Research Article
Salubrinal Regulates the Apoptosis of Adrenocortical Carcinoma Cells via the PERK/eIF2x/ATF4 Signaling Pathway
Lili Wu (D,1,2 Chunfeng Liang,1 Xuemei Huang,1 Xiujun Deng,1 Jiming Jiang,3 and Zuojie Luo D’ 1
1 Department of Endocrinology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, China
2Department of Integrated Medicine, Guangxi Medical University Cancer Hospital, Nanning, Guangxi 530021, China
3Department of Analysis for Cosmetics (Dietary Supplements), Guangxi Institute for Food and Drug Control, Nanning, Guangxi 530021, China
Correspondence should be addressed to Zuojie Luo; luozuojie2021@163.com
Received 18 May 2021; Revised 15 August 2021; Accepted 19 August 2021; Published 7 September 2021
Academic Editor: Raffaele Pezzani
Copyright @ 2021 Lili Wu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The protein-kinase-R- (PKR-) like endoplasmic reticulum kinase (PERK) signaling pathway is a well-known promoter of cell apoptosis. In this study, we aimed to determine whether salubrinal (Sal), a selective activator of eukaryotic translation initiation factor 2 (eIF2a), can induce apoptosis of human adrenocortical carcinoma (ACC) cell via activating the PERK/eIF2x/ATF4 signaling pathway, and the potential mechanisms of this action were explored. The ACC cell lines, including SW-13 and NCI-H295 R, were used. 3-(4,5)-Di- methylthiazol(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, cell scratch experiments, flow cytometry, and JC-1 staining assays were performed to detect the cell viability, cell migration, and cell apoptosis. The expression of PERK/eIF2a/ATF4 signaling-pathway- related proteins and apoptosis-related proteins was detected by western blot (WB). Intracellular Ca2+ ion concentration was determined by a confocal laser scanning microscope. The results showed that Sal inhibited the migration and proliferation of ACC cells. Sal remarkably increased the influx of Ca2+ ion and the apoptosis rate of ACC cells in vitro. Furthermore, the expression levels of PERK/ eIF2a/ATF4 signaling-related proteins and apoptosis-related proteins were upregulated in the treatment of Sal. The research dem- onstrated that Sal reduces the cell viability, increases the intracellular calcium concentration, and promotes the apoptosis of ACC cells in vitro through increasing the phosphorylation level of eIF2x and activating the PERK/eIF2x/ATF4 signaling. PERK/eIF2x/ATF4 is expected to act as a potential therapeutic target for the treatment of adrenocortical carcinoma.
1. Introduction
Adrenocortical carcinoma (ACC) is a kind of malignant tumor with low incidence but poor prognosis. ACC in most patients has already metastasized when the disease is di- agnosed, and the risk of recurrence is very common after standard systemic therapies [1]. Currently, the most op- timal strategy for adrenocortical cancer is surgery, but most patients have lost the opportunity for surgical treatment by the time they are diagnosed, and the cancer is often in- sensitive to radiotherapy and chemotherapy. Drug therapy has emerged as an effective therapeutic strategy. However, it still remained challenge due to the lack of effective therapeutic target. Therefore, to explore new therapeutic
target for treatment of adrenocortical carcinoma is urgently needed. Extracted from human ACC tissue, the SW-13 cell line has been used for most of the basic research on an- ticancer drugs and cancer cell signaling pathways. The NCI-H295 R cell line, obtained from human ACC patient, has secretion function and is widely used in the study of ACC [2].
Endoplasmic reticulum (ER) stress is the main mechanism of apoptosis. The influencing balance of ER results in the production of unfolded proteins. Unfolded protein response (UPR) attempts to maintain ER homeostasis by activating a series of signaling pathways when unfolded proteins accu- mulated [3]. The three main signal pathways for maintaining ER homeostasis are PERK, ATF6 (activated transcription factor
6), and IRE1 (inositol-requiring enzyme 1). Of these three signaling mechanisms, the PERK signal pathway is activated first. The PERK/eIF2x/ATF4 signaling pathway is one of the most crucial pathways for the survival of cells exposed to a variety of stressors, such as toxic environments, malnu- trition, and oxidative stress [4]. Active phosphorylation of eukaryotic translation initiation factor 2 (eIF2x) blocks the downstream protein translation of activating transcription factor 4 (ATF4). The active pathway involves the prevention of protein phosphatase 1 (PP1) to dephosphorylate p-eif2x [5]. CCAAT enhancer-binding protein homologous protein (CHOP) is the main downstream effector of the PERK pathway, and it functions as a proapoptotic factor [6].
Salubrinal (Sal), a selective activator of eIF2a, can en- hance the phosphorylation of eIF2a, which leads to the expression of ATF4 [7]. Sal is also an inhibitor of the GADD34:PP1 holoenzyme complex which protects the neuronal cell through inhibition of eIF2alpha dephos- phorylation and interrupts the other degenerative pathways [8]. Doxorubicin induces cholangiocarcinoma cell death through initiating ROS production and DNA damage, which could be aggravated by eIF2a inhibitor Sal [9]. However, whether the PERK signaling pathway is involved in Sal- induced apoptosis of ACC cells is still unclear.
Here, the role of Sal on the proliferation, migration, and apoptosis of ACC cells was explored. And whether Sal-ac- tivated PERK/eIF2a/ATF4 signaling pathway can inhibit the proliferation of ACC cells and promote the apoptosis of ACC cells was also investigated.
2. Materials and Methods
2.1. Reagents and Antibodies. Sal was purchased from Sigma Chemical Company (USA). Fetal bovine serum (FBS) was maintained from HyClone (USA). MTT and JC-1 were ob- tained from Beyotime (Shanghai, China). Antibodies against PERK, eIF2a, ATF4, Bcl-2, p-PERK, and p-eIF2x were ob- tained from Cell Signaling Technology (USA). Fluo-3/AM was purchased from Sigma Chemical Company (USA).
2.2. Cell Cultures. The human ACC cell lines (SW-13 and NCI-H295 R) were obtained from Shanghai Institute of Life Sciences (Shanghai, China). SW-13 cells were cultured in DMEM with 10% FBS and supplemented with 1 mm glu- tamine and 1% penicillin/streptomycin (Beyotime, China). NCI-H295 R cells were cultured in DMEM/F12 (Sigma- Aldrich, Japan) with 5% FBS and 1% penicillin/strepto- mycin. The NCI-H295 R cell medium also included 0.1% ITS (BD Biosciences, USA) as described previously [2]. All cells were cultured at 37℃ and 5% CO2.
2.3. Cell Viability Assay. Cells were cultured with different concentrations (0, 50, 100, 150, and 200 um) of Sal for 12 h, 24h, 36 h, and 48 h, respectively. The viability of the SW-13 and NCI-H295 R cells was determined by MTT according to the manufacturer’s instructions as previously reported [10]. The same experiment was replicated three times.
2.4. Analysis of Cell Apoptosis. Cells were treated with 100 uM of Sal for 24 h. Then, the cells were harvested and apoptotic cells were identified by flow cytometry following the manufacturer’s instructions. The assessment was conducted for three times. The cell apoptosis rate was calculated using Cell Quest™ 3.0 software (BD, USA) as previously reported [11].
2.5. JC-1 Fluorescence Staining. The mitochondrial mem- brane potential was determined by the fluorescence probe, JC- 1, a detection indicator in the early stage of apoptosis. Cells were treated with 100 um of Sal for 24h. Then, the culture medium was removed, and the JC-1 dye was added to the working solution. The mitochondrial membrane potential of the cells was detected according to the manual of dye kit [12]. Images of the cells were taken using an inverted phase-contrast microscope (Nikon, Japan). The ratio of apoptotic cells to total cells in the visual field was calculated as the apoptotic rate.
2.6. Cell Migration Assay. Cell migration is a method to determine the ability of [13]. For the migration assay, SW-13 and NCI-H295 R cells were seeded at a density of 3 x 105 cells/ well in the plates and cultured with only conventional medium or with medium containing 100 um of Sal for 24 h. When cell confluence reached to approximately 90%, a scratch was made using a 200 µI micropipette tip. The migration area was ob- served at 0h, 24h, and 48 h, and photos were obtained using optical microscopy (Leica, Germany). The wound area was analyzed with ImageJ software.
2.7. Western Blot Analysis. Experimental cells were treated with 100 um of Sal for 24h. The cells were harvested and lysed by RIPA buffer (Beyotime, China) for one-half hour. Fifty micrograms of total extracted cell protein was added to each gel lane for SDS-PAGE and then transferred to a PVDF membrane and blocked with 5% nonfat milk for 1 h which had been dissolved in TBST at RT as previously described [14]. The membrane was incubated overnight at 4℃ with primary antibodies against PERK, p-PERK, eIF2a, p-eIF2a, ATF4, and Bcl-2 (all 1:1000 dilution). The next day, the membranes were incubated with secondary antibodies and visualized with a LI-COR platform (Odyssey, USA). GAPDH (CST, USA) was used as internal control. Image] software was used to measure the gray values of the proteins.
2.8. Statistical Analysis. Data are presented as the mean- ± standard deviation (SD). Graphs were generated by GraphPad Prism 6.0. All statistical analyses were performed using SPSS 22.0. Differences between two groups were de- termined by unpaired Student’s t-tests. * p<0.05; ** p <0.01; and *** p < 0.001 indicate significance.
3. Results
3.1. Cell Line Characterization. NCI-H295 R cells were spindle in shape and showed characteristics of hyper chromatic nuclei (Figure 1(a)), while SW-13 cells were
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round in shape and with hyper chromatic nuclei (Figure 1(b)).
3.2. Sal Reduced the Cell Viability of ACC Cells. Cells treated with different concentrations of Sal (0 um, 50 um, 100 um, 150 um, and 200 um) for different durations (12 h, 24 h, 36 h, and 48 h) were measured with MTT assays. As shown in Figures 1(c) and 1(d), 50 um, 100 um, 150 um, and 200 um of Sal significantly decreased the cell viability of the ACC cells treated with Sal for 24h, 36 h, and 48 h (p<0.05, compared to control). The one-half maximal inhibition concentration of Sal in the NCI-H295 R and SW-13 cells were 104.177 um and 102.341 um, respectively. Sal significantly decreased the cell viability of the ACC cells when the cells were treated with Sal for 24h (the viability of SW-13 was 52.34±7.03% in 100 um of Sal intervention group and 92.65 ± 14.27% in the control group; the viability of H295 R was 51.06 ± 6.45% in the 100 um Sal intervention group and 94.13 ± 13.79% in control group) (p <0.05 compared to control). Sal has no significant effect on ACC cells at low does (<75 um) (Figure S1). Therefore, we used Sal at a concentration of 100 um in subsequent investigations.
3.3. Sal Inhibits the Migration of ACC Cells. To explore the role of Sal in migration of ACC cells, wound healing assay was carried out. It demonstrated that incubation with
100 um of Sal for 24h significantly inhibited the migration capacity of the ACC cells (SW-13 and H295 R), while inverse results were observed in a routine culture of ACC cells (Figures 2(a) and 2(b)). Sal significantly decreased the cell migration of the ACC cells when the cells were treated with Sal for 24h (the migration of SW-13 is 40.17 ±6.82% in 100 um of Sal intervention group and 97.24 ± 15.45% in control group; the migration of H295 R is 47.39 ±6.18% in 100 um Sal intervention group and 96.72 ± 17.58% in control group) (p<0.001 compared to control) (Figures 2(c) and 2(d)).
3.4. Sal Induces Apoptosis of Human ACC Cell. To investigate whether the ACC cells underwent apoptosis in the treatment of Sal, flow cytometry was performed to detect the apoptosis rate, WB was used to detect the expression of the apoptosis- related protein Bcl-2, and JC-1 staining was used to detect changes in the in-mitochondrial membrane potential of the cells to confirm the occurrence of apoptosis. As shown in Figures 3(a) and 3(c), the results of JC-1 staining revealed that the color of the cells staining changed from red to green, indicating changes to the mitochondrial membrane po- tential. Therefore, the cells underwent apoptosis, and the apoptosis rate of SW-13 is 12.31 ±7.81% (0h) and 62.45 ± 6.52% (24h) in 100 um of Sal intervention group and 6.37 ± 2.45% (0h) and 14.57 ± 4.66% (24h) in control group;
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the apoptosis of H295 R is 14.96±5.13% (0h) and 78.32 ± 12.76% (24h) in 100 um of Sal intervention group and 9.46 ± 3.14% (0h) and 15.89 ±5.24% (24h) in control group. Significant differences were detected in apoptosis rates in the Sal intervention group and control group after 24h (p<0.001) (Figures 3(b) and 3(d)).
The rate of apoptosis in the Sal-treated cells was further evaluated by flow cytometry. As shown in Figure 4, the ap- optosis rate of the cells in the Sal (100 um) treatment group for 24 h was significantly higher than that of the nonintervention group (the apoptosis rate in SW-13 is 45.67 ± 6.48% in 100 um of Sal intervention group and 5.78 ± 1.74% in control group; the apoptosis rate in H295 R is 43.49 ±7.44% in 100 um Sal intervention group and 4.82 + 1.03% in control group).
Bcl-2 family proteins are associated with the process of apoptosis, and the change in Bcl-2 protein level indicates the occurrence of apoptosis. Therefore, to compare the incidence of apoptosis in the two groups, the change in the Bcl-2 protein level was detected. The results showed that the Bcl-2 protein in the Sal (100 um) treatment group was significantly inhibited, indicating that the rate of apoptosis was significantly increased due to Sal (100 um) treatment as compared with the control group (Figures 5(a)-5(c)).
3.5. Sal Induces ACC Cell Apoptosis via the PERK Pathway. To analyze whether the PERK/eIF2« signaling pathway was involved in the mechanism of Sal-induced ACC cell apoptosis, the protein levels of p-PERK, p-eIF2a, and ATF4 were mea- sured via WB. At a concentration of 100 um, Sal markedly increased the protein expression levels of p-PERK, p-eIF2a, and ATF4 in the two lineages of ACC cells (p < 0.05) (Figures 5(a)- 5(c)).
4. Discussion
In this study, we investigated the role of Sal in the regulation of the proliferation, invasion, migration, and apoptosis of
ACC cells through regulating the PERK/eIF2« signaling pathway. The results indicated that Sal inhibited the pro- liferation, invasion, and migration and induced the apo- ptosis of ACC cells by upregulating the PERK/eIF2x signaling pathway.
It is generally accepted that changes in the cellular en- vironment, such as the induction of oxidative stress, cyto- toxicity, and tumorigenesis, can lead to cell metabolism disorders, as indicated by increases in endoplasmic reticu- lum (ER) stress and unfolded protein response (UPR). UPR either promote stability of the intracellular environment or initiate a cell death process [15]. In our previous studies, the ER was found to play an important role in apoptosis of ACC cells [16]. The UPR is based on three important ER stress- related proteins: PERK, IRE1, and ATF6.
PERK is the most important sensor for ER stress. When ER stress occurs, it activates ATF4 and protein translation to enable cells to adapt to the new environment and undergo self-repair. PERK signaling is based on activating phos- phorylated PERK to activate the downstream translation promoter eIF2a, which blocks most protein translation, including ATF4.
Under ER stress, eIF2a is phosphorylated by eIF2 kinases and inhibits protein translation. In addition, p-eIF2« in- duces the stress response by activating signaling downstream of ATF4 [17]. Of the four proteins (PERK, GCN, PKR, and HRI) known to affect eIF2a, PERK has the greatest acti- vation effect [18]. The eIF2a-ATF4 pathway not only maintains the stability of the intracellular redox environ- ment but also regulates cellular metabolism and nutrient uptake [4, 19]. This pathway is also important for the ad- aptation of tumor cells in a hypoxic microenvironment and contributes to tumor growth [20] and the development of resistance to chemotherapy drugs [21-23].
Sal, a selective activator of eIF2a, can prevent eIF2x dephosphorylation by inhibiting the formation of protein complex GADD34/PP [24, 25]. It has been reported that
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Sal showed anticancer properties in cancer cells by ma- nipulating the endoplasmic reticulum stress pathway or through promoting DNA damage [9, 26]. It also dem- onstrated that it could offer neuroprotection through UPR-related signaling factors along with other bio- chemical events [8]. The concentrations of Sal utilized in different organs or tissues showed varying difference [27-29]. In the present study, we have observed that the viability of the ACC cells was inhibited by Sal at the given dose. Sal had no significant effect on ACC cells at low does (<75 um) (Figure S1). The major reason is supposed to be that the concentration of Sal action in different organ cells is different [30].
Bcl-2 family proteins are important in regulating apo- ptosis signaling pathways [31]. Overexpression of Bcl-2 makes cells resistant to apoptosis, a phenomenon found in breast cancer and many other cancers [32]. Our results show that Bcl-2 was decreased by Sal in adrenocortical carcinoma
cells (Figure 5), a finding that is consistent with previous research.
Sal inhibited the proliferation (Figure 1) and migration (Figure 2) of SW-13 and NCI-H295 R cells. The migration of H295 R cells is different from SW13 since the growth of H295 R cells is Spindle shaped and foot shaped. Further- more, Sal promoted SW-13 and NCI-H295 R cell apoptosis (Figures 3-5). Yu et al. [9] revealed that Sal can promote ROS-mediated DNA damage to enhance the sensitivity of cells to doxorubicin to induce apoptosis in chol- angiocarcinoma cells; similar results were obtained in this study. Some studies have shown that Sal can protect cells from the endoplasmic reticulum (ER) stress-induced apo- ptosis [24], which is somewhat inconsistent with the results of this study. One possible reason for this finding is that Sal added at low concentrations can have a protective effect on cells, while Sal added at high concentrations promotes apoptosis.
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Cells protrude through the front end, locally form focal spots, surrounding hydrolyzed fibers, cell body contraction, and rear end separation to complete a migration cycle. Formation of cell protrusion (also known as pseudopod) is an essential step for cell migration. The formation of pro- trusion mainly depends on the flow of actin or the poly- merization of actin fibers, the contraction of the cytoskeleton, and increased concentration of calcium ions and Rho GTPase. Focal adhesion between cells induces movement and cell advancement by transmitting sliding motion of actin stress fiber bundles, thereby exerting signal and mechanical conduction functions [33-35]. Podosomes are other structures which play an important role in cell migration while Ca2+ signaling is essential in the assembly of podosomes. Our results show that Sal diminished cell mi- gration in ACC cells (Figure 2). Indicating Sal may inhibit cell migration through increased cell apoptosis.
The present study revealed that Sal could inhibit the pro- liferation of ACC cells by inducing apoptosis. Additionally, Sal suppresses the expression of apoptosis-related protein Bcl-2 through activated PERK/eIF2a/ATF4 signaling pathway.
5. Conclusion
In summary, the sensitivity of ACC cells to Sal is related to the regulation of apoptosis by activating the PERK/eIF2a/ ATF4 signaling pathway.
Abbreviations
Sal: Salubrinal
PERK: Protein-kinase-R- (PKR-) like endoplasmic reticulum kinase
eIF2a: ATF4: Activated transcription factor 4
Eukaryotic translation initiation factor 2
ACC: Adrenocortical carcinomas
ER: Endoplasmic reticulum
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
JC-1: Mitochondrial membrane potential assay kit with JC-1
CLSM: Confocal laser scanning microscope.
Data Availability
The data are available upon request (data contact details: wuliligxmu@126.com).
Conflicts of Interest
The authors report they have no conflicts of interest related to this work.
Authors’ Contributions
L. W. and Z. L. substantially contributed to the conception and design. C. L. and J. J. performed the experiments. X. H. and X. D. contributed to the acquisition and analysis of the data. All the authors agreed and approved the final version of this manuscript for publication.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (grant numbers: 81660138 and
81860146); Innovation Project of Guangxi Graduate Edu- cation (grant number: YCBZ2018037); Guangxi Autono- mous Region Administration of Traditional Chinese Medicine Self-Financing Project (grant number: GZZC2019136); and Guangxi Medical and Health Self-Fi- nancing Project (grant number: Z20180636).
Supplementary Materials
Figure S1. flow cytometry was used to explore the apoptosis rate of the ACC cells induced by low concentrations of Sal. The apoptosis rate of SW-13 and H295 R cells was detected after being incubated with 0 um, 5 um, 10 um, 15 um, 20 um, 25 um, 50 um, or 75 um Sal for 24h. (Supplementary Materials)
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