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The ERK-MNK-eIF4F signaling pathway mediates TPDHT-induced A549 cell death in vitro and in vivo
Food and Chemical Toxicology 137 (2020) 111158
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The ERK-MNK-eIF4F signaling pathway mediates TPDHT-induced A549 cell death in vitro and in vivo
T
Chuanlong Guoa,b, Yuzhen Houa,b, Xuemin Yuc, Fan Zhanga,b, Xiaochen Wua,b, Xianggen Wua,b,∗, Lijun Wangd,∗∗ a
Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China Key Laboratory of Pharmaceutical Research for Metabolic Diseases, Qingdao University of Science and Technology, China c Department of Otorhinolaryngology, Qilu Hospital of Shandong University, NHC Key Laboratory of Otorhinolaryngology, Qingdao, Shandong, 266035, China d Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China b
ARTICLE INFO
ABSTRACT
Keywords: Apoptosis eIF4F ERK MNK A549 cells
The eIF4E/eIF4G complex plays a central role in gene expression regulation during the initial stage of translation. This study aimed to determine if the novel small molecule compound, TPDHT, could disrupt the interaction of eIF4E/eIF4G, and if it could exhibit excellent antitumor activity in vivo without causing apparent toxicity effect. This study investigated the antitumor mechanism of TPDHT in vitro and in vivo. TPDHT showed significant anti-proliferative activity on human lung cancer A549 cells, and it induced G0/G1 cycle arrest. Moreover, TPDHT also induced A549 cell apoptosis through the mitochondria-mediated apoptotic pathway. Our results indicate that TPDHT could disrupt the interaction of eIF4E/eIF4G, and the activity of eIF4F plays an important role in TPDHT-induced cell proliferation inhibition and apoptosis. Further research showed that TPDHT could inhibit the Ras/ERK/MNK pathway, and activation of the ERK pathway reversed TPDHT-induced cell proliferation inhibition and apoptosis. Taken together, our study findings indicated that TPDHT exerts an antitumor effect through an intrinsic apoptotic pathway controlled by the ERK/MNK/eIF4F pathway.
1. Introduction Non-small cell lung cancer (NSCLC) is the most comment type of human lung cancer and account for 85% of all human lung cancers (Rami-Porta et al., 2017). Although lung cancer treatments have progressed, rates of five-year survival remain low. Therefore, developing new drugs to treat lung cancer is very important for improving the living conditions of patients (Farhan et al., 2019; Hong et al., 2015; Tetsu et al., 2016). Eukaryotic initiation factor 4E (eIF4E) plays a key role in regulating the translation of eukaryotic cells. eIF4E, also known as cap-binding protein, affects the processing, metabolism, and translation of eukaryotic mRNA, and it plays an important role in the initial stages of protein synthesis (Mamane et al., 2004). Studies have found that when the level of eIF4E expression in cells increases, it causes translation of some tumor-related mRNAs, such as proto-oncogenes (c-Myc, Cyclin D1, etc.), angiogenic factors (vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), etc.), and matrix metalloproteinases (Mamane et al., 2004). eIF4E is critical for the regulation of
∗
apoptosis, therefore, targeting eIF4E may be a rational anticancer therapy (Descamps et al., 2012; Moerke et al., 2007). eIF4E can form an eIF4F complex with eIF4A and eIF4G to participate in eukaryotic translation initiation. The activity of eIF4F is mainly regulated by two signals: the PI3K/Akt/mTOR pathway and the Ras/MNK pathway (Proud, 2015a; Richter and Sonenberg, 2005). mTOR signals control eIF4F assembly by liberating both eIF4E and eIF4A from their respective inhibitory binding proteins: eIF4E-binding proteins (4E-BPs). The 4E-BPs (mainly 4E-BP1) compete with eIF4G for a binding site on eIF4E. Acting as a substrate for mTOR, the phosphorylation level of 4E-BP1 is regulated by mTOR (Matsumoto et al., 2015). However, the activity of eIF4F is also regulated by the MNK signaling pathway. Studies have shown that phosphorylated eIF4E (Ser 209) enhances its ability to bind to the 5′-end of the mRNA structure, while also enhancing the interaction of eIF4E/eIF4G (Feoktistova et al., 2013). Importantly, the only kinases that phosphorylate eIF4E are the mitogen-activated protein kinase (MAPK)-interacting kinases MNK1 and MNK2. MNK is activated by P38 and ERK 1/2 MAPK (Proud,
Corresponding author. Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China. Corresponding author. E-mail addresses: [email protected] (X. Wu), [email protected] (L. Wang).
∗∗
https://doi.org/10.1016/j.fct.2020.111158 Received 8 November 2019; Received in revised form 20 January 2020; Accepted 22 January 2020 Available online 25 January 2020 0278-6915/ © 2020 Elsevier Ltd. All rights reserved.
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2015b; Pyronnet, 2000). The role of eIF4E in the malignant transformation of cells has received much attention. It has been reported that the expression of eIF4E is low level in normal tissues (Qiuyuan Wen et al., 2017). However, eIF4E is overexpressed in a variety of human cancers, and its expression level is related to tumor invasion ability. Moreover, it has been found that the expression of phosphorylated eIF4E is increased in cancer tissues, and it is associated with poor prognosis in patients, especially those with non-small cell lung cancer (NSCLC) (Cencic et al., 2011b; Jacobson et al., 2013). In our previous studies, a series of bromophenol-thiazolylhydrazone hybrids were synthesized, and their potential activity for targeting eIF4E was demonstrated (Chen et al., 2016). Among these hybrid compounds, (E)-4-(3,5-bis(trifluoromethyl)phenyl)-2-(2-(3,4-difluorobenzylidene)hydrazinyl)thiazole (TPDHT) exhibited excellent antitumor activity in vitro and in vivo. In the present study, we further explored the antitumor mechanism of TPDHT, especially for its ability to regulate eIF4E and related signaling pathways.
2.5. siRNA transfection The eIF4E siRNA and control siRNA were obtained from Cell Signaling Technology (Beverly, MA, USA). In brief, the A549 cells were plated in 6-well plate at the density of 4 × 105 cells/well. After 24 h, 75 pmol siRNA was transfected into the cells using Lipofectamine™ 2000 (Life Technologies, Carlsbad, CA, USA) reagent, according to the manufacturer's instructions. Then, 24 h after transfection, the cells were exposed to TPDHT for 48 h, after which they were harvested for western blotting or cellular assays. 2.6. m7 GTP pull-down assay The A549 cells were treated with TPDHT for 48 h. Then, the total proteins were extracted, and 700 mg aliquots of the cell lysates were incubated with 7-methylguanosine (m7-GTP)-Sepharose beads at 4 °C for 2 h with constant shaking. After washing the resin, the bound proteins were eluted with free m7-GTP, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed using western blotting using antibodies against eIF4G, eIF4E, and 4EBP1.
2. Materials and methods 2.1. Chemicals and materials
2.7. Analysis of apoptosis
TPDHT was synthesized in our lab (with a purity of > 95%, Supplemental Fig. S1). TPDHT was dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. The Duolink® In Situ Red Starter Kit Mouse/Rabbit Kit (DUO92101-1 KT) was obtained from Sigma-Aldrich. The mediums were obtained from Hyclone Laboratories (USA). Fetal bovine serum (FBS) was obtained from ExCell Bio (China). The Reactive Oxygen Species (ROS) assay kit, JC-1 assay kit, apoptosis assay kit, Hoechst 33258 staining kit, and the pan-caspase inhibitor (Z-VAD-FMK) were obtained from Beyotime Biotechnology (Nanjing, China). The antibodies are listed in Supplemental Table S1. The ERK activator, Honokiol, was obtained from Dalian Meilun Biotechnology Co., LTD.
The A549 cells were seeded in a 6-well plate and incubated for 24 h. The cells were exposed to TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h, then cells were harvested and stained with Annexin V/PI for 15 min in the dark. The cells were analyzed in three different experiments using flow cytometry (FACScan™ flow cytometry, Becton Dickinson, USA). 2.8. Cell cycle analysis The A549 cells were seeded in a 6-well plate and incubated for 24 h. The cells were exposed to TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h, then cells were harvested and fixed with 70% ethanol at −20 °C overnight. Following fixation, the cells were stained with Propidium iodide (PI) for 30 min, and analyzed in three different experiments using flow cytometry.
2.2. Cell culture The cells were purchased from Cell Bank, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in mediums supplemented with 10% FBS at 37 °C and in an atmosphere containing 5% CO2.
2.9. Analysis of ROS generation The A549 cells were seeded in a 6-well plate, and incubated for 24 h. The cells were exposed to TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h, then cells were stained with peroxide-sensitive fluorescent probe DCFH-DA (2′,7-dichlorofluorescein diacetate) for 30 min. After being washed for three times, cells were harvested and analyzed in three different experiments using flow cytometry.
2.3. Analysis of cell viability Cell viability was determined by MTT assay. A549 cells were plated into a 96-well culture plate at the density of 3 × 103 cells per well. After 24 h incubation, the cells were exposed to TPDHT (0, 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM) for 12 h, 24 h and 48 h. Following treatment, the cells were further incubated with MTT (5 mg/mL) for 4 h. The medium was carefully removed, and 150 μL DMSO was added to each well. The samples were thoroughly agitated for 10 min on a shaker. Finally, the absorbance of the samples at 490 nm was measured using a microplate reader.
2.10. Morphological observation For transmission electron microscopy (TEM) analysis, the A549 cells were exposed to TPDHT (5 μM) for 48 h, and then fixed, dehydrated, and embedded in resin. Images were obtained from the Transmission Electron Microscopy Facility (JEM-1200EX, Japan). For Hoechst 33258 staining, the A549 cells were seeded in a 6-well plate and allowed to settle overnight. The cells were treated with TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h. After treatment, cells were stained with Hoechst 33258 for 5 min at room temperature, and then cells were assessed using a fluorescence microscope.
2.4. Immunofluorescence detection The A549 cells were seeded on glass bottom dishes and incubated for 24 h. After 48 h of treatment with TPDHT, the cells were fixed with 4% paraformaldehyde and blocked with bovine serum albumin (BSA) for 1 h. The cells were incubated with primary antibody at 4 °C, overnight. After being washed three times in TBST, the cells were incubated with fluorescence-conjugated second antibody at 37 °C for 1 h. The nuclei were labeled with DAPI, and the cells were visualized under a fluorescence microscope (Olympus, Japan).
2.11. Analysis of cell mitochondrial transmembrane potential (Δψm) The A549 cells were seeded in a 6-well plate and incubated for 24 h. The cells were exposed to TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h, then cells were harvested and stained with JC-1 at 37 °C for 30 min. After being washed for three times, the relative fluorescence intensities 2
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were analyzed using flow cytometry.
Table 1 IC50 values of TPDHT against four human cancer cell lines.
2.12. Western blotting analysis After treatment with TPDHT, at the previously indicated concentrations, the A549 cells were harvested, and the tumor tissues were homogenized. The proteins were harvested and separated using SDSPAGE, and then they were transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in blocking solution (containing 5% non-fat milk) and subsequently probed with primary antibodies (Supplemental Table S1) at 4 °C, overnight. After being washed in TBST for 15 min, the membranes were incubated with a secondary antibody for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence system (BeyoECL Plus, Beyotime Technology).
Cell Line
IC50 (μM)
A549 NCI–H460 PANC-1 SK-OV-3 CaCo-2 MCF-7 MDA-MB-231 HCC-1937 HepG2 U87 MG HL-7702
2.81 ± 0.25 4.23 ± 0.26 11.80 ± 1.15 16.31 ± 1.02 38.25 ± 8.70 22.40 ± 6.75 11.21 ± 3.79 22.65 ± 8.24 7.53 ± 2.74 13.4 ± 8.24 11.90 ± 6.24
a
IC50: Concentration of the compound producing 50% cell growth inhibition after 48 h of drug exposure, as determined by the MTT assay. Each experiment was run at least three times.
2.13. In vivo studies All the animal experiments were performed in accordance with the guidelines established by the Institute of Oceanology Committee for the Care and Use of Laboratory Animals. Female congenital athymic BALB/ c nude (nu/nu) mice were purchased from the Model Animal Research Center of Nanjing University. They were maintained under specificpathogen-free (SPF) conditions, and they were provided with sterile food and water. All experiments were carried out using 6–8-week-old mice weighing 18–22 g. In brief, the A549 cells (1 × 107) were subcutaneously (s.c.) injected into the back of the mice. When the tumor reached 150 mm3 in volume, the animals were randomly assigned into test groups, each consisting of six mice, and then 25 mg/kg and 50 mg/ kg TPDHT (dissolved by 0.5% CMC-Na) was administered intraperitoneally (i.p.) for 21 consecutive days. The tumor volumes and body weights of the animals were measured every three days. Tumor volumes were calculated according to the following equation: length × (width) 2/2. The tumor tissue was removed for immunohistochemistry, western blotting, or hematoxylin and eosin (H&E) staining.
comparisons were performed using one-way analysis of variance. P < 0.05 was considered to be statistically significant. 3. Results 3.1. TPDHT inhibits cell proliferation We measured the proliferation activities of TPDHT using MTT assay. The following types of tumor cells were tested in this study: human lung cancer cells, A549 and NCI–H460, human pancreatic cancer cell PANC1, human ovarian cancer cell SK-OV-3, human colon cancer cell CaCo-2, human breast cancer cells MCF-7, MDA-MB-231, and HCC-1937, human hepatoma cell HepG2, human glioma cell U87 MG, and one normal cell HL-7702. As shown in Table 1, TPDHT inhibited proliferation of the tumor cells and the normal cells. Among these cells, TPDHT showed excellent activity for the inhibition of A549 proliferation with an IC50 value of 2.81 μM. The MTT assay also indicated that TPDHT could decrease cell viability in a dose-dependent and time-dependent manner (12 h, 24 h, and 48 h) (Fig. 1B). The effect of TPDHT on the cell colony formation was also detected. The results indicated that TPDHT could inhibit the clonogenicity of the A549 cells (Fig. 1C and D).
2.14. Safety of TPDHT in vivo All the animal experiments were performed in accordance with the guidelines established by the Institute of Oceanology Committee for the Care and Use of Laboratory Animals. Forty SPF Kunming mice (18–22 g), 10 males and 10 females, were used for the acute toxicity analysis. The mice were randomly assigned to four groups, each consisting of five males and five females. The four groups of mice were orally administered TPDHT at single doses of 0 (blank group, the same volume of physiological saline), 500 mg/kg, 1000 mg/kg, and 2000 mg/kg, respectively. The mice were housed with free access to water and food in stainless steel cages in a room with controlled temperature (25 ± 1 °C) and a 12-h light/dark cycle. The survival rate and body weight of the mice were monitored and recorded up to 14 days post-treatment. Twenty SPF Kunming mice (18–22 g), 10 males and 10 females, were used for the subacute toxicity analysis. The mice were randomly assigned to two groups, each consisting of five males and five females. The treatment group was treated with 2000 mg/kg, orally, once a day for 14 days. The body weight of the mice was measured every week, and the mice were closely monitored for any signs of toxicity or discomfort. After 14 days of treatment, the mice were anesthetized with isoflurane and killed by decapitation, and the tissues were collected for further analysis.
3.2. TPDHT induces cell apoptosis through the mitochondrial-mediated apoptotic pathway To evaluate the effect of TPDHT on the induction of apoptosis, the A549 cells were treated with TPDHT for 48 h, then cells were stained with Annexin V/PI and analyzed using flow cytometry. The results showed that TPDHT could induce apoptosis of the A549 cells in a concentration-dependent manner (Fig. 2A and B). We also observed the apoptotic features in the A549 cells after staining with Hoechst 33258 (Fig. 1E). Moreover, Z-VAD-FMK (the pan-caspase inhibitor) was used in our study. The results showed that Z-VAD-FMK could inhibit TPDHTinduced cell proliferation inhibition and apoptosis (Fig. 2G and H) in A549 cells, indicating that TPDHT-induced cell apoptosis was caspasedependent. Mitochondrial damage and a decrease in mitochondrial membrane potential (MMP) are characteristics of early apoptosis. In this study, we used TEM to detect the effect of TPDHT on the mitochondrial damage of the A549 cells. As shown in Fig. 3A, after being exposed to TPDHT for 48 h, changes in mitochondrial morphology, such as mitochondrial swelling and vacuolization, were observed. The JC-1 results also showed that the MMP was disrupted in the TPDHT-treated groups (Fig. 3B and C). The mitochondrial respiratory chain is a major source of ROS, and ROS is a switch that regulates the mitochondrial
2.15. Statistical analysis Statistical analysis was performed using GraphPad Prism 5.0 (San Diego, CA, USA). The data were presented as mean ± SD. Statistical 3
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Fig. 1. TPDHT inhibits cell proliferation in A549 cells. (A) Structure of TPDHT. (B) A549 cells were treated with TPDHT for 12 h, 24 h and 48 h. Cell viability was determined by MTT assay. (C, D) A549 cells were treated with TPDHT for 10 days and colony formation was determined by staining with crystal violet. (E) A549 cells were treated with TPDHT for 48 h, and cells were stained with Hoechst 33258 and photographed using a fluorescence microscopy. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
permeability transition pore (MPTP) (Ma et al., 2017). In this study, TPDHT was also found to increase ROS generation in a dose-dependent manner (Fig. 3D and E). Proteins in the Bcl-2 family of proteins are localized in the mitochondrial membrane, and they are involved in the activation of caspase enzymes. Western blotting analysis showed that the expression of Bcl-2 was decreased while the expression of Bax was increased. Furthermore, caspase-3 and poly(ADP-ribose)polymerase (PARP) were also activated by TPDHT (Fig. 2E and F).
3.3. TPDHT induces G0/G1 cell cycle arrest in A549 cells To determine the effect of TPDHT on cell-cycle distribution, the A549 cells were treated with TPDHT for 48 h and analyzed using flow cytometry. The results showed that the cells were arrested in the G0/G1 phase. In comparison to the control group, the ratio of the G0/G1 phase in the TPDHT-treated groups was increased in a dose-dependent manner (Fig. 2C and D).
Fig. 2. TPDHT induces cell apoptosis in A549 cells. (A, B) A549 cells were treated with TPDHT for 48 h. Cells were harvested, stained with Annexin V/PI and analyzed by flow cytometry (FACS). (C, D) A549 cells were treated with TPDHT for 48 h. Cells were harvested and fixed in 70% ethanol overnight, and then cells were stained with PI and analysis by FACS. (E) A549 cells were treated with TPDHT for 48 h. Proteins were extracted and analyzed by western blotting. (F) Protein bands were analyzed by Image J. (G) A549 cells were treated with 5 μM TPDHT alone or in combination with Z-VAD-FMK (10 μM) for 48 h. The percentages of apoptotic cells were determined by FACS via Annexin V/PI staining. (H) A549 cells were treated with 5 μM TPDHT alone or in combination with Z-VAD-FMK (10 μM) for 48 h. The cell viability was detected by MTT assay. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group. ##P < 0.01. 4
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Fig. 3. TPDHT induces mitochondrial damage and ROS generation in A549 cells. (A) A549 cells were treated with TPDHT for 48 h. The internal morphology of A549 cells were observed by transmission electron microscopy (TEM). (B, C) A549 cells were treated with TPDHT for 48 h. Cells were stained with JC-1 and analyzed by FCAS. (D, E) A549 cells were treated with TPDHT for 48 h. Cells were stained with FCFH-DA and analyzed by FCAS. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group.
3.4. TPDHT disrupts the activity of eIF4F
apoptosis was inhibited (Fig. 5C and D). Moreover, TPDHT-induced mitochondrial damage and MMP reduction were also inhibited by 4EBP1 siRNA (Fig. 5E). These results indicated that the activity of eIF4F was involved in TPDHT-induced anti-apoptotic signaling.
In eukaryotic cells, eIF4E interacts with eIF4G to form an eIF4F complex that initiates the translation process and induces expression of several oncogenes, such as c-Myc and Cyclin D1. Therefore, inhibiting the interaction between eIF4E and eIF4G is a new strategy for the treatment of cancer. The activity of eIF4F is controlled by two signaling pathways: the PI3K/Akt/mTOR-4E-BP1 pathway and the Ras/ERK/ MNK-eIF4E pathway. First, we used the in situ proximity ligation assay (PLA) (DuoLink PLA) to detect the interference of TPDHT between eIF4E and eIF4G. The phosphor dots were reduced significantly, indicating that the interaction of eIF4E/eIF4G was disrupted by TPDHT (Fig. 4A and B). We then used the cap-binding assay to further analyze the effect of TPDHT on the interaction of eIF4E/eIF4G. Cell extracts were affinity-chromatized on m7 GTP-Sepharose beads for western blotting analysis. As shown in Fig. 4C, the amount of eIF4E-bound 4E-BP1 was significantly increased, whereas the amount of eIF4E-bound eIF4G was decreased. These results indicated that TPDHT induced the disruption of eIF4E/ eIF4G.
3.6. TPDHT inhibits ERK/MNK-mediated eIF4E phosphorylation eIF4E phosphorylation is elevated in a variety of tumor cells. eIF4E acts as the only substrate for MAPK-interacting kinases, MNK1 and MNK2, and phosphorylation is regulated by MNK (Fischer, 2017; Zhao et al., 2016). First, we analyzed the phosphorylation level of eIF4E in the TPDHT-treated A549 cells. We found that TPDHT decreased the phosphorylation level of eIF4E in a concentration-dependent manner (Fig. 6A and B). Moreover, the expression of Cyclin D1 and c-Myc was also decreased in the TPDHT-treated cells. It has been reported that the activated Ras/ERK/MNK signaling is critical for the activation of eIF4F(Lewinska et al., 2017). We found that TPDHT decreased the phosphorylation level of ERK and MNK (Fig. 6D and E). These results suggested that the ERK/MNK signaling pathway may be involved in TPDHT-induced eIF4F disruption. Honokiol, a potent stimulator of ERK (Liu et al., 2019; Wang et al., 2011), was used in this study to employ the ERK/MNK pathway in the TPDHT-induced disruption of eIF4F. As shown in Fig. 7D, honokiol was able to reverse the dephosphorylation of ERK and eIF4E induced by TPDHT. Moreover, honokiol could also prevent TPDHT-induced mitochondrial membrane dysfunction and cell apoptosis (Fig. 7A,B,C). Taken together, the results from our study indicated that TPDHT-induced apoptosis was mediated by the ERK/MNK signaling pathway.
3.5. eIF4F plays an important role in TPDHT-induced apoptosis eIF4F is essential for the growth and survival of cancer cells, and it can regulate apoptosis. First, we transfected the A549 cells with 4E-BP1 siRNA. The phosphor dots were increased in the 4E-BP1 siRNA group, indicating the enhanced eIF4F activity (Fig. 5A and B). In the 4E-BP1 siRNA group, the TPDHT-induced cell proliferation inhibition and 5
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Fig. 4. TPDHT disrupts the eIF4E/ eIF4G interaction. (A, B) A549 cells were treated with TPDHT. The eIF4E/ eIF4G interactions were detected using a proximity ligation assay (PLA). (C) A549 cells were treated with TPDHT and lysed. Cap-affinity chromatography and SDS-PAGE immunoblotting were used to detect eIF4E, eIF4G, and 4E-BP1. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group.
3.7. TPDHT exhibits antitumor activity in vivo
growth was observed for the mice treated with TPDHT; the tumor growth inhibition (TGI) values were 59.56% and 74.07% at dosages of 25 mg/kg/day and 50 mg/kg/day, respectively. Moreover, no significant differences were found in terms of the body weight of the mice (as a surrogate marker of toxicity). The animals’ visceral tissues, including the liver, spleen, kidney, heart, and lung, were examined using
We also examined the antitumor activity of TPDHT in A549 xenograft models. The animals were repeatedly administered with a vehicle or TPDHT (25 mg/kg and 50 mg/kg) once daily via oral gavage for 21 days. In comparison to the control groups, a significant delay in tumor
Fig. 5. eIF4F plays an important role in TPDHT-induced cell proliferation inhibition and apoptosis. A549 cells were interfered with 4E-BP1 siRNA or control nontarget siRNA 24 h before exposure to TPDHT for 48 h. (A, B) The eIF4E/eIF4G interactions were detected using a proximity ligation assay (PLA). (B) Cell viability was detected by MTT assay. (C) Cell apoptosis was detected by FACS via Annexin V/PI staining. (D) The Δψm was detected by FACS via JC-1 staining. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group, ##P < 0.01. 6
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Fig. 6. TPDHT inhibits Ras/MNK/ERK-eIF4E signal pathway. (A, B, C) A549 cells were treated with TPDHT for 48 h, the phosphorylation of eIF4E was determined using western blotting and immunofluorescence. (D) A549 cells were treated with TPDHT for 48 h. The levels of Ras, p-MNK, MNK, p-ERK, ERK were determined using western blotting. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group.
Fig. 7. ERK pathway plays an important role in TPDHT-indunced apoptosis. A549 cells were treated with TPDHT alone or in combination with Honokiol. (A) Cell viability was detected by MTT assay. (B) Cell apoptosis was detected by FACS via Annexin V/PI staining. (C) The Δψm was detected by FACS via JC-1 staining. (D) The levels of p-ERK and p-eIF4E were detected by western blotting. (F) eIF4E/eIF4G interaction was detected by cap-affinity chromatography and SDS-PAGE immunoblotting. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group. ##P < 0.01. 7
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Fig. 8. TPDHT inhibits tumor growth in A549-xenografted athymic mice. (A) Mice (n = 6) were administered with TPDHT (25 mg/kg) once per day for 21 days. The mice were anesthetized, and the tumor tissues were collected and photographed. (B) The tumor volumes were recorded every 3 days. (C) Body weights. (D) Immunohistochemical analysis Ki 67, p-ERK, p-MNK and p-eIF4E in tumor tissues. (E) The organs from treated and control groups were stained with H&E to evaluate the toxicity of TPDHT.
H&E staining, and the results showed no significant morphological changes in the vehicle and the TPDHT-treated mice. On the other hand, morphological changes in mouse tumor tissue, such as irregular cell morphology and vacuolization were also detected after H&E staining (Supplemental Fig. S2). To investigate the antitumor mechanism of TPDHT in vivo, tumor samples were analyzed using western blotting and immunochemistry. As shown in Fig. 8D, the immunochemistry results indicated that the level of p-eIF4E and Ki 67 was decreased in the TPDHT-treated tumor samples. Western blotting results showed that the levels of p-MNK, pERK, and p-eIF4E were also decreased in the TPDHT-treated tumor samples.
there were no significant changes in the morphology of tissues (liver, heart, and kidney) (Supplemental Fig. S3). These results demonstrated that TPDHT was safe, in vivo. 4. Discussion eIF4E is closely related to the occurrence, development, and metastasis of tumors. It is an important factor that promotes the transformation of tumor cells. Therefore, eIF4E is an attractive new target for the treatment of neoplastic diseases. eIF4E, together with eIF4A and eIF4G, form an eIF4F complex to participate in eukaryotic translation initiation. eIF4F binds to 7-methylguanylate cap (m7G) at the 5′ end of mRNA and, ultimately, transports it to the ribosomes (Burz et al., 2009; Hassan et al., 2014; Lewinska et al., 2017). Assembly of active eIF4F complex depends on interaction between eIF4E/eIF4G (Moerke et al., 2007). A variety of small molecules, such as 4EGI-1 and 4E1RCat, have been shown to disrupt the interaction of eIF4E/eIF4G (Cencic et al., 2011b; Moerke et al., 2007). 4EGI-1 is the first eIF4E/eIF4G interaction inhibitor with a Kd of 25 μM against eIF4E binding (Cencic et al., 2011a; Moerke et al., 2007). And 4EGI-1 can inhibit the proliferation of various tumor cells, such as human lung cancer cells, human breast cancer cells, glioma cells, etc (Fan et al., 2010; Mahalingam et al., 2014; Takrouri et al., 2014). However, eIF4E activity can be inhibited by preventing phosphorylation of MNK kinase. Small molecules, such as cercosporamide and CGP 57380, are MNK inhibitors that could also regulate the activity of eIF4E (Qiuyuan Wen et al., 2017). In our study, we found that a small molecule, TPDHT, disrupted the eIF4E/eIF4G interaction by inhibiting the Ras/MAPK pathway. First, we demonstrated that TPDHT could inhibit the proliferation of a variety of tumor cells, especially for the human lung cancer cell A549 (with a lowest IC50 of 2.81 ± 0.25 μM). Moreover, we also found that TPDHT could inhibit colony formation and induce apoptosis and G0/G1 phase arrest in A549 cells. TPDHT also exhibited antitumor activity in vivo, with low toxicity. Mitochondria play a key role in the process of apoptosis; changes in the structure and function of mitochondria play an important role in
3.8. TPDHT exhibits low toxicity in vivo To investigate the safety of TPDHT in vivo, acute toxicity and subacute toxicity tests were performed in the mice. In the acute toxicity test, the mice were orally administered single doses of TPDHT: 500 mg/ kg, 1000 mg/kg, and 2000 mg/kg, respectively. The experimental results showed that, after 14 days of monitoring, no mortality and abnormalities (including body weight) were observed (Table 2). In the subacute toxicity test, the mice were orally administered a dose of 2000 mg/kg TPHDT for 14 days. During the 14 days of administration, no deaths or abnormalities (including body weight) were observed (Supplemental Table S2). Furthermore, the H&E analysis showed that Table 2 Acute toxicity test of TPDHT. Dosage (mg/kg)
Number of mice
Deaths of mice 1–7 day
8–14 day
500 1000 2000 Blank
20 20 20 20
0 0 0 0
0 0 0 0
Mortality rate
0/0 0/0 0/0 0/0
Weight (g) 0 day
7 day
20.8 20.4 20.8 20.6
25.7 26.8 28.1 27.7
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many different cellular processes, including apoptosis (Gupta, 2001). We found that TPDHT caused a decrease in MMP in A549 cells, and TPDHT also destroyed the morphology of mitochondria. MMP is regulated by the Bcl-2 family of proteins; when mitochondrial damage or MMP decreases, cytochrome c is released from the mitochondria. This, in turn, activates some apoptotic proteins, such as caspase-3 and PARP, which ultimately leads to apoptosis (Shan et al., 2016). The western blotting results indicated that the expression of Bax increased and the expression of Bcl-2 decreased, while PARP and caspase-3 were activated by TPDHT (Fig. 2E). Moreover, we found that Z-VAD-FMK, the pancaspase inhibitor, could inhibit TPDHT-induced apoptosis and cell hypoproliferation (Fig. 2G and H). These results indicated that TPDHTinduced apoptosis occurred through the mitochondrial-dependent pathway. The activity of eIF4F is controlled by a family of proteins known as eIF4E-bingding proteins, 4E-BPs (mainly 4E-BP1). The phosphorylated 4E-BP1 competes with eIF4G for a binding site on eIF4E, and the interaction of eIF4E/eIF4G is essential for eIF4F activity (Piao et al., 2018; Tang et al., 2019). In our study, we used the in situ PLA and capbinding assay to detect the eIF4E/eIF4G interactions. The in situ PLA results showed that the fluorescence spots of the TPDHT-treated group were significantly decreased, indicating that the eIF4E/eIF4G interaction was inhibited by TPDHT (Fig. 4A). We then used m7-GTP-Sepharose beads to capture eIF4E and its binding partners (eIF4G and 4EBP1). The pull-down assay showed that TPDHT decreased the amount of eIF4G and increased the amount of 4E-BP1 (Fig. 4C). These results indicated that TPDHT could disrupt the interaction of eIF4E/eIF4G. Activated eIF4F promotes downstream signaling factors, including the expression of Cyclin D1, c-Myc, etc., and it regulates cell proliferation and death (Anzell et al., 2018; Sifuentes-Franco et al., 2018). To explore the role of the eIF4F complex in TPDHT-induced cell proliferation inhibition, cell cycle arrest, and apoptosis, we used siRNA 4EBP1 to knockdown 4E-BP1 (manufacturing an eIF4F activation model). The results showed that TPDHT-induced apoptosis and MMP disruption was inhibited by 4E-BP1 siRNA (Fig. 5). These results indicated that eIF4F was involved in TPDHT-induced anti-apoptotic signaling. The activity of eIF4F is also regulated by the Ras/ERK/MNK pathway (Gao et al., 2012; Hall et al., 2014; Jing et al., 2018). eIF4E is the only substrate of the MAP kinase-interacting kinases, MNK1 and MNK2, and its phosphorylation is only affected by MNKs (Cheng et al., 2020; Zhao et al., 2016). MNKs are located at key points in the intracellular signaling pathway, and they can also be activated as a downstream consequence of ERK and MAPK signaling (Altman et al., 2013; Fischer, 2017; Ramalingam et al., 2014). ERK lies downstream of oncoproteins, such as the receptor tyrosine kinases, Ras and Raf (He et al., 2015; Kim et al., 2018). In our study, the immunofluorescence and western blotting results showed that phosphorylation of eIF4E was inhibited by TPDHT (Fig. 6A,C). The expression levels of Ras, p-ERK, and p-MNK were also inhibited by TPDHT (Fig. 6D). To verify that the ERK signaling pathway plays a key role in TPDHT-induced apoptosis, we used honokiol, an ERK stimulator. Honokiol was able to reverse the dephosphorylation of ERK and eIF4E induced by TPDHT (Fig. 7). Moreover, we found that honokiol also attenuated the TPDHT-mediated intrinsic apoptotic pathway. Interestingly, the western blotting results also showed that the expression levels of p-ERK, p-MNK, and p-eIF4E were inhibited in the TPDHT-treated tumor groups (Fig. 8D). These results indicated that the ERK/MNK/eIF4E pathway was involved in TPDHT-exhibited antitumor activity in vitro and in vivo. Above all, this study identified that TPDHT had a potent antitumor effect against human lung cancer cells in vitro and in vivo. Furthermore, TPDHT could induce apoptosis through the mitochondria-induced apoptotic pathway. The mechanism study results indicated that TPDHT could inhibit the activity of eIF4F through the Ras/ERK/MNK-eIF4F signal pathway. Therefore, these findings provide evidence that TPDHT may demonstrate potentially useful anti-cancer activity against human lung cancer.
CRediT authorship contribution statement Chuanlong Guo: Conceptualization, Methodology, Software, Writing - review & editing. Yuzhen Hou: Data curation, Writing original draft. Xuemin Yu: Data curation, Writing - original draft. Fan Zhang: Visualization, Investigation, Visualization, Investigation. Xiaochen Wu: Supervision, Software, Validation. Xianggen Wu: Writing - review & editing, Visualization. Lijun Wang: Conceptualization, Software, Writing - review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81773586) and the Talent Fund of Shandong Collaborative Innovation Center of Eco-Chemical Engineering (project no. XTCXQN19). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fct.2020.111158. References Altman, J.K., Szilard, A., Konicek, B.W., Iversen, P.W., Kroczynska, B., Glaser, H., Sassano, A., Vakana, E., Graff, J.R., Platanias, L.C., 2013. Inhibition of Mnk kinase activity by cercosporamide and suppressive effects on acute myeloid leukemia precursors. Blood 121, 3675–3681. Anzell, A.R., Maizy, R., Przyklenk, K., Sanderson, T.H., 2018. Mitochondrial quality control and disease: insights into ischemia-reperfusion injury. Mol. Neurobiol. 55, 2547–2564. Burz, C., Berindan-Neagoe, I., Balacescu, O., Irimie, A., 2009. Apoptosis in cancer: key molecular signaling pathways and therapy targets. Acta Oncol. 48, 811–821. Cencic, R., Hall, D.R., Robert, F., Du, Y.H., Min, J., Li, L., Qui, M., Lewis, I., Kurtkaya, S., Dingledine, R., Fu, H.A., Kozakov, D., Vajda, S., Pelletier, J., 2011a. Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F (vol 108, pg 1046, 2010). Proc. Natl. Acad. Sci. U. S. A. 108 6689-6689. Cencic, R., Hall, D.R., Robert, F., Du, Y.H., Min, J.K., Li, L.A., Qui, M., Lewis, I., Kurtkaya, S., Dingledine, R., Fu, H.A., Kozakov, D., Vajda, S., Pelletier, J., 2011b. Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F. Proc. Natl. Acad. Sci. U. S. A. 108, 1046–1051. Chen, W.Q., Zheng, R.S., Baade, P.D., Zhang, S.W., Zeng, H.M., Bray, F., Jemal, A., Yu, X.Q., He, J., 2016. Cancer statistics in China, 2015. Ca - Cancer J. Clin. 66, 115–132. Cheng, C.H., Su, Y.L., Ma, H.L., Deng, Y.Q., Feng, J., Chen, X.L., Jie, Y.K., Guo, Z.X., 2020. Effect of nitrite exposure on oxidative stress, DNA damage and apoptosis in mud crab (Scylla paramamosain). Chemosphere 239. Descamps, G., Gomez-Bougie, P., Tamburini, J., Green, A., Bouscary, D., Maiga, S., Moreau, P., Le Gouill, S., Pellat-Deceunynck, C., Amiot, M., 2012. The cap-translation inhibitor 4EGI-1 induces apoptosis in multiple myeloma through Noxa induction. Br. J. Canc. 106, 1660–1667. Fan, S., Li, Y., Yue, P., Khuri, F.R., Sun, S.-Y., 2010. The eIF4E/eIF4G interaction inhibitor 4EGI-1 augments TRAIL-mediated apoptosis through c-FLIP down-regulation and DR5 induction independent of inhibition of cap-dependent protein translation. Neoplasia 12, 346–IN347. Farhan, M., Malik, A., Ullah, M.F., Afaq, S., Faisal, M., Farooqi, A.A., Biersack, B., Schobert, R., Ahmad, A., 2019. Garcinol sensitizes NSCLC cells to standard therapies by regulating EMT-modulating miRNAs. Int. J. Mol. Sci. 20. Feoktistova, K., Tuvshintogs, E., Do, A., Fraser, C.S., 2013. Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity. Proc. Natl. Acad. Sci. U. S. A. 110, 13339–13344. Fischer, M., 2017. Census and evaluation of p53 target genes. Oncogene 36, 3943–3956. Gao, N., Cheng, S., Budhraja, A., Gao, Z., Chen, J., Liu, E.H., Huang, C., Chen, D., Yang, Z., Liu, Q., Li, P., Shi, X., Zhang, Z., 2012. Ursolic acid induces apoptosis in human leukaemia cells and exhibits anti-leukaemic activity in nude mice through the PKB pathway. Br. J. Pharmacol. 165, 1813–1826. Gupta, S., 2001. Molecular steps of death receptor and mitochondrial pathways of apoptosis. Life Sci. 69, 2957–2964. Hall, C., Dumstorf, C., Konicek, B., Robichaud, N., McNulty, A., Parsons, S., Pelltier, J., Sonenberg, N., Graff, J.R., 2014. eIF4E phosphorylation is Mnk-dependent but does not require assembly of the eIF4F translation initiation complex. Canc. Res. 74.
9
Food and Chemical Toxicology 137 (2020) 111158
C. Guo, et al. Hassan, M., Watari, H., AbuAlmaaty, A., Ohba, Y., Sakuragi, N., 2014. Apoptosis and molecular targeting therapy in cancer. BioMed Res. Int. 2014, 150845. He, W., Shi, F., Zhou, Z.W., Li, B., Zhang, K., Zhang, X., Ouyang, C., Zhou, S.F., Zhu, X., 2015. A bioinformatic and mechanistic study elicits the antifibrotic effect of ursolic acid through the attenuation of oxidative stress with the involvement of ERK, PI3K/ Akt, and p38 MAPK signaling pathways in human hepatic stellate cells and rat liver. Drug Des. Dev. Ther. 9, 3989–4104. Hong, Q.Y., Wu, G.M., Qian, G.S., Hu, C.P., Zhou, J.Y., Chen, L.A., Li, W.M., Li, S.Y., Wang, K., Wang, Q., Zhang, X.J., Li, J., Gong, X., Bai, C.X., Lung Cancer Group of Chinese Thoracic, S, Chinese Alliance Against Lung, C, 2015. Prevention and management of lung cancer in China. Cancer 121 (Suppl. 17), 3080–3088. Jacobson, B.A., Thumma, S.C., Jay-Dixon, J., Patel, M.R., Kroening, K.D., Kratzke, M.G., Etchison, R.G., Konicek, B.W., Graff, J.R., Kratzke, R.A., 2013. Targeting eukaryotic translation in mesothelioma cells with an eIF4E-specific antisense oligonucleotide. PloS One 8. Jing, B., Liu, M., Yang, L., Cai, H.Y., Chen, J.B., Li, Z.X., Kou, X., Wu, Y.Z., Qin, D.J., Zhou, L., Jin, J., Lei, H., Xu, H.Z., Wang, W.W., Wu, Y.L., 2018. Characterization of naturally occurring pentacyclic triterpenes as novel inhibitors of deubiquitinating protease USP7 with anticancer activity in vitro. Acta Pharmacol. Sin. 39, 492–498. Kim, K., Shin, E.A., Jung, J.H., Park, J.E., Kim, D.S., Shim, B.S., Kim, S.H., 2018. Ursolic acid induces apoptosis in colorectal cancer cells partially via upregulation of MicroRNA-4500 and inhibition of JAK2/STAT3 phosphorylation. Int. J. Mol. Sci. 20. Lewinska, A., Adamczyk-Grochala, J., Kwasniewicz, E., Deregowska, A., Wnuk, M., 2017. Ursolic acid-mediated changes in glycolytic pathway promote cytotoxic autophagy and apoptosis in phenotypically different breast cancer cells. Apoptosis : an international journal on programmed cell death 22, 800–815. Liu, P., Du, R., Yu, X., 2019. Ursolic acid exhibits potent anticancer effects in human metastatic melanoma cancer cells (SK-MEL-24) via apoptosis induction, inhibition of cell migration and invasion, cell cycle arrest, and inhibition of mitogen-activated protein kinase (MAPK)/ERK signaling pathway. Med. Sci. Mon. Int. Med. J. Exp. Clin. Res. : Int. Med. J. Exp. Clin. Res. 25, 1283–1290. Ma, X., Zhang, Y., Wang, Z., Shen, Y., Zhang, M., Nie, Q., Hou, Y., Bai, G., 2017. Ursolic acid, a natural nutraceutical agent, targets Caspase3 and alleviates inflammationassociated downstream signal transduction. Mol. Nutr. Food Res. 61. Mahalingam, P., Takrouri, K., Chen, T., Sahoo, R., Papadopoulos, E., Chen, L., Wagner, G., Aktas, B.H., Halperin, J.A., Chorev, M., 2014. Synthesis of rigidified eIF4E/eIF4G inhibitor-1 (4EGI-1) mimetic and their in vitro characterization as inhibitors of protein-protein interaction. J. Med. Chem. 57, 5094–5111. Mamane, Y., Petroulakis, E., Rong, L.W., Yoshida, K., Ler, L.W., Sonenberg, N., 2004. eIF4E - from translation to transformation. Oncogene 23, 3172–3179. Matsumoto, M., Seike, M., Noro, R., Soeno, C., Sugano, T., Takeuchi, S., Miyanaga, A., Kitamura, K., Kubota, K., Gemma, A., 2015. Control of the MYC-eIF4E axis plus mTOR inhibitor treatment in small cell lung cancer. BMC Canc. 15, 241. Moerke, N.J., Aktas, H., Chen, H., Cantel, S., Reibarkh, M.Y., Fahmy, A., Gross, J.D., Degterev, A., Yuan, J.Y., Chorev, M., Halperin, J.A., Wagner, G., 2007. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and
eIF4G. Cell 128, 257–267. Piao, M.J., Ahn, M.J., Kang, K.A., Ryu, Y.S., Hyun, Y.J., Shilnikova, K., Zhen, A.X., Jeong, J.W., Choi, Y.H., Kang, H.K., Koh, Y.S., Hyun, J.W., 2018. Particulate matter 2.5 damages skin cells by inducing oxidative stress, subcellular organelle dysfunction, and apoptosis. Arch. Toxicol. 92, 2077–2091. Proud, C.G., 2015a. Mnks, eIF4E phosphorylation and cancer. Bba-Gene Regul Mech 1849, 766–773. Proud, C.G., 2015b. Mnks, eIF4E phosphorylation and cancer. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1849, 766–773. Pyronnet, S., 2000. Phosphorylation of the cap-binding protein eIF4E by the MAPK-activated protein kinase Mnk1. Biochem. Pharmacol. 60, 1237–1243. Ramalingam, S., Gediya, L., Kwegyir-Afful, A.K., Ramamurthy, V.P., Purushottamachar, P., Mbatia, H., Njar, V.C.O., 2014. First Mnks degrading agents block phosphorylation of eIF4E, induce apoptosis, inhibit cell growth, migration and invasion in triple negative and Her2-overexpressing breast cancer cell lines. Oncotarget 5, 530–543. Rami-Porta, R., Asamura, H., Travis, W.D., Rusch, V.W., 2017. Lung cancer - major changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA A Cancer J. Clin. 67, 138–155. Richter, J.D., Sonenberg, N., 2005. Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433, 477–480. Shan, J.Z., Xuan, Y.Y., Zhang, Q., Zhu, C.P., Liu, Z., Zhang, S.Z., 2016. Ursolic acid synergistically enhances the therapeutic effects of oxaliplatin in colorectal cancer. Protein Cell 7, 571–585. Sifuentes-Franco, S., Padilla-Tejeda, D.E., Carrillo-Ibarra, S., Miranda-Diaz, A.G., 2018. Oxidative stress, apoptosis, and mitochondrial function in diabetic nephropathy. Internet J. Endocrinol. 2018, 1875870. Takrouri, K., Chen, T., Papadopoulos, E., Sahoo, R., Kabha, E., Chen, H., Cantel, S., Wagner, G., Halperin, J.A., Aktas, B.H., Chorev, M., 2014. Structure-activity relationship study of 4EGI-1, small molecule eIF4E/eIF4G protein-protein interaction inhibitors. Eur. J. Med. Chem. 77, 361–377. Tang, J.Y., Fu, O.Y., Hou, M.F., Huang, H.W., Wang, H.R., Li, K.T., Fayyaz, S., Shu, C.W., Chang, H.W., 2019. Oxidative stress-modulating drugs have preferential anticancer effects - involving the regulation of apoptosis, DNA damage, endoplasmic reticulum stress, autophagy, metabolism, and migration. Semin. Canc. Biol. 58, 109–117. Tetsu, O., Hangauer, M.J., Phuchareon, J., Eisele, D.W., McCormick, F., 2016. Drug resistance to EGFR inhibitors in lung cancer. Chemotherapy 61, 223–235. Wang, X., Zhang, F., Yang, L., Mei, Y., Long, H., Zhang, X., Zhang, J., Qimuge, S., Su, X., 2011. Ursolic acid inhibits proliferation and induces apoptosis of cancer cells in vitro and in vivo. J. Biomed. Biotechnol. 2011, 419343. Wen, Qiuyuan, W, W., Luo, Jiadi, 2017. CGP57380 enhances efficacy of RAD001 in nonsmall cell lung cancer through abrogating mTOR inhibition-induced phosphorylation of eIF4E and activating mitochondrial apoptotic pathway. oncotarget 7. Zhao, X.S., Ren, X., Zhu, R., Luo, Z.Y., Ren, B.X., 2016. Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria-mediated apoptosis in zebrafish embryos. Aquat. Toxicol. 180, 56–70.
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The ERK-MNK-eIF4F signaling pathway mediates TPDHT-induced A549 cell death in vitro and in vivo
T
Chuanlong Guoa,b, Yuzhen Houa,b, Xuemin Yuc, Fan Zhanga,b, Xiaochen Wua,b, Xianggen Wua,b,∗, Lijun Wangd,∗∗ a
Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China Key Laboratory of Pharmaceutical Research for Metabolic Diseases, Qingdao University of Science and Technology, China c Department of Otorhinolaryngology, Qilu Hospital of Shandong University, NHC Key Laboratory of Otorhinolaryngology, Qingdao, Shandong, 266035, China d Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China b
ARTICLE INFO
ABSTRACT
Keywords: Apoptosis eIF4F ERK MNK A549 cells
The eIF4E/eIF4G complex plays a central role in gene expression regulation during the initial stage of translation. This study aimed to determine if the novel small molecule compound, TPDHT, could disrupt the interaction of eIF4E/eIF4G, and if it could exhibit excellent antitumor activity in vivo without causing apparent toxicity effect. This study investigated the antitumor mechanism of TPDHT in vitro and in vivo. TPDHT showed significant anti-proliferative activity on human lung cancer A549 cells, and it induced G0/G1 cycle arrest. Moreover, TPDHT also induced A549 cell apoptosis through the mitochondria-mediated apoptotic pathway. Our results indicate that TPDHT could disrupt the interaction of eIF4E/eIF4G, and the activity of eIF4F plays an important role in TPDHT-induced cell proliferation inhibition and apoptosis. Further research showed that TPDHT could inhibit the Ras/ERK/MNK pathway, and activation of the ERK pathway reversed TPDHT-induced cell proliferation inhibition and apoptosis. Taken together, our study findings indicated that TPDHT exerts an antitumor effect through an intrinsic apoptotic pathway controlled by the ERK/MNK/eIF4F pathway.
1. Introduction Non-small cell lung cancer (NSCLC) is the most comment type of human lung cancer and account for 85% of all human lung cancers (Rami-Porta et al., 2017). Although lung cancer treatments have progressed, rates of five-year survival remain low. Therefore, developing new drugs to treat lung cancer is very important for improving the living conditions of patients (Farhan et al., 2019; Hong et al., 2015; Tetsu et al., 2016). Eukaryotic initiation factor 4E (eIF4E) plays a key role in regulating the translation of eukaryotic cells. eIF4E, also known as cap-binding protein, affects the processing, metabolism, and translation of eukaryotic mRNA, and it plays an important role in the initial stages of protein synthesis (Mamane et al., 2004). Studies have found that when the level of eIF4E expression in cells increases, it causes translation of some tumor-related mRNAs, such as proto-oncogenes (c-Myc, Cyclin D1, etc.), angiogenic factors (vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), etc.), and matrix metalloproteinases (Mamane et al., 2004). eIF4E is critical for the regulation of
∗
apoptosis, therefore, targeting eIF4E may be a rational anticancer therapy (Descamps et al., 2012; Moerke et al., 2007). eIF4E can form an eIF4F complex with eIF4A and eIF4G to participate in eukaryotic translation initiation. The activity of eIF4F is mainly regulated by two signals: the PI3K/Akt/mTOR pathway and the Ras/MNK pathway (Proud, 2015a; Richter and Sonenberg, 2005). mTOR signals control eIF4F assembly by liberating both eIF4E and eIF4A from their respective inhibitory binding proteins: eIF4E-binding proteins (4E-BPs). The 4E-BPs (mainly 4E-BP1) compete with eIF4G for a binding site on eIF4E. Acting as a substrate for mTOR, the phosphorylation level of 4E-BP1 is regulated by mTOR (Matsumoto et al., 2015). However, the activity of eIF4F is also regulated by the MNK signaling pathway. Studies have shown that phosphorylated eIF4E (Ser 209) enhances its ability to bind to the 5′-end of the mRNA structure, while also enhancing the interaction of eIF4E/eIF4G (Feoktistova et al., 2013). Importantly, the only kinases that phosphorylate eIF4E are the mitogen-activated protein kinase (MAPK)-interacting kinases MNK1 and MNK2. MNK is activated by P38 and ERK 1/2 MAPK (Proud,
Corresponding author. Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China. Corresponding author. E-mail addresses: [email protected] (X. Wu), [email protected] (L. Wang).
∗∗
https://doi.org/10.1016/j.fct.2020.111158 Received 8 November 2019; Received in revised form 20 January 2020; Accepted 22 January 2020 Available online 25 January 2020 0278-6915/ © 2020 Elsevier Ltd. All rights reserved.
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2015b; Pyronnet, 2000). The role of eIF4E in the malignant transformation of cells has received much attention. It has been reported that the expression of eIF4E is low level in normal tissues (Qiuyuan Wen et al., 2017). However, eIF4E is overexpressed in a variety of human cancers, and its expression level is related to tumor invasion ability. Moreover, it has been found that the expression of phosphorylated eIF4E is increased in cancer tissues, and it is associated with poor prognosis in patients, especially those with non-small cell lung cancer (NSCLC) (Cencic et al., 2011b; Jacobson et al., 2013). In our previous studies, a series of bromophenol-thiazolylhydrazone hybrids were synthesized, and their potential activity for targeting eIF4E was demonstrated (Chen et al., 2016). Among these hybrid compounds, (E)-4-(3,5-bis(trifluoromethyl)phenyl)-2-(2-(3,4-difluorobenzylidene)hydrazinyl)thiazole (TPDHT) exhibited excellent antitumor activity in vitro and in vivo. In the present study, we further explored the antitumor mechanism of TPDHT, especially for its ability to regulate eIF4E and related signaling pathways.
2.5. siRNA transfection The eIF4E siRNA and control siRNA were obtained from Cell Signaling Technology (Beverly, MA, USA). In brief, the A549 cells were plated in 6-well plate at the density of 4 × 105 cells/well. After 24 h, 75 pmol siRNA was transfected into the cells using Lipofectamine™ 2000 (Life Technologies, Carlsbad, CA, USA) reagent, according to the manufacturer's instructions. Then, 24 h after transfection, the cells were exposed to TPDHT for 48 h, after which they were harvested for western blotting or cellular assays. 2.6. m7 GTP pull-down assay The A549 cells were treated with TPDHT for 48 h. Then, the total proteins were extracted, and 700 mg aliquots of the cell lysates were incubated with 7-methylguanosine (m7-GTP)-Sepharose beads at 4 °C for 2 h with constant shaking. After washing the resin, the bound proteins were eluted with free m7-GTP, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed using western blotting using antibodies against eIF4G, eIF4E, and 4EBP1.
2. Materials and methods 2.1. Chemicals and materials
2.7. Analysis of apoptosis
TPDHT was synthesized in our lab (with a purity of > 95%, Supplemental Fig. S1). TPDHT was dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. The Duolink® In Situ Red Starter Kit Mouse/Rabbit Kit (DUO92101-1 KT) was obtained from Sigma-Aldrich. The mediums were obtained from Hyclone Laboratories (USA). Fetal bovine serum (FBS) was obtained from ExCell Bio (China). The Reactive Oxygen Species (ROS) assay kit, JC-1 assay kit, apoptosis assay kit, Hoechst 33258 staining kit, and the pan-caspase inhibitor (Z-VAD-FMK) were obtained from Beyotime Biotechnology (Nanjing, China). The antibodies are listed in Supplemental Table S1. The ERK activator, Honokiol, was obtained from Dalian Meilun Biotechnology Co., LTD.
The A549 cells were seeded in a 6-well plate and incubated for 24 h. The cells were exposed to TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h, then cells were harvested and stained with Annexin V/PI for 15 min in the dark. The cells were analyzed in three different experiments using flow cytometry (FACScan™ flow cytometry, Becton Dickinson, USA). 2.8. Cell cycle analysis The A549 cells were seeded in a 6-well plate and incubated for 24 h. The cells were exposed to TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h, then cells were harvested and fixed with 70% ethanol at −20 °C overnight. Following fixation, the cells were stained with Propidium iodide (PI) for 30 min, and analyzed in three different experiments using flow cytometry.
2.2. Cell culture The cells were purchased from Cell Bank, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in mediums supplemented with 10% FBS at 37 °C and in an atmosphere containing 5% CO2.
2.9. Analysis of ROS generation The A549 cells were seeded in a 6-well plate, and incubated for 24 h. The cells were exposed to TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h, then cells were stained with peroxide-sensitive fluorescent probe DCFH-DA (2′,7-dichlorofluorescein diacetate) for 30 min. After being washed for three times, cells were harvested and analyzed in three different experiments using flow cytometry.
2.3. Analysis of cell viability Cell viability was determined by MTT assay. A549 cells were plated into a 96-well culture plate at the density of 3 × 103 cells per well. After 24 h incubation, the cells were exposed to TPDHT (0, 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM) for 12 h, 24 h and 48 h. Following treatment, the cells were further incubated with MTT (5 mg/mL) for 4 h. The medium was carefully removed, and 150 μL DMSO was added to each well. The samples were thoroughly agitated for 10 min on a shaker. Finally, the absorbance of the samples at 490 nm was measured using a microplate reader.
2.10. Morphological observation For transmission electron microscopy (TEM) analysis, the A549 cells were exposed to TPDHT (5 μM) for 48 h, and then fixed, dehydrated, and embedded in resin. Images were obtained from the Transmission Electron Microscopy Facility (JEM-1200EX, Japan). For Hoechst 33258 staining, the A549 cells were seeded in a 6-well plate and allowed to settle overnight. The cells were treated with TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h. After treatment, cells were stained with Hoechst 33258 for 5 min at room temperature, and then cells were assessed using a fluorescence microscope.
2.4. Immunofluorescence detection The A549 cells were seeded on glass bottom dishes and incubated for 24 h. After 48 h of treatment with TPDHT, the cells were fixed with 4% paraformaldehyde and blocked with bovine serum albumin (BSA) for 1 h. The cells were incubated with primary antibody at 4 °C, overnight. After being washed three times in TBST, the cells were incubated with fluorescence-conjugated second antibody at 37 °C for 1 h. The nuclei were labeled with DAPI, and the cells were visualized under a fluorescence microscope (Olympus, Japan).
2.11. Analysis of cell mitochondrial transmembrane potential (Δψm) The A549 cells were seeded in a 6-well plate and incubated for 24 h. The cells were exposed to TPDHT (1 μM, 2.5 μM and 5 μM) for 48 h, then cells were harvested and stained with JC-1 at 37 °C for 30 min. After being washed for three times, the relative fluorescence intensities 2
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were analyzed using flow cytometry.
Table 1 IC50 values of TPDHT against four human cancer cell lines.
2.12. Western blotting analysis After treatment with TPDHT, at the previously indicated concentrations, the A549 cells were harvested, and the tumor tissues were homogenized. The proteins were harvested and separated using SDSPAGE, and then they were transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in blocking solution (containing 5% non-fat milk) and subsequently probed with primary antibodies (Supplemental Table S1) at 4 °C, overnight. After being washed in TBST for 15 min, the membranes were incubated with a secondary antibody for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence system (BeyoECL Plus, Beyotime Technology).
Cell Line
IC50 (μM)
A549 NCI–H460 PANC-1 SK-OV-3 CaCo-2 MCF-7 MDA-MB-231 HCC-1937 HepG2 U87 MG HL-7702
2.81 ± 0.25 4.23 ± 0.26 11.80 ± 1.15 16.31 ± 1.02 38.25 ± 8.70 22.40 ± 6.75 11.21 ± 3.79 22.65 ± 8.24 7.53 ± 2.74 13.4 ± 8.24 11.90 ± 6.24
a
IC50: Concentration of the compound producing 50% cell growth inhibition after 48 h of drug exposure, as determined by the MTT assay. Each experiment was run at least three times.
2.13. In vivo studies All the animal experiments were performed in accordance with the guidelines established by the Institute of Oceanology Committee for the Care and Use of Laboratory Animals. Female congenital athymic BALB/ c nude (nu/nu) mice were purchased from the Model Animal Research Center of Nanjing University. They were maintained under specificpathogen-free (SPF) conditions, and they were provided with sterile food and water. All experiments were carried out using 6–8-week-old mice weighing 18–22 g. In brief, the A549 cells (1 × 107) were subcutaneously (s.c.) injected into the back of the mice. When the tumor reached 150 mm3 in volume, the animals were randomly assigned into test groups, each consisting of six mice, and then 25 mg/kg and 50 mg/ kg TPDHT (dissolved by 0.5% CMC-Na) was administered intraperitoneally (i.p.) for 21 consecutive days. The tumor volumes and body weights of the animals were measured every three days. Tumor volumes were calculated according to the following equation: length × (width) 2/2. The tumor tissue was removed for immunohistochemistry, western blotting, or hematoxylin and eosin (H&E) staining.
comparisons were performed using one-way analysis of variance. P < 0.05 was considered to be statistically significant. 3. Results 3.1. TPDHT inhibits cell proliferation We measured the proliferation activities of TPDHT using MTT assay. The following types of tumor cells were tested in this study: human lung cancer cells, A549 and NCI–H460, human pancreatic cancer cell PANC1, human ovarian cancer cell SK-OV-3, human colon cancer cell CaCo-2, human breast cancer cells MCF-7, MDA-MB-231, and HCC-1937, human hepatoma cell HepG2, human glioma cell U87 MG, and one normal cell HL-7702. As shown in Table 1, TPDHT inhibited proliferation of the tumor cells and the normal cells. Among these cells, TPDHT showed excellent activity for the inhibition of A549 proliferation with an IC50 value of 2.81 μM. The MTT assay also indicated that TPDHT could decrease cell viability in a dose-dependent and time-dependent manner (12 h, 24 h, and 48 h) (Fig. 1B). The effect of TPDHT on the cell colony formation was also detected. The results indicated that TPDHT could inhibit the clonogenicity of the A549 cells (Fig. 1C and D).
2.14. Safety of TPDHT in vivo All the animal experiments were performed in accordance with the guidelines established by the Institute of Oceanology Committee for the Care and Use of Laboratory Animals. Forty SPF Kunming mice (18–22 g), 10 males and 10 females, were used for the acute toxicity analysis. The mice were randomly assigned to four groups, each consisting of five males and five females. The four groups of mice were orally administered TPDHT at single doses of 0 (blank group, the same volume of physiological saline), 500 mg/kg, 1000 mg/kg, and 2000 mg/kg, respectively. The mice were housed with free access to water and food in stainless steel cages in a room with controlled temperature (25 ± 1 °C) and a 12-h light/dark cycle. The survival rate and body weight of the mice were monitored and recorded up to 14 days post-treatment. Twenty SPF Kunming mice (18–22 g), 10 males and 10 females, were used for the subacute toxicity analysis. The mice were randomly assigned to two groups, each consisting of five males and five females. The treatment group was treated with 2000 mg/kg, orally, once a day for 14 days. The body weight of the mice was measured every week, and the mice were closely monitored for any signs of toxicity or discomfort. After 14 days of treatment, the mice were anesthetized with isoflurane and killed by decapitation, and the tissues were collected for further analysis.
3.2. TPDHT induces cell apoptosis through the mitochondrial-mediated apoptotic pathway To evaluate the effect of TPDHT on the induction of apoptosis, the A549 cells were treated with TPDHT for 48 h, then cells were stained with Annexin V/PI and analyzed using flow cytometry. The results showed that TPDHT could induce apoptosis of the A549 cells in a concentration-dependent manner (Fig. 2A and B). We also observed the apoptotic features in the A549 cells after staining with Hoechst 33258 (Fig. 1E). Moreover, Z-VAD-FMK (the pan-caspase inhibitor) was used in our study. The results showed that Z-VAD-FMK could inhibit TPDHTinduced cell proliferation inhibition and apoptosis (Fig. 2G and H) in A549 cells, indicating that TPDHT-induced cell apoptosis was caspasedependent. Mitochondrial damage and a decrease in mitochondrial membrane potential (MMP) are characteristics of early apoptosis. In this study, we used TEM to detect the effect of TPDHT on the mitochondrial damage of the A549 cells. As shown in Fig. 3A, after being exposed to TPDHT for 48 h, changes in mitochondrial morphology, such as mitochondrial swelling and vacuolization, were observed. The JC-1 results also showed that the MMP was disrupted in the TPDHT-treated groups (Fig. 3B and C). The mitochondrial respiratory chain is a major source of ROS, and ROS is a switch that regulates the mitochondrial
2.15. Statistical analysis Statistical analysis was performed using GraphPad Prism 5.0 (San Diego, CA, USA). The data were presented as mean ± SD. Statistical 3
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Fig. 1. TPDHT inhibits cell proliferation in A549 cells. (A) Structure of TPDHT. (B) A549 cells were treated with TPDHT for 12 h, 24 h and 48 h. Cell viability was determined by MTT assay. (C, D) A549 cells were treated with TPDHT for 10 days and colony formation was determined by staining with crystal violet. (E) A549 cells were treated with TPDHT for 48 h, and cells were stained with Hoechst 33258 and photographed using a fluorescence microscopy. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
permeability transition pore (MPTP) (Ma et al., 2017). In this study, TPDHT was also found to increase ROS generation in a dose-dependent manner (Fig. 3D and E). Proteins in the Bcl-2 family of proteins are localized in the mitochondrial membrane, and they are involved in the activation of caspase enzymes. Western blotting analysis showed that the expression of Bcl-2 was decreased while the expression of Bax was increased. Furthermore, caspase-3 and poly(ADP-ribose)polymerase (PARP) were also activated by TPDHT (Fig. 2E and F).
3.3. TPDHT induces G0/G1 cell cycle arrest in A549 cells To determine the effect of TPDHT on cell-cycle distribution, the A549 cells were treated with TPDHT for 48 h and analyzed using flow cytometry. The results showed that the cells were arrested in the G0/G1 phase. In comparison to the control group, the ratio of the G0/G1 phase in the TPDHT-treated groups was increased in a dose-dependent manner (Fig. 2C and D).
Fig. 2. TPDHT induces cell apoptosis in A549 cells. (A, B) A549 cells were treated with TPDHT for 48 h. Cells were harvested, stained with Annexin V/PI and analyzed by flow cytometry (FACS). (C, D) A549 cells were treated with TPDHT for 48 h. Cells were harvested and fixed in 70% ethanol overnight, and then cells were stained with PI and analysis by FACS. (E) A549 cells were treated with TPDHT for 48 h. Proteins were extracted and analyzed by western blotting. (F) Protein bands were analyzed by Image J. (G) A549 cells were treated with 5 μM TPDHT alone or in combination with Z-VAD-FMK (10 μM) for 48 h. The percentages of apoptotic cells were determined by FACS via Annexin V/PI staining. (H) A549 cells were treated with 5 μM TPDHT alone or in combination with Z-VAD-FMK (10 μM) for 48 h. The cell viability was detected by MTT assay. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group. ##P < 0.01. 4
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Fig. 3. TPDHT induces mitochondrial damage and ROS generation in A549 cells. (A) A549 cells were treated with TPDHT for 48 h. The internal morphology of A549 cells were observed by transmission electron microscopy (TEM). (B, C) A549 cells were treated with TPDHT for 48 h. Cells were stained with JC-1 and analyzed by FCAS. (D, E) A549 cells were treated with TPDHT for 48 h. Cells were stained with FCFH-DA and analyzed by FCAS. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group.
3.4. TPDHT disrupts the activity of eIF4F
apoptosis was inhibited (Fig. 5C and D). Moreover, TPDHT-induced mitochondrial damage and MMP reduction were also inhibited by 4EBP1 siRNA (Fig. 5E). These results indicated that the activity of eIF4F was involved in TPDHT-induced anti-apoptotic signaling.
In eukaryotic cells, eIF4E interacts with eIF4G to form an eIF4F complex that initiates the translation process and induces expression of several oncogenes, such as c-Myc and Cyclin D1. Therefore, inhibiting the interaction between eIF4E and eIF4G is a new strategy for the treatment of cancer. The activity of eIF4F is controlled by two signaling pathways: the PI3K/Akt/mTOR-4E-BP1 pathway and the Ras/ERK/ MNK-eIF4E pathway. First, we used the in situ proximity ligation assay (PLA) (DuoLink PLA) to detect the interference of TPDHT between eIF4E and eIF4G. The phosphor dots were reduced significantly, indicating that the interaction of eIF4E/eIF4G was disrupted by TPDHT (Fig. 4A and B). We then used the cap-binding assay to further analyze the effect of TPDHT on the interaction of eIF4E/eIF4G. Cell extracts were affinity-chromatized on m7 GTP-Sepharose beads for western blotting analysis. As shown in Fig. 4C, the amount of eIF4E-bound 4E-BP1 was significantly increased, whereas the amount of eIF4E-bound eIF4G was decreased. These results indicated that TPDHT induced the disruption of eIF4E/ eIF4G.
3.6. TPDHT inhibits ERK/MNK-mediated eIF4E phosphorylation eIF4E phosphorylation is elevated in a variety of tumor cells. eIF4E acts as the only substrate for MAPK-interacting kinases, MNK1 and MNK2, and phosphorylation is regulated by MNK (Fischer, 2017; Zhao et al., 2016). First, we analyzed the phosphorylation level of eIF4E in the TPDHT-treated A549 cells. We found that TPDHT decreased the phosphorylation level of eIF4E in a concentration-dependent manner (Fig. 6A and B). Moreover, the expression of Cyclin D1 and c-Myc was also decreased in the TPDHT-treated cells. It has been reported that the activated Ras/ERK/MNK signaling is critical for the activation of eIF4F(Lewinska et al., 2017). We found that TPDHT decreased the phosphorylation level of ERK and MNK (Fig. 6D and E). These results suggested that the ERK/MNK signaling pathway may be involved in TPDHT-induced eIF4F disruption. Honokiol, a potent stimulator of ERK (Liu et al., 2019; Wang et al., 2011), was used in this study to employ the ERK/MNK pathway in the TPDHT-induced disruption of eIF4F. As shown in Fig. 7D, honokiol was able to reverse the dephosphorylation of ERK and eIF4E induced by TPDHT. Moreover, honokiol could also prevent TPDHT-induced mitochondrial membrane dysfunction and cell apoptosis (Fig. 7A,B,C). Taken together, the results from our study indicated that TPDHT-induced apoptosis was mediated by the ERK/MNK signaling pathway.
3.5. eIF4F plays an important role in TPDHT-induced apoptosis eIF4F is essential for the growth and survival of cancer cells, and it can regulate apoptosis. First, we transfected the A549 cells with 4E-BP1 siRNA. The phosphor dots were increased in the 4E-BP1 siRNA group, indicating the enhanced eIF4F activity (Fig. 5A and B). In the 4E-BP1 siRNA group, the TPDHT-induced cell proliferation inhibition and 5
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Fig. 4. TPDHT disrupts the eIF4E/ eIF4G interaction. (A, B) A549 cells were treated with TPDHT. The eIF4E/ eIF4G interactions were detected using a proximity ligation assay (PLA). (C) A549 cells were treated with TPDHT and lysed. Cap-affinity chromatography and SDS-PAGE immunoblotting were used to detect eIF4E, eIF4G, and 4E-BP1. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group.
3.7. TPDHT exhibits antitumor activity in vivo
growth was observed for the mice treated with TPDHT; the tumor growth inhibition (TGI) values were 59.56% and 74.07% at dosages of 25 mg/kg/day and 50 mg/kg/day, respectively. Moreover, no significant differences were found in terms of the body weight of the mice (as a surrogate marker of toxicity). The animals’ visceral tissues, including the liver, spleen, kidney, heart, and lung, were examined using
We also examined the antitumor activity of TPDHT in A549 xenograft models. The animals were repeatedly administered with a vehicle or TPDHT (25 mg/kg and 50 mg/kg) once daily via oral gavage for 21 days. In comparison to the control groups, a significant delay in tumor
Fig. 5. eIF4F plays an important role in TPDHT-induced cell proliferation inhibition and apoptosis. A549 cells were interfered with 4E-BP1 siRNA or control nontarget siRNA 24 h before exposure to TPDHT for 48 h. (A, B) The eIF4E/eIF4G interactions were detected using a proximity ligation assay (PLA). (B) Cell viability was detected by MTT assay. (C) Cell apoptosis was detected by FACS via Annexin V/PI staining. (D) The Δψm was detected by FACS via JC-1 staining. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group, ##P < 0.01. 6
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Fig. 6. TPDHT inhibits Ras/MNK/ERK-eIF4E signal pathway. (A, B, C) A549 cells were treated with TPDHT for 48 h, the phosphorylation of eIF4E was determined using western blotting and immunofluorescence. (D) A549 cells were treated with TPDHT for 48 h. The levels of Ras, p-MNK, MNK, p-ERK, ERK were determined using western blotting. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group.
Fig. 7. ERK pathway plays an important role in TPDHT-indunced apoptosis. A549 cells were treated with TPDHT alone or in combination with Honokiol. (A) Cell viability was detected by MTT assay. (B) Cell apoptosis was detected by FACS via Annexin V/PI staining. (C) The Δψm was detected by FACS via JC-1 staining. (D) The levels of p-ERK and p-eIF4E were detected by western blotting. (F) eIF4E/eIF4G interaction was detected by cap-affinity chromatography and SDS-PAGE immunoblotting. Data were expressed as the mean ± SD obtained from triplicate experiments. **P < 0.01 vs. control group. ##P < 0.01. 7
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Fig. 8. TPDHT inhibits tumor growth in A549-xenografted athymic mice. (A) Mice (n = 6) were administered with TPDHT (25 mg/kg) once per day for 21 days. The mice were anesthetized, and the tumor tissues were collected and photographed. (B) The tumor volumes were recorded every 3 days. (C) Body weights. (D) Immunohistochemical analysis Ki 67, p-ERK, p-MNK and p-eIF4E in tumor tissues. (E) The organs from treated and control groups were stained with H&E to evaluate the toxicity of TPDHT.
H&E staining, and the results showed no significant morphological changes in the vehicle and the TPDHT-treated mice. On the other hand, morphological changes in mouse tumor tissue, such as irregular cell morphology and vacuolization were also detected after H&E staining (Supplemental Fig. S2). To investigate the antitumor mechanism of TPDHT in vivo, tumor samples were analyzed using western blotting and immunochemistry. As shown in Fig. 8D, the immunochemistry results indicated that the level of p-eIF4E and Ki 67 was decreased in the TPDHT-treated tumor samples. Western blotting results showed that the levels of p-MNK, pERK, and p-eIF4E were also decreased in the TPDHT-treated tumor samples.
there were no significant changes in the morphology of tissues (liver, heart, and kidney) (Supplemental Fig. S3). These results demonstrated that TPDHT was safe, in vivo. 4. Discussion eIF4E is closely related to the occurrence, development, and metastasis of tumors. It is an important factor that promotes the transformation of tumor cells. Therefore, eIF4E is an attractive new target for the treatment of neoplastic diseases. eIF4E, together with eIF4A and eIF4G, form an eIF4F complex to participate in eukaryotic translation initiation. eIF4F binds to 7-methylguanylate cap (m7G) at the 5′ end of mRNA and, ultimately, transports it to the ribosomes (Burz et al., 2009; Hassan et al., 2014; Lewinska et al., 2017). Assembly of active eIF4F complex depends on interaction between eIF4E/eIF4G (Moerke et al., 2007). A variety of small molecules, such as 4EGI-1 and 4E1RCat, have been shown to disrupt the interaction of eIF4E/eIF4G (Cencic et al., 2011b; Moerke et al., 2007). 4EGI-1 is the first eIF4E/eIF4G interaction inhibitor with a Kd of 25 μM against eIF4E binding (Cencic et al., 2011a; Moerke et al., 2007). And 4EGI-1 can inhibit the proliferation of various tumor cells, such as human lung cancer cells, human breast cancer cells, glioma cells, etc (Fan et al., 2010; Mahalingam et al., 2014; Takrouri et al., 2014). However, eIF4E activity can be inhibited by preventing phosphorylation of MNK kinase. Small molecules, such as cercosporamide and CGP 57380, are MNK inhibitors that could also regulate the activity of eIF4E (Qiuyuan Wen et al., 2017). In our study, we found that a small molecule, TPDHT, disrupted the eIF4E/eIF4G interaction by inhibiting the Ras/MAPK pathway. First, we demonstrated that TPDHT could inhibit the proliferation of a variety of tumor cells, especially for the human lung cancer cell A549 (with a lowest IC50 of 2.81 ± 0.25 μM). Moreover, we also found that TPDHT could inhibit colony formation and induce apoptosis and G0/G1 phase arrest in A549 cells. TPDHT also exhibited antitumor activity in vivo, with low toxicity. Mitochondria play a key role in the process of apoptosis; changes in the structure and function of mitochondria play an important role in
3.8. TPDHT exhibits low toxicity in vivo To investigate the safety of TPDHT in vivo, acute toxicity and subacute toxicity tests were performed in the mice. In the acute toxicity test, the mice were orally administered single doses of TPDHT: 500 mg/ kg, 1000 mg/kg, and 2000 mg/kg, respectively. The experimental results showed that, after 14 days of monitoring, no mortality and abnormalities (including body weight) were observed (Table 2). In the subacute toxicity test, the mice were orally administered a dose of 2000 mg/kg TPHDT for 14 days. During the 14 days of administration, no deaths or abnormalities (including body weight) were observed (Supplemental Table S2). Furthermore, the H&E analysis showed that Table 2 Acute toxicity test of TPDHT. Dosage (mg/kg)
Number of mice
Deaths of mice 1–7 day
8–14 day
500 1000 2000 Blank
20 20 20 20
0 0 0 0
0 0 0 0
Mortality rate
0/0 0/0 0/0 0/0
Weight (g) 0 day
7 day
20.8 20.4 20.8 20.6
25.7 26.8 28.1 27.7
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many different cellular processes, including apoptosis (Gupta, 2001). We found that TPDHT caused a decrease in MMP in A549 cells, and TPDHT also destroyed the morphology of mitochondria. MMP is regulated by the Bcl-2 family of proteins; when mitochondrial damage or MMP decreases, cytochrome c is released from the mitochondria. This, in turn, activates some apoptotic proteins, such as caspase-3 and PARP, which ultimately leads to apoptosis (Shan et al., 2016). The western blotting results indicated that the expression of Bax increased and the expression of Bcl-2 decreased, while PARP and caspase-3 were activated by TPDHT (Fig. 2E). Moreover, we found that Z-VAD-FMK, the pancaspase inhibitor, could inhibit TPDHT-induced apoptosis and cell hypoproliferation (Fig. 2G and H). These results indicated that TPDHTinduced apoptosis occurred through the mitochondrial-dependent pathway. The activity of eIF4F is controlled by a family of proteins known as eIF4E-bingding proteins, 4E-BPs (mainly 4E-BP1). The phosphorylated 4E-BP1 competes with eIF4G for a binding site on eIF4E, and the interaction of eIF4E/eIF4G is essential for eIF4F activity (Piao et al., 2018; Tang et al., 2019). In our study, we used the in situ PLA and capbinding assay to detect the eIF4E/eIF4G interactions. The in situ PLA results showed that the fluorescence spots of the TPDHT-treated group were significantly decreased, indicating that the eIF4E/eIF4G interaction was inhibited by TPDHT (Fig. 4A). We then used m7-GTP-Sepharose beads to capture eIF4E and its binding partners (eIF4G and 4EBP1). The pull-down assay showed that TPDHT decreased the amount of eIF4G and increased the amount of 4E-BP1 (Fig. 4C). These results indicated that TPDHT could disrupt the interaction of eIF4E/eIF4G. Activated eIF4F promotes downstream signaling factors, including the expression of Cyclin D1, c-Myc, etc., and it regulates cell proliferation and death (Anzell et al., 2018; Sifuentes-Franco et al., 2018). To explore the role of the eIF4F complex in TPDHT-induced cell proliferation inhibition, cell cycle arrest, and apoptosis, we used siRNA 4EBP1 to knockdown 4E-BP1 (manufacturing an eIF4F activation model). The results showed that TPDHT-induced apoptosis and MMP disruption was inhibited by 4E-BP1 siRNA (Fig. 5). These results indicated that eIF4F was involved in TPDHT-induced anti-apoptotic signaling. The activity of eIF4F is also regulated by the Ras/ERK/MNK pathway (Gao et al., 2012; Hall et al., 2014; Jing et al., 2018). eIF4E is the only substrate of the MAP kinase-interacting kinases, MNK1 and MNK2, and its phosphorylation is only affected by MNKs (Cheng et al., 2020; Zhao et al., 2016). MNKs are located at key points in the intracellular signaling pathway, and they can also be activated as a downstream consequence of ERK and MAPK signaling (Altman et al., 2013; Fischer, 2017; Ramalingam et al., 2014). ERK lies downstream of oncoproteins, such as the receptor tyrosine kinases, Ras and Raf (He et al., 2015; Kim et al., 2018). In our study, the immunofluorescence and western blotting results showed that phosphorylation of eIF4E was inhibited by TPDHT (Fig. 6A,C). The expression levels of Ras, p-ERK, and p-MNK were also inhibited by TPDHT (Fig. 6D). To verify that the ERK signaling pathway plays a key role in TPDHT-induced apoptosis, we used honokiol, an ERK stimulator. Honokiol was able to reverse the dephosphorylation of ERK and eIF4E induced by TPDHT (Fig. 7). Moreover, we found that honokiol also attenuated the TPDHT-mediated intrinsic apoptotic pathway. Interestingly, the western blotting results also showed that the expression levels of p-ERK, p-MNK, and p-eIF4E were inhibited in the TPDHT-treated tumor groups (Fig. 8D). These results indicated that the ERK/MNK/eIF4E pathway was involved in TPDHT-exhibited antitumor activity in vitro and in vivo. Above all, this study identified that TPDHT had a potent antitumor effect against human lung cancer cells in vitro and in vivo. Furthermore, TPDHT could induce apoptosis through the mitochondria-induced apoptotic pathway. The mechanism study results indicated that TPDHT could inhibit the activity of eIF4F through the Ras/ERK/MNK-eIF4F signal pathway. Therefore, these findings provide evidence that TPDHT may demonstrate potentially useful anti-cancer activity against human lung cancer.
CRediT authorship contribution statement Chuanlong Guo: Conceptualization, Methodology, Software, Writing - review & editing. Yuzhen Hou: Data curation, Writing original draft. Xuemin Yu: Data curation, Writing - original draft. Fan Zhang: Visualization, Investigation, Visualization, Investigation. Xiaochen Wu: Supervision, Software, Validation. Xianggen Wu: Writing - review & editing, Visualization. Lijun Wang: Conceptualization, Software, Writing - review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81773586) and the Talent Fund of Shandong Collaborative Innovation Center of Eco-Chemical Engineering (project no. XTCXQN19). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fct.2020.111158. References Altman, J.K., Szilard, A., Konicek, B.W., Iversen, P.W., Kroczynska, B., Glaser, H., Sassano, A., Vakana, E., Graff, J.R., Platanias, L.C., 2013. Inhibition of Mnk kinase activity by cercosporamide and suppressive effects on acute myeloid leukemia precursors. Blood 121, 3675–3681. Anzell, A.R., Maizy, R., Przyklenk, K., Sanderson, T.H., 2018. Mitochondrial quality control and disease: insights into ischemia-reperfusion injury. Mol. Neurobiol. 55, 2547–2564. Burz, C., Berindan-Neagoe, I., Balacescu, O., Irimie, A., 2009. Apoptosis in cancer: key molecular signaling pathways and therapy targets. Acta Oncol. 48, 811–821. Cencic, R., Hall, D.R., Robert, F., Du, Y.H., Min, J., Li, L., Qui, M., Lewis, I., Kurtkaya, S., Dingledine, R., Fu, H.A., Kozakov, D., Vajda, S., Pelletier, J., 2011a. Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F (vol 108, pg 1046, 2010). Proc. Natl. Acad. Sci. U. S. A. 108 6689-6689. Cencic, R., Hall, D.R., Robert, F., Du, Y.H., Min, J.K., Li, L.A., Qui, M., Lewis, I., Kurtkaya, S., Dingledine, R., Fu, H.A., Kozakov, D., Vajda, S., Pelletier, J., 2011b. Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F. Proc. Natl. Acad. Sci. U. S. A. 108, 1046–1051. Chen, W.Q., Zheng, R.S., Baade, P.D., Zhang, S.W., Zeng, H.M., Bray, F., Jemal, A., Yu, X.Q., He, J., 2016. Cancer statistics in China, 2015. Ca - Cancer J. Clin. 66, 115–132. Cheng, C.H., Su, Y.L., Ma, H.L., Deng, Y.Q., Feng, J., Chen, X.L., Jie, Y.K., Guo, Z.X., 2020. Effect of nitrite exposure on oxidative stress, DNA damage and apoptosis in mud crab (Scylla paramamosain). Chemosphere 239. Descamps, G., Gomez-Bougie, P., Tamburini, J., Green, A., Bouscary, D., Maiga, S., Moreau, P., Le Gouill, S., Pellat-Deceunynck, C., Amiot, M., 2012. The cap-translation inhibitor 4EGI-1 induces apoptosis in multiple myeloma through Noxa induction. Br. J. Canc. 106, 1660–1667. Fan, S., Li, Y., Yue, P., Khuri, F.R., Sun, S.-Y., 2010. The eIF4E/eIF4G interaction inhibitor 4EGI-1 augments TRAIL-mediated apoptosis through c-FLIP down-regulation and DR5 induction independent of inhibition of cap-dependent protein translation. Neoplasia 12, 346–IN347. Farhan, M., Malik, A., Ullah, M.F., Afaq, S., Faisal, M., Farooqi, A.A., Biersack, B., Schobert, R., Ahmad, A., 2019. Garcinol sensitizes NSCLC cells to standard therapies by regulating EMT-modulating miRNAs. Int. J. Mol. Sci. 20. Feoktistova, K., Tuvshintogs, E., Do, A., Fraser, C.S., 2013. Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity. Proc. Natl. Acad. Sci. U. S. A. 110, 13339–13344. Fischer, M., 2017. Census and evaluation of p53 target genes. Oncogene 36, 3943–3956. Gao, N., Cheng, S., Budhraja, A., Gao, Z., Chen, J., Liu, E.H., Huang, C., Chen, D., Yang, Z., Liu, Q., Li, P., Shi, X., Zhang, Z., 2012. Ursolic acid induces apoptosis in human leukaemia cells and exhibits anti-leukaemic activity in nude mice through the PKB pathway. Br. J. Pharmacol. 165, 1813–1826. Gupta, S., 2001. Molecular steps of death receptor and mitochondrial pathways of apoptosis. Life Sci. 69, 2957–2964. Hall, C., Dumstorf, C., Konicek, B., Robichaud, N., McNulty, A., Parsons, S., Pelltier, J., Sonenberg, N., Graff, J.R., 2014. eIF4E phosphorylation is Mnk-dependent but does not require assembly of the eIF4F translation initiation complex. Canc. Res. 74.
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C. Guo, et al. Hassan, M., Watari, H., AbuAlmaaty, A., Ohba, Y., Sakuragi, N., 2014. Apoptosis and molecular targeting therapy in cancer. BioMed Res. Int. 2014, 150845. He, W., Shi, F., Zhou, Z.W., Li, B., Zhang, K., Zhang, X., Ouyang, C., Zhou, S.F., Zhu, X., 2015. A bioinformatic and mechanistic study elicits the antifibrotic effect of ursolic acid through the attenuation of oxidative stress with the involvement of ERK, PI3K/ Akt, and p38 MAPK signaling pathways in human hepatic stellate cells and rat liver. Drug Des. Dev. Ther. 9, 3989–4104. Hong, Q.Y., Wu, G.M., Qian, G.S., Hu, C.P., Zhou, J.Y., Chen, L.A., Li, W.M., Li, S.Y., Wang, K., Wang, Q., Zhang, X.J., Li, J., Gong, X., Bai, C.X., Lung Cancer Group of Chinese Thoracic, S, Chinese Alliance Against Lung, C, 2015. Prevention and management of lung cancer in China. Cancer 121 (Suppl. 17), 3080–3088. Jacobson, B.A., Thumma, S.C., Jay-Dixon, J., Patel, M.R., Kroening, K.D., Kratzke, M.G., Etchison, R.G., Konicek, B.W., Graff, J.R., Kratzke, R.A., 2013. Targeting eukaryotic translation in mesothelioma cells with an eIF4E-specific antisense oligonucleotide. PloS One 8. Jing, B., Liu, M., Yang, L., Cai, H.Y., Chen, J.B., Li, Z.X., Kou, X., Wu, Y.Z., Qin, D.J., Zhou, L., Jin, J., Lei, H., Xu, H.Z., Wang, W.W., Wu, Y.L., 2018. Characterization of naturally occurring pentacyclic triterpenes as novel inhibitors of deubiquitinating protease USP7 with anticancer activity in vitro. Acta Pharmacol. Sin. 39, 492–498. Kim, K., Shin, E.A., Jung, J.H., Park, J.E., Kim, D.S., Shim, B.S., Kim, S.H., 2018. Ursolic acid induces apoptosis in colorectal cancer cells partially via upregulation of MicroRNA-4500 and inhibition of JAK2/STAT3 phosphorylation. Int. J. Mol. Sci. 20. Lewinska, A., Adamczyk-Grochala, J., Kwasniewicz, E., Deregowska, A., Wnuk, M., 2017. Ursolic acid-mediated changes in glycolytic pathway promote cytotoxic autophagy and apoptosis in phenotypically different breast cancer cells. Apoptosis : an international journal on programmed cell death 22, 800–815. Liu, P., Du, R., Yu, X., 2019. Ursolic acid exhibits potent anticancer effects in human metastatic melanoma cancer cells (SK-MEL-24) via apoptosis induction, inhibition of cell migration and invasion, cell cycle arrest, and inhibition of mitogen-activated protein kinase (MAPK)/ERK signaling pathway. Med. Sci. Mon. Int. Med. J. Exp. Clin. Res. : Int. Med. J. Exp. Clin. Res. 25, 1283–1290. Ma, X., Zhang, Y., Wang, Z., Shen, Y., Zhang, M., Nie, Q., Hou, Y., Bai, G., 2017. Ursolic acid, a natural nutraceutical agent, targets Caspase3 and alleviates inflammationassociated downstream signal transduction. Mol. Nutr. Food Res. 61. Mahalingam, P., Takrouri, K., Chen, T., Sahoo, R., Papadopoulos, E., Chen, L., Wagner, G., Aktas, B.H., Halperin, J.A., Chorev, M., 2014. Synthesis of rigidified eIF4E/eIF4G inhibitor-1 (4EGI-1) mimetic and their in vitro characterization as inhibitors of protein-protein interaction. J. Med. Chem. 57, 5094–5111. Mamane, Y., Petroulakis, E., Rong, L.W., Yoshida, K., Ler, L.W., Sonenberg, N., 2004. eIF4E - from translation to transformation. Oncogene 23, 3172–3179. Matsumoto, M., Seike, M., Noro, R., Soeno, C., Sugano, T., Takeuchi, S., Miyanaga, A., Kitamura, K., Kubota, K., Gemma, A., 2015. Control of the MYC-eIF4E axis plus mTOR inhibitor treatment in small cell lung cancer. BMC Canc. 15, 241. Moerke, N.J., Aktas, H., Chen, H., Cantel, S., Reibarkh, M.Y., Fahmy, A., Gross, J.D., Degterev, A., Yuan, J.Y., Chorev, M., Halperin, J.A., Wagner, G., 2007. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and
eIF4G. Cell 128, 257–267. Piao, M.J., Ahn, M.J., Kang, K.A., Ryu, Y.S., Hyun, Y.J., Shilnikova, K., Zhen, A.X., Jeong, J.W., Choi, Y.H., Kang, H.K., Koh, Y.S., Hyun, J.W., 2018. Particulate matter 2.5 damages skin cells by inducing oxidative stress, subcellular organelle dysfunction, and apoptosis. Arch. Toxicol. 92, 2077–2091. Proud, C.G., 2015a. Mnks, eIF4E phosphorylation and cancer. Bba-Gene Regul Mech 1849, 766–773. Proud, C.G., 2015b. Mnks, eIF4E phosphorylation and cancer. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1849, 766–773. Pyronnet, S., 2000. Phosphorylation of the cap-binding protein eIF4E by the MAPK-activated protein kinase Mnk1. Biochem. Pharmacol. 60, 1237–1243. Ramalingam, S., Gediya, L., Kwegyir-Afful, A.K., Ramamurthy, V.P., Purushottamachar, P., Mbatia, H., Njar, V.C.O., 2014. First Mnks degrading agents block phosphorylation of eIF4E, induce apoptosis, inhibit cell growth, migration and invasion in triple negative and Her2-overexpressing breast cancer cell lines. Oncotarget 5, 530–543. Rami-Porta, R., Asamura, H., Travis, W.D., Rusch, V.W., 2017. Lung cancer - major changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA A Cancer J. Clin. 67, 138–155. Richter, J.D., Sonenberg, N., 2005. Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433, 477–480. Shan, J.Z., Xuan, Y.Y., Zhang, Q., Zhu, C.P., Liu, Z., Zhang, S.Z., 2016. Ursolic acid synergistically enhances the therapeutic effects of oxaliplatin in colorectal cancer. Protein Cell 7, 571–585. Sifuentes-Franco, S., Padilla-Tejeda, D.E., Carrillo-Ibarra, S., Miranda-Diaz, A.G., 2018. Oxidative stress, apoptosis, and mitochondrial function in diabetic nephropathy. Internet J. Endocrinol. 2018, 1875870. Takrouri, K., Chen, T., Papadopoulos, E., Sahoo, R., Kabha, E., Chen, H., Cantel, S., Wagner, G., Halperin, J.A., Aktas, B.H., Chorev, M., 2014. Structure-activity relationship study of 4EGI-1, small molecule eIF4E/eIF4G protein-protein interaction inhibitors. Eur. J. Med. Chem. 77, 361–377. Tang, J.Y., Fu, O.Y., Hou, M.F., Huang, H.W., Wang, H.R., Li, K.T., Fayyaz, S., Shu, C.W., Chang, H.W., 2019. Oxidative stress-modulating drugs have preferential anticancer effects - involving the regulation of apoptosis, DNA damage, endoplasmic reticulum stress, autophagy, metabolism, and migration. Semin. Canc. Biol. 58, 109–117. Tetsu, O., Hangauer, M.J., Phuchareon, J., Eisele, D.W., McCormick, F., 2016. Drug resistance to EGFR inhibitors in lung cancer. Chemotherapy 61, 223–235. Wang, X., Zhang, F., Yang, L., Mei, Y., Long, H., Zhang, X., Zhang, J., Qimuge, S., Su, X., 2011. Ursolic acid inhibits proliferation and induces apoptosis of cancer cells in vitro and in vivo. J. Biomed. Biotechnol. 2011, 419343. Wen, Qiuyuan, W, W., Luo, Jiadi, 2017. CGP57380 enhances efficacy of RAD001 in nonsmall cell lung cancer through abrogating mTOR inhibition-induced phosphorylation of eIF4E and activating mitochondrial apoptotic pathway. oncotarget 7. Zhao, X.S., Ren, X., Zhu, R., Luo, Z.Y., Ren, B.X., 2016. Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria-mediated apoptosis in zebrafish embryos. Aquat. Toxicol. 180, 56–70.
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