. In the present study, a decrease in MMP was observed by using
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the JC-10 probe in flow cytometry analysis. DAPI staining, DNA fragmentation assay, and the annexin V-FITC/PI probe provided evidence for EFL1-induced apoptosis, which was characterized
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by condensed and fragmented nuclei, DNA breakage, and annexin V-FITC/PI stained cells. In this
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study, the total percentage of apoptotic cells in EFL1 200 μM group is lower than 100 μM group,
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especially the early apoptotic cells, potentially due to that a plenty of cells in 200 μM group went to death, and were difficult to fully collect for flow cytometry detection.
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Molecular docking was used to analyse the docking sites and interaction forces between EFL1 and seven target proteins involved in mitochondria-mediated apoptosis (Bcl-2, cytochrome c, caspase-9, and caspase-3) and PI3K/AKT/mTOR pathway. The docking scores, hydrogen bonds, and hydrophobic contacts indicated the stable interactions between EFL1 and these proteins. The binding ability of EFL1 for these proteins should be confirmed by further experimental evidence, and how EFL1 precisely affected the function of these proteins need to be verified by molecular dynamics computation.
ACCEPTED MANUSCRIPT Our results demonstrated that EFL1 induced apoptosis in GES-1 cells and theoretically interacted with the proteins in the mitochondrial apoptotic pathway. Under physiological conditions, the anti-apoptotic protein Bcl-2 moves from the cytosol to the outer mitochondrial membrane, which prevented the release of cytochrome c (Song et al., 2008). Decreased Bcl-2 expression triggered a fail-safe mechanism to activate apoptosis (Delbridge et al., 2016). cytochrome c bound Apaf-1 and
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caspase-9 to form a complex, which led to the activation of caspase-3 and resulted in cell apoptosis (Barzilai, 2010). By using RT-qPCR and Western blotting, we confirmed abnormal mRNA and protein expression of the signaling transduction factors Bcl-2, cytochrome c, caspase-9, and
the EFL1-induced cytotoxicity to GES-1 cells.
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caspase-3. Our results suggested that mitochondria-mediated apoptosis was a key participant in
In the PI3K/AKT/mTOR pathway, PI3K enabled the membrane protein PIP2 to be
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phosphorylated into PIP3, and then recruited AKT to be phosphorylated at the Thr308 and Ser473 sites (Sarbassov et al., 2005). Activated AKT inhibited the pro-apoptotic factors Bad, Bax,
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caspase-3, and caspase-8, and simultaneously promoted the anti-apoptotic factor Bcl-2.
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Furthermore, activated AKT mediated downstream mTOR signaling to regulate cell growth, protein
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translation, and ribosome biogenesis (Roos and Kaina, 2013). In the present study, EFL1 downregulated the mRNA and phosphorylated protein expression of PI3K, AKT, and mTOR in
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GES-1 cells. The inhibition of the PI3K/AKT/mTOR pathway might be unfavourable to cell survival and proliferation, and simultaneously contributed to apoptosis. Moreover, inhibition of PI3K/ AKT/mTOR pathway was also involved in autophagy. During autophagy, LC3-l was converted to LC3-ll through lipidation by an ubiquitin-like system. Then the formation of autophagosome was initiated by PI3K type III-Atg6/Beclin-1 complex, and lysosomal degradation of autophagosomes led to a decrease level of p62 (Dong et al., 2019). The present study revealed EFL1-activated
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Conclusion In summary, this study investigated EFL1-induced cytotoxicity and the underlying mechanism in gastric
mucosa
epithelium
cells.
Oxidative
stress
was
the
initial
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event
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mitochondria-mediated apoptosis, and the inhibited PI3K/AKT/mTOR pathway was involved in the autophagy of GES-1 cells. Our study has therefore revealed a possible mechanism for the gastric
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cytotoxicity of EFL1.
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The research was financially supported by funding from the National Natural Science Foundation of China (81673685) and the Special Research Project of Traditional Chinese Medicine (201507004-1).
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Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication
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and there has been no significant financial support for this work that could have influenced its
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outcome.
ACCEPTED MANUSCRIPT References Adolf, W., Hecker, E., Balmain, A., Lhomme, M.F., Nakatani, Y., Ourisson, G., Ponsinet, G., Pryce, R.J., Santhanakrishnan, T.S., Matyukhina, L.G., 1970. “Euphorbiasteroid” (Epoxy-lathyrol) A new tricyclic diterpene from Euphorbia lathyris L. Tetrahedron Lett. 11, 2241-2244. Barzilai, A., 2010. DNA damage, neuronal and glial cell death and neurodegeneration. Apoptosis
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15, 1371-1381. Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G., Franceschi, C., 1993. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming
AN US
lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide JC-1. Biochem. Biophys. Res. Commun. 197, 40-45.
Cui, J., Xing, L., Li, Z., Wu, S., Wang, J., Liu, J., Wang, J., Yan, X., Zhang, X., 2010. Ochratoxin A
M
induces G(2) phase arrest in human gastric epithelium GES-1 cells in vitro. Toxicol. Lett. 193, 152-158.
ED
Delbridge, A.R., Grabow, S., Strasser, A., Vaux, D.L., 2016. Thirty years of BCL-2: translating cell
PT
death discoveries into novel cancer therapies. Nat. Rev. Cancer 16, 99-109. Dong, M.X., Ye, T., Bi, Y.Y., Wang, Q., Kuerban, K., Li, J.Y., Feng, M.Q., Wang, K., Chen, Y., Ye, L.,
CE
2019. A novel hybrid of 3-benzyl coumarin seco-B-ring derivative and phenylsulfonylfuroxan
AC
induces apoptosis and autophagy in non-small-cell lung cancer. Phytomedicine 52, 79-88. Fuchs, Y., Steller, H., 2011. Programmed cell death in animal development and disease. Cell 147, 742-758.
Gong, K., Li, W., 2011. Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: A potential new treatment for hepatocellular carcinoma. Free Radic. Biol. Med. 51, 2259-2271. Ho, E., Karimi Galougahi, K., Liu, C.C., Bhindi, R., Figtree, G.A., 2013. Biological markers of
ACCEPTED MANUSCRIPT oxidative stress: Applications to cardiovascular research and practice. Redox Biol. 1, 483-491. Hong, S.E., Lee, J., Seo, D.H., In Lee, H., Ri Park, D., Lee, G.R., Jo, Y.J., Kim, N., Kwon, M., Shon, H., Kyoung Seo, E., Kim, H.S., Young Lee, S., Jeong, W., 2017. Euphorbia factor L1 inhibits osteoclastogenesis by regulating cellular redox status and induces Fas-mediated apoptosis in osteoclast. Free Radic. Biol. Med. 112, 191-199.
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Huang, P., Feng, L., Oldham, E.A., Keating, M.J., Plunkett, W., 2000. Superoxide dismutase as a target for the selective killing of cancer cells. Nature 407, 390-395.
Kapuscinski, J., 1995. DAPI: a DNA-specific fluorescent probe. Biotech. Histochem. 70, 220-233.
AN US
Kuo, H.C., Lee, H.J., Hu, C.C., Shun, H.I., Tseng, T.H., 2006. Enhancement of esculetin on Taxol-induced apoptosis in human hepatoma HepG2 cells. Toxicol. Appl. Pharmacol. 210, 55-62.
Lai, Z.Q., Ip, S.P., Liao, H.J., Lu, Z., Xie, J.H., Su, Z.R., Chen, Y.L., Xian, Y.F., Leung, P.S., Lin, Z.X.,
M
2017. Brucein D, a naturally occurring tetracyclic triterpene quassinoid, induces apoptosis in
ED
pancreatic cancer through ROS-associated PI3K/Akt signaling pathway. Front. Pharmacol. 8, 936.
PT
Lin, M., Tang, S., Zhang, C., Chen, H., Huang, W., Liu, Y., Zhang, J., 2017. Euphorbia factor L2
AC
59-64.
CE
induces apoptosis in A549 cells through the mitochondrial pathway. Acta. Pharm. Sin. B 7,
Liu, Q., Wang, Q., Yang, X.W., Shen, X.F., Zhang, B.X., 2009. Differential cytotoxic effects of denitroaristolochic acid II and aristolochic acids on renal epithelial cells. Toxicol. Lett. 184, 5-12. Park, S.J., Park, J.H., Han, A., Davaatseren, M., Kim, H.J., Kim, M.S., Hur, H.J., Sung, M.J., Hwang, J.T., Yang, H.J., Kwon, D.Y., 2015. Euphorbiasteroid, a component of Euphorbia lathyris L., inhibits adipogenesis of 3T3-L1 cells via activation of AMP-activated protein kinase. Cell Biochem. Funct. 33, 220-225.
ACCEPTED MANUSCRIPT Roos, W.P., Kaina, B., 2013. DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett. 332, 237-248. Sarbassov, D.D., Guertin, D.A., Ali, S.M., Sabatini, D.M., 2005. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098-1101. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative CT method.
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Nat. Protoc. 3, 1101-1108. Schutte, B., Nuydens, R., Geerts, H., Ramaekers, F., 1998. Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells. J. Neurosci. Methods 86, 63-69.
AN US
Seca, A.M.L., Pinto, D., 2018. Plant secondary metabolites as anticancer agents: successes in clinical trials and therapeutic application. Int. J. Mol. Sci. 19, 263.
Shinojima, N., Yokoyama, T., Kondo, Y., Kondo, S., 2014. Roles of the Akt/mTOR/p70S6K and ERK1/2 signaling pathways in curcumin-induced autophagy. Autophagy 3, 635-637.
M
Sinha, K., Das, J., Pal, P.B., Sil, P.C., 2013. Oxidative stress: the mitochondria-dependent and
ED
mitochondria-independent pathways of apoptosis. Arch. Toxicol. 87, 1157-1180. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto,
PT
E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic
CE
acid. Anal. Biochem. 150, 76-85.
AC
Song, Y., Liang, X., Hu, Y., Wang, Y., Yu, H., Yang, K., 2008. p,p'-DDE induces mitochondria-mediated apoptosis of cultured rat Sertoli cells. Toxicology 253, 53-61. Teng, Y.N., Wang, Y., Hsu, P.L., Xin, G., Zhang, Y., Morris-Natschke, S.L., Goto, M., Lee, K.H., 2018. Mechanism of action of cytotoxic compounds from the seeds of Euphorbia lathyris. Phytomedicine 41, 62-66. Thorpe, L.M., Yuzugullu, H., Zhao, J.J., 2015. PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting. Nat. Rev. Cancer 15, 7-24.
ACCEPTED MANUSCRIPT Wang, J.X., Wang, Q., Zhen, Y.Q., Zhao, S.M., Gao, F., Zhou, X.L., 2018. Cytotoxic lathyrane-type diterpenes from seeds of Euphorbia lathyris. Chem. Pharm. Bull. 66, 674-677. Zhang, J.Y., Liang, Y.J., Chen, H.B., Zheng, L.S., Mi, Y.J., Wang, F., Zhao, X.Q., Wang, X.K., Zhang, H., Fu, L.W., 2011. Structure identification of Euphorbia Factor L3 and its induction of apoptosis through the mitochondrial pathway. Molecules 16, 3222-3231.
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Zhang, J.Y., Lin, M.T., Yi, T., Tang, Y.N., Fan, L.L., He, X.C., Zhao, Z.Z., Chen, H.B., 2013. Apoptosis sensitization by Euphorbia factor L1 in ABCB1-mediated multidrug resistant K562/ADR cells. Molecules 18, 12793-12808.
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Zhu, A., Zhang. T., Wang, Q., 2018. The phytochemistry, pharmacokinetics, pharmacology and
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toxicity of Euphorbia semen. J. Ethnopharmacol. 227, 41-55.
ACCEPTED MANUSCRIPT Figure Legends Fig. 1. (A) The chemical structure of EFL1. (B) The purity of EFL1 was detected by High Performance Liquid Chromatography. The retention time of EFL1 was 8.50 min, and the purity of EFL1 was 98.75%. Fig. 2. Cytotoxicity of EFL1 in GES-1 cells. (A) Concentration effect and time effect of EFL1 on cell
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viability were determined by the CCK-8 assay, the cells viability of each EFL1 group was expressed in percentage in comparison with vehicle control group. (B) Concentration effect and time effect of EFL1 on LDH activity of GES-1 cells. Data were presented as mean ± standard deviation from
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three independent experiments. *p < 0.05 or **p < 0.01 compared with vehicle control.
Fig. 3. EFL1-induced oxidative stress in GES-1 cells. (A) ROS content was determined by DCFH-DA fluorescence probe, in FL1 channel of flow cytometry. (B) The ROS content in GES-1
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cells after various concentrations of EFL1 exposure for 72 h. The MDA content (C) and SOD activity (D) was assayed by biochemical kit. Data were presented as mean ± standard deviation
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from five independent experiments. *p < 0.05 or **p < 0.01 compared with vehicle control.
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Fig. 4. EFL1-induced mitochondria-mediated apoptosis in GES-1 cells. Cells were exposed to 0, 50, 100 and 200 μM EFL1 for 72 h. (A) The mitochondrial membrane potential (MMP) was detected by
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JC-10 fluorescence probe in FL1 channel of flow cytometry, and results were from five independent
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experiments. (B) Morphology of GES-1 cells stained with DAPI, and the white arrows indicates nuclear fragmentation and chromatin condensation, characteristics of apoptosis; typical images were from one of three independent experiments. (C) DNA were extracted from cells exposed to EFL1, and separated on 2% agarose gel containing ethidium bromide; typical images were from one of five independent experiments. (D) Apoptosis was evaluated by annexin V-FITC and PI staining, and percent of early and late stage of apoptotic cells was quantified (E); results were from five independent experiments. Data were presented as mean ± standard deviation. *p < 0.05 or **p
ACCEPTED MANUSCRIPT < 0.01 compared with vehicle control. Fig. 5. Molecular interactions between EFL1 and human proteins of apoptotic pathway and PI3K/AKT/mTOR pathway. 3D and 2D interaction models were presented. (A) Bcl-2, (B) cytochrome c, (C) caspase-9, (D) caspase-3, (E) PI3K, (F) AKT and (G) mTOR. Fig. 6. Effect of EFL1 on the mRNA and protein expression of Bcl-2, cytochrome c, caspase-9 and
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caspase-3 in GES-1 cells, after exposure for 72 h. Results of statistical analysis for mRNA and protein expressions were normalized to β-actin. Relative mRNA expression levels in GES-1 cells by RT-qPCR (A). Relative protein expression levels by Western blotting (B) and semi-quantitative
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analysis (C). Data were presented as mean ± standard deviation from five independent experiments. *p < 0.05 or **p < 0.01 compared with vehicle control.
Fig. 7. Effect of EFL1 on the mRNA and protein expression of PI3K, AKT and mTOR in GES-1 cells, after exposure for 72 h. Results of statistical analysis for mRNA and protein expressions were
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normalized to β-actin. Relative mRNA expression levels in GES-1 cells by RT-qPCR (A). Relative
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protein expression levels by Western blotting (B) and semi-quantitative analysis (C). Data were presented as mean ± standard deviation from five independent experiments. *p < 0.05 or **p < 0.01
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compared with vehicle control.
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Fig. 8. Effect of EFL1 on the expression of autophagy related proteins LC3, Beclin-1 and p62 in
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GES-1 cells, after exposure for 72 h. Results of statistical analysis for protein expression were normalized to β-actin. Relative protein expression levels by Western blotting (A) and semi-quantitative analysis (B). Data were presented as mean ± standard deviation from three independent experiments. *p < 0.05 or **p < 0.01 compared with vehicle control.
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Table 1. Primer sequences and theoretical amplification length Primer sequences (5’ to 3’)
Gene name
Bcl-2
caspase-9
caspase-3
PI3K
AKT
Reverse
CTAAGTCATAGTCCGCCTAGAAGCA
Forward
CTTTGGGCGGAAGACAGGTC
Reverse
TTATTGGCGGCTGTGTAAGAG
Forward
AACATCGCCCTGTGGATGAC
Reverse
AGAGTCTTCAGAGACAGCCAGGAG
Forward
GAACTAACAGGCAAGCAGCAAA
Reverse
GACATCACCAAATCCTCCAGAAC
Forward
CTCCACAGCACCTGGTTATTATTCT
Reverse
GAAAAGTAGCGTCAAAGGAAAAGG
Forward
TGCAGCAGCCTTCAACAAAGA
Reverse
AGCTACACAGTAGCCAGCACAGGA
Forward
AGCGACGTGGCTATTGTGAA
Reverse
CACGTTGGTCCACATCCTG
Forward
GGCCTGGATGGCAACTACAGA
Reverse
TGACTGGCCAGCAGAGTAGGAA
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mTOR
TGGCACCCAGCACAATGAA
186
54
146
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cytochrome c
Forward
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β-actin
Product lengths (bp)
37
149
179
118
142
192
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Table 2. Primary and secondary antibodies for Western blotting analysis Host species
Clonality
Dilution
Brand
Product number
β-actin Bcl-2 cytochrome c caspase-9 caspase-3 PI3K p110 beta p-AKT (Thr308) AKT p-mTOR (Ser2448) mTOR LC3 Beclin-1 p62 Rabbit IgG (HPR)
Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Goat
Polyclonal Polyclonal Polyclonal Polyclonal Monoclonal Monoclonal Polyclonal Polyclonal Monoclonal Monoclonal Monoclonal Polyclonal Monoclonal Polyclonal
1:2000 1:500 1:500 1:2000 1:2000 1:1000 1:500 1:400 1:2000 1:1000 1:1000 1:400 1:1000 1:2000
Abcam Abcam Abcam Abcam Abcam Abcam Abcam Boster Abcam CST CST Boster CST Abcam
ab8227 ab59348 ab53056 ab25758 ab32351 ab151549 ab38449 BA2138 ab109268 2983T 3868S BA3123-2 8025S ab6721
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Product name
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Table 3. Molecular interactions between EFL1 and human proteins Bcl-2, cytochrome c, caspase-9, caspase-3, PI3K, AKT and mTOR RMSD (Å)
Total score
H-bond number
Residues involved in H-bond formation
4LVT 3NWV
1.6468 1.8395
8.5061 6.6789
1 2
A/Tyr105.HH A/Cys17.H, A/Gln16.H
4RHW 5IAB 4WAF 4GV1 4DRH
0.2059 1.2952 0.5950 1.5312 0.8685
5.4125 7.1806 8.0823 8.5364 7.5213
1 1 1 1 2
E/Gln21.HE21 A/Arg86.HH11 A/Gln859.HE21 A/Lys276.HZ2 B/Arg2086.HH21, B/Arg2086.HE
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Bcl-2 cytochrome c caspase-9 caspase-3 PI3K AKT mTOR
PDB ID
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Protein
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Graphical abstract
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Effect of Euphorbia factor L1 on oxidative stress, apoptosis and autophagy in human gastric epithelial cells An Zhu , Yuqing Sun , Qianwen Zhong , Jinlan Yang , Tao Zhang , Jingwei Zhao , Qi Wang PII: DOI: Article Number: Reference:
S0944-7113(19)30098-4 https://doi.org/10.1016/j.phymed.2019.152929 152929 PHYMED 152929
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
1 February 2019 11 April 2019 15 April 2019
Please cite this article as: An Zhu , Yuqing Sun , Qianwen Zhong , Jinlan Yang , Tao Zhang , Jingwei Zhao , Qi Wang , Effect of Euphorbia factor L1 on oxidative stress, apoptosis and autophagy in human gastric epithelial cells, Phytomedicine (2019), doi: https://doi.org/10.1016/j.phymed.2019.152929
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Effect of Euphorbia factor L1 on oxidative stress, apoptosis and autophagy in human gastric epithelial cells
An Zhua, Yuqing Suna, Qianwen Zhonga, Jinlan Yanga, Tao Zhanga, Jingwei Zhaoa, Qi
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Wanga,b,c,*
a
Department of Toxicology, School of Public Health, Peking University, Beijing 100191, China
b
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Key Laboratory of State Administration of Traditional Chinese Medicine for Compatibility
Toxicology, Beijing 100191, China
c
Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing
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100191, China
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*Correspondence author at: Department of Toxicology, School of Public Health, Peking University,
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82801527
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No. 38 Xueyuan Road, Haidian District, Beijing 100191, People’s Republic of China. Tel.: +86 10
E-mail address: [email protected] (Q. Wang)
ACCEPTED MANUSCRIPT Abstract Background: Euphorbia factor L1 (EFL1), a lathyrane-type diterpenoid from the medicinal herb Euphorbia lathyris L. (Euphorbiaceae), has been reported for many decades to induce gastric irritation, but the underlying mechanisms remain unclear. Purpose: The objective of this study was to investigate EFL1-induced cytotoxicity and the potential
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mechanisms of action on the human normal gastric epithelial cell GES-1.
Methods: GES-1 cells were treated with EFL1 (12.5-200 μM) for different time intervals, and cell survival, LDH release, intracellular reactive oxygen species (ROS), malondialdehyde (MDA)
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content, and superoxide dismutase (SOD) activity were detected. Mitochondrial membrane potential (MMP) assay, DAPI staining, DNA fragment assay, and annexin V-FITC/PI staining were performed. The interaction between EFL1 and Bcl-2, cytochrome c, caspase-9, caspase-3, PI3K,
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AKT, and mTOR proteins was simulated by molecular docking. The mRNA and protein expression of apoptosis and autophagy factors were detected by RT-qPCR and Western blotting.
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Results: EFL1 decreased survival, increased LDH leakage, and induced abnormal production of
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ROS, MDA and SOD in GES-1 cells. Mitochondria-mediated apoptosis was characterized by
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decreased MMP, condensed nuclei, fragmented DNA, and increased apoptosis rate. EFL1 interacted with proteins via hydrogen bonding. The mRNA, total or phosphorylated protein
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expression of Bcl-2, mitochondrial cytochrome c, PI3K, AKT, mTOR and p62 were downregulated; in contrast, those of cytoplasmic cytochrome c, cleaved caspase-9, cleaved caspase-3, LC3-ll and Beclin-1 were upregulated. Conclusion: These findings indicated that EFL1 decreased the survival of GES-1 cells through EFL1-induced oxidative stress, activation of the mitochondria-mediated apoptosis as well as autophagy via inhibition of the PI3K/AKT/mTOR pathway.
ACCEPTED MANUSCRIPT Keywords Euphorbia factor L1; Gastric toxicity; Oxidative stress; Mitochondria; Apoptosis; Autophagy.
Abbreviations EFL1, Euphorbia factor L1
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LDH, lactate dehydrogenase ROS, reactive oxygen species MDA, malondialdehyde
MMP, mitochondrial membrane potential Bcl-2, B-cell lymphoma-2
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PI3K, phosphatidylinositol 3-kinase
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SOD, superoxide dismutase
AKT, protein kinase B
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mTOR, mammalian target of rapamycin
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LC3B, microtubule-associated protein light chain-3
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p62/SQSTM1, sequestosome-1.
ACCEPTED MANUSCRIPT Introduction Euphorbia lathyris L. (Euphorbiaceae) is widely used as a medicinal herb in East Asia, Europe and the USA (Seca and Pinto, 2018). Euphorbia factor L1 (EFL1) is a lathyrane-type diterpenoid mainly found in the seeds of E. lathyris (Adolf et al., 1970). It is one of the main active constituents isolated from E. lathyris and exerts multiple activities, including anticancer, antiadipogenesis,
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antiosteoclastogenesis and multidrug resistance-modulating effects (Hong et al., 2017; Park et al., 2015).
It was recently reported that EFL1 could inhibit the proliferation of tumour cell lines (Wang et al.,
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2018). EFL1 was also reported as a gastrointestinal toxic ingredient, and caused severe toxicity in the gastrointestinal system, manifested by hyperemesis, stomachache, and diarrhoea (Zhu et al., 2018). EFL1 is toxic to tumour cells, but whether it exerts toxicity in normal gastric epithelial cells is
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still unknown, as the mechanisms underlying EFL1-induced gastric toxicity have not been clarified.
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Previous studies suggested that the anticancer mechanisms of lathyrane-type diterpenoids may
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be related to apoptosis (Lin et al., 2017; Zhang et al., 2011). In this study, we hypothesized that EFL1 induced apoptosis in normal gastric cells; consequently, we performed further investigations
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of the interrelated signaling pathways. Cell apoptosis, the process of programmed cell death, plays
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a vital role in maintenance of organism homeostasis (Fuchs and Steller, 2011). Endogenous and exogenous toxicants increase the generation of intracellular reactive oxygen species (ROS), and excessive ROS causes a loss of mitochondrial membrane potential (MMP). During the early stage of mitochondria-mediated apoptosis, the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) was downregulated and therefore unable to inhibit the release of cytochrome c (Lai et al., 2017). After cytochrome c was released from the mitochondria to the cytoplasm, caspase-9 was activated, resulting in the upregulation of cleaved poly ADP-ribose polymerase (PARP) and cleaved
ACCEPTED MANUSCRIPT caspase-3 proteins, involved in the execution of apoptosis (Kuo et al., 2006). The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway is a convergence point of multiple upstream signals; in turn, these regulate various downstream effectors and are therefore involved in the regulation of cell metabolism, survival, proliferation, migration, and protein synthesis, in response to exogenous stimuli (Thorpe et al.,
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2015). Autophagy is a cellular “self-digestion” manner for cell survival in response to extrinsic stress, and PI3K/AKT/mTOR pathway is a negative regulator of autophagy (Shinojima et al., 2014).
The present study used the human normal gastric epithelial cell line GES-1 to investigate the
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potential gastric toxicity of EFL1. The mechanisms underlying the cytotoxicity, including the role of oxidative stress, mitochondria-mediated apoptosis and autophagy via PI3K/AKT/mTOR pathway, were explored by using molecular biology experiments and computational toxicological methods.
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Chemical reagents
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Materials and methods
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EFL1 was purchased from Spring & Autumn Biological Engineering Co., Ltd (Nanjing, China). 13
C and 1H NMR
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The chemical structure (Fig. 1A) of EFL1 was confirmed from the 400 MHz
spectra (Bruker, Rheinstetten, Germany) (Fig. S1). The purity of EFL1 was 98.75% (Fig. 1B) as
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determined by HPLC (Agilent, Santa Clara, CA, USA).
Cell culture and treatments
GES-1 cells derived from the normal epithelium of human gastric mucosa (Cui et al., 2010), were obtained from the Cell Culture Center of Peking Union Medical College (Beijing, China). The cells were cultured in high-glucose Dulbecco's modified Eagle’s medium (DMEM, GE Healthcare HyClone, Logan, UT, USA) supplemented with 10% foetal bovine serum (Gibco, New York, NY
ACCEPTED MANUSCRIPT USA), 100 U/ml penicillin G sodium salt, and 100 μg/ml streptomycin sulphate and maintained in an incubator (Thermo Fisher, Langenselbold, Germany) with an atmosphere of 5% CO 2 at 37°C. EFL1 was dissolved in dimethyl sulfoxide (DMSO), then diluted in DMEM medium to the concentrations 12.5, 25, 50, 100, and 200 μM. The final working concentration of DMSO in cell culture experiments was ≤1%.
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Measurement of cytotoxicity by CCK-8 assay
The cell counting kit (CCK-8) method was used to analyse the cytotoxicity of EFL1 on GES-1 cells. Briefly, the cells were plated into 96-well plates at a density of 1×104 cells/well in a total
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volume of 100 μl, and left to attach and grow for 24 h. The EFL1 concentrations used in this study, 0, 12.5, 25, 50, 100, and 200 μM, were added in a volume of 10 μl to each well and then incubated for 24, 48, and 72 h. Subsequently, 10 μl of CCK-8 solution was added to the cells, incubated 4 h
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at 37°C, and the absorbance at 450 nm was measured by using a microplate reader (Omega,
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Ortenberg, Germany). The EFL1-induced half maximal inhibitory concentration (IC 50) in GES-1
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cells were calculated by probit regression using SPSS 20.0 software (IBM, New York, NY, USA).
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Lactate dehydrogenase release assay
The integrity of the cell membrane was examined by a previously described method (Liu et al.,
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2009) through the measurement of lactate dehydrogenase (LDH) release. After exposure for 24, 48, and 72 h, the 96-well plate was centrifuged at 500 ×g for 5 min; 20 μl of the culture supernatant was transferred to another 96-well culture plate and reacted with solutions of 2,4-dinitrophenylhydrazine and NaOH in succession. After incubation at room temperature for 5 min, LDH activity was measured by using a microplate reader at 450 nm.
ACCEPTED MANUSCRIPT Intracellular ROS detection by flow cytometry
The intracellular ROS levels were detected by using a dichlorofluorescein (DCF) probe. After the addition of 10 μM DCFH-DA and incubation of the GES-1 cells for 1 h at 37°C in the dark, the cells were washed twice with PBS, collected and centrifuged at 500 ×g for 5 min, and washed twice with PBS prior to resuspension to obtain 1×10 6 cells per tube. The cell solutions were then analysed on
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fluorescence channel 1 (FL1) by using flow cytometry (Beckman Coulter, Brea, CA, USA).
Detection of MDA content and SOD activity
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GES-1 cells were treated with various concentrations of EFL1 for 72 h and collected for protein quantification using the bicinchoninic acid (BCA) method (Smith et al., 1985). Malondialdehyde (MDA) was reacted with thiobarbituric acid (TBA) to generate the MDA-TBA adduct, which was
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easily quantifiable at 535 nm. Superoxide dismutase (SOD) catalysed the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, and WST-8 produced
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water-soluble formazan once reacted with a superoxide anion. Formazan was detected at 450 nm.
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MMP detection by flow cytometry
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After the treatment of GES-1 cells with EFL1, JC-10 mitochondrial membrane potential was
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detected by using a previously reported method (Cossarizza et al., 1993). The cells were incubated with JC-10 solution in the dark at 37°C for 20 min, washed twice with PBS, detached by incubation with 0.25% trypsin-0.53 mM EDTA, and collected. The collected cells were centrifuged at 500 ×g for 5 min and then 1×106 cells per tube were resuspended in JC-10 dye buffer prior to analysis on the FL1 channel.
DAPI staining assay by confocal microscope
Nuclear DNA staining was performed to observe EFL1-induced apoptosis through morphological
ACCEPTED MANUSCRIPT nucleolus deformation (Kapuscinski, 1995). GES-1 cells were cultured in glass-bottomed culture dishes and treated with 50, 100 and 200 μM EFL1 for 72 h. After the cells were fixed with 4% formaldehyde for 15 min, the cells were stained with 10 μg/ml DAPI. After 10 min incubation in the dark, the DAPI solution was removed and the cells were washed three times with PBS. The deformation of the nuclei was observed by using laser scanning confocal microscope (Nikon, Tokyo,
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Japan).
DNA fragmentation assay
DNA fragmentation assay was performed to detect the fragmentation of DNA induced by EFL1.
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GES-1 cells were collected, resuspended for lysis, and 200 μl of 100% ethanol was added, centrifuged at 13000 ×g for 2 min, and washed twice with 70% ethanol (v/v). Finally, the DNA was dissolved in 30 μl of eluent. Equal amounts of DNA (1 μg per sample) were migrated in 2%
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horizontal agarose gels at 225 V for 30 min. The gels were premixed with ethidium bromide to
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enable visualization of the fluorescence of the DNA fragments under UV light.
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Annexin V-FITC/PI analysis of apoptosis
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The annexin V-FITC/propidium iodide (PI) probe was used for the detection of apoptosis, as described previously (Schutte et al., 1998). GES-1 cells were seeded in 25-cm2 flasks, pretreated
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with different concentrations of EFL1, and then incubated for 72 h. Thereafter, cells were harvested and washed twice with PBS, and resuspended in 500 μl binding buffer at a density of 5×10 5 cells/ml. Annexin V-FITC (5 μl) and PI (5 μl) were added to the samples and incubated at room temperature for 15 min in the dark, and then samples were processed immediately in flow cytometry. The annexin V-FITC (green) and PI (red) signals were analysed on the FL1 and FL2 channels, respectively.
ACCEPTED MANUSCRIPT Molecular docking
To predict whether EFL1 could interact with proteins involved in apoptosis, molecular docking was performed by SYBYL-X 2.0 software (Tripos, St Louis, MO, USA). The 3D structures of proteins were retrieved from the Protein Data Bank (PDB). The ligand and proteins were prepared prior to docking. The energy of EFL1 was minimized via the addition of Gasteiger-Hückel charges
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in the Tripos force field. To prepare the proteins, we removed the water molecules, metal ions, and solvent molecules, repaired the side chain, fixed the side chain amides, and added the hydrogen atoms. Ligand and proteins were docked in Surflex-Dock Geom mode. Root-mean-square
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deviation (RMSD) was the average distance between the reference structure and the highest ranking docked structure, and RMSD less than 2 Å was deemed to be a proper protein for molecular docking. The total score, a comprehensive evaluation of hydrophobic complementarity,
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RNA isolation and RT-qPCR
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when the value was higher than 5.
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polar complementarity, solvation terms, and entropic terms, was deemed as a stable interaction
RT-qPCR was performed to identify the expression of genes related to the mitochondrial
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apoptotic pathway and the PI3K/AKT/mTOR pathway. Total RNA from GES-1 cells was isolated by
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using TRIzol reagent (Invitrogen, Carlsbad, CA, USA); subsequently, cDNA was synthesized from 10 μl of total RNA by using a reverse transcriptase reaction. To quantify the relative mRNA expression, the primers were synthesized, as shown in Table 1, with β-actin used as the endogenous control. RT-qPCR was performed using a 20 μl reaction volume; relative mRNA expression was calculated by using the 2 -ΔΔCt method (Schmittgen and Livak, 2008) and the fold change was normalized to that observed in the control group.
ACCEPTED MANUSCRIPT Western blotting
Mitochondrial and cytoplasmic proteins contained cytochrome c were extracted using a Mitochondrial Isolation Kit (Applygen, Beijing, China). Total protein was extracted from the GES-1 cells by using the RIPA lysate containing proteinase inhibitors and phosphatase inhibitors and then centrifuged at 14000 ×g for 5 min at 4°C. The protein concentration in the supernatant was
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measured by using the BCA method, and denatured at 100°C for 10 min. Equal amounts of protein (15 μg) were separated on a 4%-15% precast gel and then electrophoretically transferred to polyvinylidene difluoride membranes at 220 mA for 120 min. The membranes were blocked with 5%
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bovine serum albumin in Tris-saline-Tween 20 (TBST) buffer for 120 min at room temperature, and then rinsed three times in TBST for 10 min. The membranes were incubated with primary antibodies (Table 2) in TBST overnight at 4°C and then blots were probed by incubation with a
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secondary antibody for 120 min at room temperature. Finally, the membranes were exposed to ECL
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chemiluminescence solution and imaged by using a Tanon 4500 System (Tanon, Shanghai, China).
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Statistical analysis
The data were analysed by using SPSS software (IBM) and expressed as the mean ± standard
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deviation. One-way analysis of variance (ANOVA) was performed, and the differences between the
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two groups were determined by the least-significant difference test. Values of *p﹤0.05 or **p﹤0.01 were considered statistically significant.
Results Effects of EFL1 on the survival and LDH release of GES-1 cells
The cytotoxic effects of EFL1 on GES-1 cells were evaluated at different time intervals by using the CCK-8 assay and the LDH release assay.
ACCEPTED MANUSCRIPT EFL1 significantly decreased cell viability in a concentration-dependent manner (Fig. 2A). After exposed to 50, 100, and 200 μM EFL1 for 24 h, the cell viability decreased to 90.76%, 86.48% and 82.67% of control, respectively; after exposure for 48 h, the cell viability decreased to 83.39%, 75.89% and 66.95%; and further decreased to 67.89%, 53.90% and 42.15% for 72 h. The IC50 of EFL1 in GES-1 cells following exposure for 72 h was 128.82 ± 14.55 μM, and exceeded 200 μM for
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24 and 48 h.
LDH is a stable cytosolic enzyme that is released into extracellular culture media when the plasma membrane is damaged. The LDH assay was used to confirm the cytotoxicity of EFL1. The
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extracellular LDH activity was 97.36 U/L in control group, after exposure to 50, 100, and 200 μM EFL1 for 72 h, exhibited significant increase (p﹤0.01) in extracellular LDH activity to 321.39, 416.62 and 511.23 U/L, respectively (Fig. 2B). The LDH leakage increased in a concentration- and
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time-dependent manner in response to EFL1 exposure.
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Effects of EFL1 on the oxidative stress in GES-1 cells
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EFL1-induced oxidative injury in GES-1 cells was evaluated based on intracellular ROS, MDA
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content, and SOD activity.
After exposure for 72 h, DCFH-DA fluorescence intensity, representing ROS production,
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significantly increased from 4.93% of the control group to 12.01%, 28.36% and 63.10% of the 50, 100 and 200 μM EFL1 groups (Fig. 3A and B). Excess ROS accumulation in cells was regarded as an adverse molecular event. MDA content significantly increased from 4.43 μM/mg protein of the control group to 7.19, 11.71 and 14.93 μM/mg protein of the 50, 100 and 200 μM EFL1 groups (Fig. 3C), which indicated the oxidative degradation of lipids in GES-1 cells. SOD activities decreased from 17.66 units/mg
ACCEPTED MANUSCRIPT protein in the control group to 16.26, 12.47, and 8.30 units/mg protein in the 50, 100, and 200 μM EFL1 groups, respectively. These results implied that the antioxidant defence system was imbalanced; namely, detoxification capabilities were decreased.
Effects of EFL1 on apoptosis in GES-1 cells
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Several methods were used to identify EFL1-induced apoptosis in GES-1 cells (Fig. 4). The JC-10 probe was used to quantify the MMP of cells; and after exposure for 72 h, green fluorescence intensity of 100 and 200 μM EFL1 groups increased to 1.56 and 2.32 fold of the
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control group (Fig. 4A).
After staining with DAPI, the morphology of the nuclei of GES-1 cells was presented (Fig. 4B). Cells with chromatin condensation and nuclear fragmentation were considered as an indication of
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the induction of apoptosis. In the control group, the nuclei were uniformly distributed with normal morphology, but nuclear fragmentation and chromatin condensation appeared in the EFL1 groups.
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DNA fragmentation assay indicated that the exposure of GES-1 cells to EFL1 for 72 h produced a
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concentration-dependent increase in the degradation of DNA into small fragments (Fig. 4C). DAPI
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staining and DNA fragment analysis provided convincing evidence for EFL1-induced cell apoptosis.
Annexin V-FITC/PI staining was used to estimate the apoptosis rate induced by EFL1 in GES-1
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cells. Early apoptotic cells were determined by counting the percentage of annexin V-FITC+/PIcells, and late apoptotic cells were detected by counting the percentage of annexin V-FITC+/PI+ cells. Annexin V-FITC-/PI+ stained cells were considered to be damaged and the annexin V-FITC-/PI- cells were considered to be surviving cells. After exposure for 72 h, the early apoptosis rate increased from 0.41% of the control group to 6.10%, 25.42% and 12.50% of the 50, 100 and 200 μM EFL1 groups; the late apoptosis rate increased from 0.20% of the control group to 1.60%, 13.72% and 21.54% of the 50, 100 and 200 μM EFL1 groups (Fig. 4D and E).
ACCEPTED MANUSCRIPT Molecular interactions of EFL1 with human proteins in the apoptotic and PI3K/AKT/mTOR pathways
We evaluated the molecular interactions between EFL1 and several key regulatory proteins, including Bcl-2, cytochrome c, caspase-9, caspase-3, PI3K, AKT and mTOR, by using a computational approach. As shown in Fig. 5 and Table 3, all seven proteins had reasonable RMSD
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less than 2 and total score over 5, which indicated potential interactions between EFL1 and these proteins.
Theoretically, EFL1 bound Bcl-2 via the formation of one hydrogen bond at Tyr105, and six
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hydrophobic contacts with Leu198, Tyr199, Gly142, Arg104, Asp100 and Phe101 (Fig. 5A). EFL1 bound cytochrome c via the formation of two hydrogen bonds at Cys17 and Gln16, and nine hydrophobic contacts with Thr28, Met80, Gly29, Lys79, Phe82, His18, Cys14, Lys13 and Il81 (Fig.
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5B). EFL1 bound to caspase-9 via the formation of one hydrogen bond at Gln21, and seven
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hydrophobic contacts at Met84, Glu19, Gln82, Gly81, Asp83, Thr80, and Gln24 (Fig. 5C). EFL1 bound to caspase-3 via the formation of one hydrogen bond at Arg86, and two hydrophobic
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contacts with Leu99 and Lys88 (Fig. 5D).
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EFL1 bound PI3K via the formation of one hydrogen bond at Gln859 and eight hydrophobic
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contacts at Ser774, Ser773, Ile932, Met922, Trp780, Ser854, Thr856, and Met 858 (Fig. 5E). EFL1 bound to AKT via the formation of one hydrogen bond at Lys276 and ten hydrophobic contacts at Glu191, Gly294, His194, Asp292, Glu234, Phe442, Gly157, Gly159, Lys158, Gly162, and Val164 (Fig. 5E). EFL1 bound mTOR via the formation of two hydrogen bonds at Arg2086.HH21 and Arg2086.HE, and four hydrophobic contacts at Cys2085, Phe2048, Glu2052, and Gln2082 (Fig. 5G).
ACCEPTED MANUSCRIPT Effect of EFL1 on the activation of mitochondria-mediated apoptotic pathway in GES-1 cells
The role of oxidative stress in the response of mitochondria related apoptotic molecules in GES-1 cells after EFL1 exposure was further investigated by using RT-qPCR and Western blotting to analyse the mRNA and protein expression of Bcl-2, cytochrome c, caspase-9, and caspase-3 (Fig. 6A and B). EFL1 downregulated the mRNA expression of Bcl-2 to 51.64% and 41.30% of control in
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the 100 and 200 μM groups, but upregulated the mRNA expression of cytochrome c, caspase-9, and caspase-3 in the 50, 100, and 200 μM EFL1 groups. The content of the anti-apoptotic protein Bcl-2 significantly decreased to 43.72% of control after exposed to 200 μM EFL1 (Fig. 6C). The
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elevated protein content of cytoplasmic cytochrome c showed that it was released from the mitochondria to the cytosol, leading to the activation of caspase cascades, namely the upregulation of cleaved caspase-9 and caspase-3. Meanwhile, the proteins of pro-caspase-9 and pro-caspase-3 not
significant
changes.
These
results
indicated
that
EFL1
activated
the
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had
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mitochondria-mediated apoptosis pathway.
Effect of EFL1 on the inhibition of PI3K/AKT/mTOR pathway and activation of autophagy in GES-1
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cells
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The participation of the PI3K/AKT/mTOR pathway in EFL1-induced cytotoxicity was investigated
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by using RT-qPCR and Western blotting. As shown in Fig. 7, the expression of PI3K, AKT, and mTOR mRNA and protein were downregulated in a concentration-dependent manner. The mRNA expression of PI3K, AKT and mTOR decreased to 28.82%, 62.92% and 37.53% of control in the 200 μM EFL1 group. The phosphorylated protein expression of p-AKT and p-mTOR was significantly decreased to 21.53% and 19.33% of control in the 200 μM EFL1 groups, without changes of total AKT and mTOR. The results suggested that EFL1 could inhibit the PI3K/AKT/mTOR pathway, contributing to the suppression of cell survival and proliferation.
ACCEPTED MANUSCRIPT To investigate the effect of the inhibition of PI3K/AKT/mTOR pathway on the autophagy, we detected the protein expression of autophagy markers including microtubule-associated protein light chain-3 (LC3), Beclin-1 and sequestosome-1 (SQSTM1, p62). As shown in Fig. 8, the expression of LC3-ll increased to 2.27 and 2.33 fold of control in 100 and 200 μM EFL1 groups; and Beclin-1 increased to 1.68 and 2.60 fold of control. Whereas the p62 decreased to 34.84% of
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control in the 200 μM EFL1 group. These results indicated that EFL1 activated autophagy in GES-1 cells.
Discussion
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The cytotoxicity of EFL1, EFL2, and EFL3 was recently investigated in A549, MDA-MB-231, KB, KB-VIN, and MCF-7 cell lines (Teng et al., 2018). After exposure for 72 h, it was shown that EFL1 had the highest LC50 (>40 μM) in several tumour cells. In K562 human leukaemia cells, EFL1 was
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cytotoxic with an IC50 value of 33.86 ± 2.51 μM after 96 h (Zhang et al., 2013). In this study, we
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evaluated the cytotoxicity of EFL1 in GES-1 cells at various concentrations and different time intervals. The cell viability and LDH release assays showed that EFL1 was cytotoxic at 50, 100, and
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200 μM after exposure for 72 h. The IC50 of EFL1 in GES-1 cells at 72 h was 128.82 ± 14.55 μM,
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which was much higher than that in tumour cells, suggesting that EFL1 may exert anticancer effects at low concentrations, but may potentially lead to gastric cytotoxicity at relatively high
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concentrations.
The detailed responses of EFL1-induced cytotoxicity in GES-1 cells were evaluated through the analysis of ROS generation, MDA content, and SOD activity. ROS are highly reactive free radicals owing to the presence of unpaired valence shell electrons, and excessive ROS generation is known to disrupt the intracellular redox balance to induce the oxidative reaction of cellular lipids, proteins and nucleic acids, and cell death (Sinha et al., 2013). MDA was generated and selected as a typical
ACCEPTED MANUSCRIPT oxidative marker of the peroxidation of polyunsaturated fatty acids (Gong and Li, 2011). SOD is known as one of the crucial enzymatic antioxidant defences against superoxide radicals; its activity is decreased in disorders of resistance to oxidative stress (Huang et al., 2000). In the present study, EFL1 induced excessive ROS accumulation in GES-1 cells. As an adverse initiation event, ROS can oxidize lipids of cellular membrane and organelle membranes into MDA. In addition, the
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antioxidant system, as represented by SOD, was also disrupted. These findings indicated that EFL1 induced oxidative injury in GES-1 cells.
The relationship between oxidative stress and mitochondria-mediated apoptosis has been
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previously described (Ho et al., 2013). During the process of cell metabolism, the mitochondrial electron transport chain continuously produces ROS, and it is known that excessive ROS can induce mitochondrial dysfunction. In the present study, a decrease in MMP was observed by using
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the JC-10 probe in flow cytometry analysis. DAPI staining, DNA fragmentation assay, and the annexin V-FITC/PI probe provided evidence for EFL1-induced apoptosis, which was characterized
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by condensed and fragmented nuclei, DNA breakage, and annexin V-FITC/PI stained cells. In this
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study, the total percentage of apoptotic cells in EFL1 200 μM group is lower than 100 μM group,
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especially the early apoptotic cells, potentially due to that a plenty of cells in 200 μM group went to death, and were difficult to fully collect for flow cytometry detection.
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Molecular docking was used to analyse the docking sites and interaction forces between EFL1 and seven target proteins involved in mitochondria-mediated apoptosis (Bcl-2, cytochrome c, caspase-9, and caspase-3) and PI3K/AKT/mTOR pathway. The docking scores, hydrogen bonds, and hydrophobic contacts indicated the stable interactions between EFL1 and these proteins. The binding ability of EFL1 for these proteins should be confirmed by further experimental evidence, and how EFL1 precisely affected the function of these proteins need to be verified by molecular dynamics computation.
ACCEPTED MANUSCRIPT Our results demonstrated that EFL1 induced apoptosis in GES-1 cells and theoretically interacted with the proteins in the mitochondrial apoptotic pathway. Under physiological conditions, the anti-apoptotic protein Bcl-2 moves from the cytosol to the outer mitochondrial membrane, which prevented the release of cytochrome c (Song et al., 2008). Decreased Bcl-2 expression triggered a fail-safe mechanism to activate apoptosis (Delbridge et al., 2016). cytochrome c bound Apaf-1 and
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caspase-9 to form a complex, which led to the activation of caspase-3 and resulted in cell apoptosis (Barzilai, 2010). By using RT-qPCR and Western blotting, we confirmed abnormal mRNA and protein expression of the signaling transduction factors Bcl-2, cytochrome c, caspase-9, and
the EFL1-induced cytotoxicity to GES-1 cells.
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caspase-3. Our results suggested that mitochondria-mediated apoptosis was a key participant in
In the PI3K/AKT/mTOR pathway, PI3K enabled the membrane protein PIP2 to be
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phosphorylated into PIP3, and then recruited AKT to be phosphorylated at the Thr308 and Ser473 sites (Sarbassov et al., 2005). Activated AKT inhibited the pro-apoptotic factors Bad, Bax,
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caspase-3, and caspase-8, and simultaneously promoted the anti-apoptotic factor Bcl-2.
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Furthermore, activated AKT mediated downstream mTOR signaling to regulate cell growth, protein
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translation, and ribosome biogenesis (Roos and Kaina, 2013). In the present study, EFL1 downregulated the mRNA and phosphorylated protein expression of PI3K, AKT, and mTOR in
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GES-1 cells. The inhibition of the PI3K/AKT/mTOR pathway might be unfavourable to cell survival and proliferation, and simultaneously contributed to apoptosis. Moreover, inhibition of PI3K/ AKT/mTOR pathway was also involved in autophagy. During autophagy, LC3-l was converted to LC3-ll through lipidation by an ubiquitin-like system. Then the formation of autophagosome was initiated by PI3K type III-Atg6/Beclin-1 complex, and lysosomal degradation of autophagosomes led to a decrease level of p62 (Dong et al., 2019). The present study revealed EFL1-activated
ACCEPTED MANUSCRIPT autophagy characterized by elevation of LC3-ll and Beclin-1, as well as reduction of p62 in GES-1 cells.
Conclusion In summary, this study investigated EFL1-induced cytotoxicity and the underlying mechanism in gastric
mucosa
epithelium
cells.
Oxidative
stress
was
the
initial
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human
event
in
mitochondria-mediated apoptosis, and the inhibited PI3K/AKT/mTOR pathway was involved in the autophagy of GES-1 cells. Our study has therefore revealed a possible mechanism for the gastric
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cytotoxicity of EFL1.
ACCEPTED MANUSCRIPT Funding of Research
The research was financially supported by funding from the National Natural Science Foundation of China (81673685) and the Special Research Project of Traditional Chinese Medicine (201507004-1).
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Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this publication
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and there has been no significant financial support for this work that could have influenced its
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outcome.
ACCEPTED MANUSCRIPT References Adolf, W., Hecker, E., Balmain, A., Lhomme, M.F., Nakatani, Y., Ourisson, G., Ponsinet, G., Pryce, R.J., Santhanakrishnan, T.S., Matyukhina, L.G., 1970. “Euphorbiasteroid” (Epoxy-lathyrol) A new tricyclic diterpene from Euphorbia lathyris L. Tetrahedron Lett. 11, 2241-2244. Barzilai, A., 2010. DNA damage, neuronal and glial cell death and neurodegeneration. Apoptosis
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15, 1371-1381. Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G., Franceschi, C., 1993. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming
AN US
lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide JC-1. Biochem. Biophys. Res. Commun. 197, 40-45.
Cui, J., Xing, L., Li, Z., Wu, S., Wang, J., Liu, J., Wang, J., Yan, X., Zhang, X., 2010. Ochratoxin A
M
induces G(2) phase arrest in human gastric epithelium GES-1 cells in vitro. Toxicol. Lett. 193, 152-158.
ED
Delbridge, A.R., Grabow, S., Strasser, A., Vaux, D.L., 2016. Thirty years of BCL-2: translating cell
PT
death discoveries into novel cancer therapies. Nat. Rev. Cancer 16, 99-109. Dong, M.X., Ye, T., Bi, Y.Y., Wang, Q., Kuerban, K., Li, J.Y., Feng, M.Q., Wang, K., Chen, Y., Ye, L.,
CE
2019. A novel hybrid of 3-benzyl coumarin seco-B-ring derivative and phenylsulfonylfuroxan
AC
induces apoptosis and autophagy in non-small-cell lung cancer. Phytomedicine 52, 79-88. Fuchs, Y., Steller, H., 2011. Programmed cell death in animal development and disease. Cell 147, 742-758.
Gong, K., Li, W., 2011. Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: A potential new treatment for hepatocellular carcinoma. Free Radic. Biol. Med. 51, 2259-2271. Ho, E., Karimi Galougahi, K., Liu, C.C., Bhindi, R., Figtree, G.A., 2013. Biological markers of
ACCEPTED MANUSCRIPT oxidative stress: Applications to cardiovascular research and practice. Redox Biol. 1, 483-491. Hong, S.E., Lee, J., Seo, D.H., In Lee, H., Ri Park, D., Lee, G.R., Jo, Y.J., Kim, N., Kwon, M., Shon, H., Kyoung Seo, E., Kim, H.S., Young Lee, S., Jeong, W., 2017. Euphorbia factor L1 inhibits osteoclastogenesis by regulating cellular redox status and induces Fas-mediated apoptosis in osteoclast. Free Radic. Biol. Med. 112, 191-199.
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Huang, P., Feng, L., Oldham, E.A., Keating, M.J., Plunkett, W., 2000. Superoxide dismutase as a target for the selective killing of cancer cells. Nature 407, 390-395.
Kapuscinski, J., 1995. DAPI: a DNA-specific fluorescent probe. Biotech. Histochem. 70, 220-233.
AN US
Kuo, H.C., Lee, H.J., Hu, C.C., Shun, H.I., Tseng, T.H., 2006. Enhancement of esculetin on Taxol-induced apoptosis in human hepatoma HepG2 cells. Toxicol. Appl. Pharmacol. 210, 55-62.
Lai, Z.Q., Ip, S.P., Liao, H.J., Lu, Z., Xie, J.H., Su, Z.R., Chen, Y.L., Xian, Y.F., Leung, P.S., Lin, Z.X.,
M
2017. Brucein D, a naturally occurring tetracyclic triterpene quassinoid, induces apoptosis in
ED
pancreatic cancer through ROS-associated PI3K/Akt signaling pathway. Front. Pharmacol. 8, 936.
PT
Lin, M., Tang, S., Zhang, C., Chen, H., Huang, W., Liu, Y., Zhang, J., 2017. Euphorbia factor L2
AC
59-64.
CE
induces apoptosis in A549 cells through the mitochondrial pathway. Acta. Pharm. Sin. B 7,
Liu, Q., Wang, Q., Yang, X.W., Shen, X.F., Zhang, B.X., 2009. Differential cytotoxic effects of denitroaristolochic acid II and aristolochic acids on renal epithelial cells. Toxicol. Lett. 184, 5-12. Park, S.J., Park, J.H., Han, A., Davaatseren, M., Kim, H.J., Kim, M.S., Hur, H.J., Sung, M.J., Hwang, J.T., Yang, H.J., Kwon, D.Y., 2015. Euphorbiasteroid, a component of Euphorbia lathyris L., inhibits adipogenesis of 3T3-L1 cells via activation of AMP-activated protein kinase. Cell Biochem. Funct. 33, 220-225.
ACCEPTED MANUSCRIPT Roos, W.P., Kaina, B., 2013. DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett. 332, 237-248. Sarbassov, D.D., Guertin, D.A., Ali, S.M., Sabatini, D.M., 2005. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098-1101. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative CT method.
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Nat. Protoc. 3, 1101-1108. Schutte, B., Nuydens, R., Geerts, H., Ramaekers, F., 1998. Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells. J. Neurosci. Methods 86, 63-69.
AN US
Seca, A.M.L., Pinto, D., 2018. Plant secondary metabolites as anticancer agents: successes in clinical trials and therapeutic application. Int. J. Mol. Sci. 19, 263.
Shinojima, N., Yokoyama, T., Kondo, Y., Kondo, S., 2014. Roles of the Akt/mTOR/p70S6K and ERK1/2 signaling pathways in curcumin-induced autophagy. Autophagy 3, 635-637.
M
Sinha, K., Das, J., Pal, P.B., Sil, P.C., 2013. Oxidative stress: the mitochondria-dependent and
ED
mitochondria-independent pathways of apoptosis. Arch. Toxicol. 87, 1157-1180. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto,
PT
E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic
CE
acid. Anal. Biochem. 150, 76-85.
AC
Song, Y., Liang, X., Hu, Y., Wang, Y., Yu, H., Yang, K., 2008. p,p'-DDE induces mitochondria-mediated apoptosis of cultured rat Sertoli cells. Toxicology 253, 53-61. Teng, Y.N., Wang, Y., Hsu, P.L., Xin, G., Zhang, Y., Morris-Natschke, S.L., Goto, M., Lee, K.H., 2018. Mechanism of action of cytotoxic compounds from the seeds of Euphorbia lathyris. Phytomedicine 41, 62-66. Thorpe, L.M., Yuzugullu, H., Zhao, J.J., 2015. PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting. Nat. Rev. Cancer 15, 7-24.
ACCEPTED MANUSCRIPT Wang, J.X., Wang, Q., Zhen, Y.Q., Zhao, S.M., Gao, F., Zhou, X.L., 2018. Cytotoxic lathyrane-type diterpenes from seeds of Euphorbia lathyris. Chem. Pharm. Bull. 66, 674-677. Zhang, J.Y., Liang, Y.J., Chen, H.B., Zheng, L.S., Mi, Y.J., Wang, F., Zhao, X.Q., Wang, X.K., Zhang, H., Fu, L.W., 2011. Structure identification of Euphorbia Factor L3 and its induction of apoptosis through the mitochondrial pathway. Molecules 16, 3222-3231.
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Zhang, J.Y., Lin, M.T., Yi, T., Tang, Y.N., Fan, L.L., He, X.C., Zhao, Z.Z., Chen, H.B., 2013. Apoptosis sensitization by Euphorbia factor L1 in ABCB1-mediated multidrug resistant K562/ADR cells. Molecules 18, 12793-12808.
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Zhu, A., Zhang. T., Wang, Q., 2018. The phytochemistry, pharmacokinetics, pharmacology and
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toxicity of Euphorbia semen. J. Ethnopharmacol. 227, 41-55.
ACCEPTED MANUSCRIPT Figure Legends Fig. 1. (A) The chemical structure of EFL1. (B) The purity of EFL1 was detected by High Performance Liquid Chromatography. The retention time of EFL1 was 8.50 min, and the purity of EFL1 was 98.75%. Fig. 2. Cytotoxicity of EFL1 in GES-1 cells. (A) Concentration effect and time effect of EFL1 on cell
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viability were determined by the CCK-8 assay, the cells viability of each EFL1 group was expressed in percentage in comparison with vehicle control group. (B) Concentration effect and time effect of EFL1 on LDH activity of GES-1 cells. Data were presented as mean ± standard deviation from
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three independent experiments. *p < 0.05 or **p < 0.01 compared with vehicle control.
Fig. 3. EFL1-induced oxidative stress in GES-1 cells. (A) ROS content was determined by DCFH-DA fluorescence probe, in FL1 channel of flow cytometry. (B) The ROS content in GES-1
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cells after various concentrations of EFL1 exposure for 72 h. The MDA content (C) and SOD activity (D) was assayed by biochemical kit. Data were presented as mean ± standard deviation
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from five independent experiments. *p < 0.05 or **p < 0.01 compared with vehicle control.
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Fig. 4. EFL1-induced mitochondria-mediated apoptosis in GES-1 cells. Cells were exposed to 0, 50, 100 and 200 μM EFL1 for 72 h. (A) The mitochondrial membrane potential (MMP) was detected by
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JC-10 fluorescence probe in FL1 channel of flow cytometry, and results were from five independent
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experiments. (B) Morphology of GES-1 cells stained with DAPI, and the white arrows indicates nuclear fragmentation and chromatin condensation, characteristics of apoptosis; typical images were from one of three independent experiments. (C) DNA were extracted from cells exposed to EFL1, and separated on 2% agarose gel containing ethidium bromide; typical images were from one of five independent experiments. (D) Apoptosis was evaluated by annexin V-FITC and PI staining, and percent of early and late stage of apoptotic cells was quantified (E); results were from five independent experiments. Data were presented as mean ± standard deviation. *p < 0.05 or **p
ACCEPTED MANUSCRIPT < 0.01 compared with vehicle control. Fig. 5. Molecular interactions between EFL1 and human proteins of apoptotic pathway and PI3K/AKT/mTOR pathway. 3D and 2D interaction models were presented. (A) Bcl-2, (B) cytochrome c, (C) caspase-9, (D) caspase-3, (E) PI3K, (F) AKT and (G) mTOR. Fig. 6. Effect of EFL1 on the mRNA and protein expression of Bcl-2, cytochrome c, caspase-9 and
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caspase-3 in GES-1 cells, after exposure for 72 h. Results of statistical analysis for mRNA and protein expressions were normalized to β-actin. Relative mRNA expression levels in GES-1 cells by RT-qPCR (A). Relative protein expression levels by Western blotting (B) and semi-quantitative
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analysis (C). Data were presented as mean ± standard deviation from five independent experiments. *p < 0.05 or **p < 0.01 compared with vehicle control.
Fig. 7. Effect of EFL1 on the mRNA and protein expression of PI3K, AKT and mTOR in GES-1 cells, after exposure for 72 h. Results of statistical analysis for mRNA and protein expressions were
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normalized to β-actin. Relative mRNA expression levels in GES-1 cells by RT-qPCR (A). Relative
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protein expression levels by Western blotting (B) and semi-quantitative analysis (C). Data were presented as mean ± standard deviation from five independent experiments. *p < 0.05 or **p < 0.01
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compared with vehicle control.
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Fig. 8. Effect of EFL1 on the expression of autophagy related proteins LC3, Beclin-1 and p62 in
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GES-1 cells, after exposure for 72 h. Results of statistical analysis for protein expression were normalized to β-actin. Relative protein expression levels by Western blotting (A) and semi-quantitative analysis (B). Data were presented as mean ± standard deviation from three independent experiments. *p < 0.05 or **p < 0.01 compared with vehicle control.
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Table 1. Primer sequences and theoretical amplification length Primer sequences (5’ to 3’)
Gene name
Bcl-2
caspase-9
caspase-3
PI3K
AKT
Reverse
CTAAGTCATAGTCCGCCTAGAAGCA
Forward
CTTTGGGCGGAAGACAGGTC
Reverse
TTATTGGCGGCTGTGTAAGAG
Forward
AACATCGCCCTGTGGATGAC
Reverse
AGAGTCTTCAGAGACAGCCAGGAG
Forward
GAACTAACAGGCAAGCAGCAAA
Reverse
GACATCACCAAATCCTCCAGAAC
Forward
CTCCACAGCACCTGGTTATTATTCT
Reverse
GAAAAGTAGCGTCAAAGGAAAAGG
Forward
TGCAGCAGCCTTCAACAAAGA
Reverse
AGCTACACAGTAGCCAGCACAGGA
Forward
AGCGACGTGGCTATTGTGAA
Reverse
CACGTTGGTCCACATCCTG
Forward
GGCCTGGATGGCAACTACAGA
Reverse
TGACTGGCCAGCAGAGTAGGAA
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mTOR
TGGCACCCAGCACAATGAA
186
54
146
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cytochrome c
Forward
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Product lengths (bp)
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149
179
118
142
192
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Table 2. Primary and secondary antibodies for Western blotting analysis Host species
Clonality
Dilution
Brand
Product number
β-actin Bcl-2 cytochrome c caspase-9 caspase-3 PI3K p110 beta p-AKT (Thr308) AKT p-mTOR (Ser2448) mTOR LC3 Beclin-1 p62 Rabbit IgG (HPR)
Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Goat
Polyclonal Polyclonal Polyclonal Polyclonal Monoclonal Monoclonal Polyclonal Polyclonal Monoclonal Monoclonal Monoclonal Polyclonal Monoclonal Polyclonal
1:2000 1:500 1:500 1:2000 1:2000 1:1000 1:500 1:400 1:2000 1:1000 1:1000 1:400 1:1000 1:2000
Abcam Abcam Abcam Abcam Abcam Abcam Abcam Boster Abcam CST CST Boster CST Abcam
ab8227 ab59348 ab53056 ab25758 ab32351 ab151549 ab38449 BA2138 ab109268 2983T 3868S BA3123-2 8025S ab6721
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Product name
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Table 3. Molecular interactions between EFL1 and human proteins Bcl-2, cytochrome c, caspase-9, caspase-3, PI3K, AKT and mTOR RMSD (Å)
Total score
H-bond number
Residues involved in H-bond formation
4LVT 3NWV
1.6468 1.8395
8.5061 6.6789
1 2
A/Tyr105.HH A/Cys17.H, A/Gln16.H
4RHW 5IAB 4WAF 4GV1 4DRH
0.2059 1.2952 0.5950 1.5312 0.8685
5.4125 7.1806 8.0823 8.5364 7.5213
1 1 1 1 2
E/Gln21.HE21 A/Arg86.HH11 A/Gln859.HE21 A/Lys276.HZ2 B/Arg2086.HH21, B/Arg2086.HE
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Bcl-2 cytochrome c caspase-9 caspase-3 PI3K AKT mTOR
PDB ID
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Protein
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Graphical abstract
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