{'fraction_non_alphanumeric': 0.03649557709624451, 'fraction_numerical': 0.021717251973091795, 'mean_word_length': 4.821769966080789, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Thapsigargin induces apoptosis in adrenocortical carcinoma by activating endoplasmic reticulum stress and the JNK signaling pathway: an in vitro and in vivo study

Objective Thapsigargin (TG) is a natural product that exists in most parts of the plant Thapsia garganica L. and possesses potential anticancer activities against variety tumor cell lines. TG induces endoplasmic reticulum (ER) stress and apoptosis by inhibiting cancer growth. However, the antineoplastic effect of TG in human adrenocortical carcinoma (ACC) cells is still unknown. Methods In this study, two human ACC cell lines including SW-13 and NCI-H295R were employed to explore the potential role of TG in ACC. A mouse xenograft model of SW-13 cells was established to verify the role of TG in vivo. The cell viability was tested using Cell Counting Kit-8 and Transwell assays. Flow cytometry and Hoechst 33,258 staining were employed to analyze cell apoptosis. RT-qPCR and Western blot (WB) were performed to explore the underlying mechanism of TG-induced apoptosis in ACC cells. Results The results indicated that TG dose-dependently inhibited proliferation, migration and invasion in human ACC cells. TG significantly increased the mitochondrial rate of apoptosis and ER stress activity in ACC cells and suppressed ACC xenograft growth in vivo. In addition, the expression of Jun N-terminal kinase (JNK) signaling-related genes and proteins was upregulated by the treatment with TG. Conclusion Our findings suggest that TG inhibits the viability of ACC cells by inducing apoptosis through the activation of JNK signaling. Thus, TG is expected to be a potential candidate for the treatment of ACC.


Introduction
Adrenocortical carcinoma (ACC) is a rare endocrine tumor with a poor prognosis. The occurrence rate of ACC ranges from approximately 1 to 2 cases per million persons per year. 1 Among early stage cases, after complete surgical resection, the 5year survival rate of ACC patients is 32% to 50%, but the risk of recurrence after surgery is as high as 50% to 85%. [2][3][4] Late stage ACC patients who present with metastatic disease have a 5-year survival rate of 15% 5 and a median survival of less than 1 year. 6 The most commonly associated endocrine disorder is Cushing's syndrome; 7 however, other syndromes, including Lynch syndrome, multiple endocrine neoplasia type 1, Beckwith-Wiedemann syndrome, and other tumor-related syndromes, are also related to ACC. 1 Mitotane (o, p'dichlorodiphenyldichloroethane, o, p'-DDD) is the most commonly used drug; nevertheless, its efficacy is limited by its narrow therapeutic index and toxicity. 8 Other systemic therapies, such as radiotherapy and chemotherapy, are also restricted by their limited efficacy. 9 Therefore, new therapeutic approaches for ACC are urgently needed. The SW-13 cell line was extracted from human ACC 10 and is currently used in anticancer drug and signaling research experiments. The H295R cell line, which also originated from human ACC, exhibits a secretion function and has been established as an appropriate model for determining the molecular mechanisms regulating ACC. 11 Thapsigargin (TG) has recently emerged as an outstanding anticancer lead compound, and prodrug derivatives of TG, such as mipsagargin (G-202), are currently in clinical trials for the treatment of multiple cancers, including hepatocellular carcinoma and prostate cancer, neovascular tissues and a wide range of other cancer cells. 12 TG is an irreversible paralyzer of the important Sarco-/ Endoplasmic Reticulum Calcium ATPase (SERCA), which blocks the reabsorption of calcium from the cytosol to the Endoplasmic Reticulum (ER). 13 This important step leads to an increase in the cytosolic calcium concentration and ultimately results in cell apoptosis. 14 The ER, which is an important organelle in cells, participates in the synthesis, folding, trafficking and secretion of a variety of chaperone proteins. However, in the presence of internal environmental disturbance in the body, the calcium ion steady state is destroyed, proteins unfold and misfold, and external stimuli in the ER could result in ER stress (ERS), therefore leading to cell apoptosis. 15,16 Serious ERS can ultimately induce cell apoptosis.
To alleviate the progression of cell apoptosis, the unfold-protein reaction (UPR) 17 is subsequently activated by stimulating three transcription factors, including inositol requiring 1 (IRE1/ERN1), activating transcription factor 4 (ATF4), 18 and PEK, such as ER kinase (PERK/PEK). PERK is a specific protein kinase that controls protein translation and mediates the terminal UPR through the regulation of ATF4, which is a vital element in the promotion of apoptosis in response to ERS and is accompanied by the upregulation of C/EBP homologous protein (CHOP). 15,19,20 Jun N-terminal kinase (JNK) is a major stress response factor and an important regulator of cell growth and death in many Drosophila tissues. [21][22][23] JNK is a key stress reaction factor and an important modulator of cell growth and death similar to mahj, 24 myc, 25 APC 6 and Minute competition 26 and has been proposed to play a role in the late stage induction of cell death. Studies have demonstrated that mitogen-activated protein kinases (MAPKs) are crucial regulators of cell migration and transformation in cancer. Within the MAPK family, extracellular signal-regulated kinase (ERK), JNK and p38 kinases are the most widely studied. However, the relationship between JNK signaling and ACC development remains unclear.
In the present study, we explored the effect of TG on ACC in vitro and in vivo. Our findings reveal that TG dose-dependently suppresses proliferation and motility in ACC cell lines in vitro and inhibits tumor growth in vivo. TG activates ERS and apoptosis. Moreover, the JNK signaling pathway is the potential mechanism underlying TGinduced apoptosis, which was confirmed by the fact that ER-related factors significantly decreased apoptosis after TG treatment in vitro and in vivo.

Cell morphological analysis
The cell morphology was analyzed by using Crystal Violet Staining Solution (Leagene Biotechnology, China). The images were obtained under an upright microscope (Olympus BX53; Japan).

Migration and invasion assays
The SW13 and NCI-H295R cells were treated with 2, 4 or 8 mM TG. The invasive captivity and motility of the cells were detected using a Transwell assay. The SW13 and NCI-H295R cells were serum starved overnight, and the cells were loaded onto the upper chambers (Corning Incorporated, USA) at a concentration of 2×10 5 cells per well. Then, 500 µL DMEM were added to the lower chambers. The upper chambers were loaded with Matrigel matrix (Corning Incorporated, USA) before the addition of the cells for the invasion analysis. After culturing for 24 h, the numbers of cells were counted and captured under an inverted microscope (Nikon Corporation, Japan).

Apoptosis assay
The SW-13 and NCI-H295R cells were cultured with different concentrations of TG or JNK inhibitor SP600125 (AbMole, USA) for 48 h, followed by harvesting and suspending in 1× Binding Buffer; the cells were incubated with 5 µL Annexin V-FITC and 5 µL PI (BD Biosciences, USA). The apoptotic SW-13 and NCI-H295R cells were analyzed by BD FACS Verse (BD Biosciences, USA). The apoptotic cells were also analyzed by Hoechst 33,258 staining. Briefly, 48 h after incubation, the SW-13 and NCI-H295R cells were immobilized with 70% ethanol for 20 min; then, the 70% ethanol was removed, and Hoechst 33,258 (Beyotime, China, 500 µL/well) was added for 10 min. The pictures were captured under a fluorescence microscope (Nikon Corporation, Japan).

Real-time quantitative polymerase chain reaction analysis
The RNA purity was detected after extracting the total RNA from ACC cells treated with TG by RNAiso Plus Reagent (TaKaRa, Shiga, Japan). Oligo dT primer and AMV reverse transcriptase were used to synthesize cDNA. The RT-PCR reactions were performed using a SYBR Green (TaKaRa, Japan) assay on an ABI 7500 (Applied Biosystems, USA). The primers used are shown in Table 1. The fold changes of each gene were calculated using the 2 −ΔΔCt method. GAPDH was used as an internal reference.

Western blot analysis
The cells were cultured with TG at concentrations of 0, 2, 4, or 8 µM for 48 h, and nude mouse xenograft tissues were obtained after treatment for TG for 15 days. The protein was separated by SDS-PAGE (Bio-Rad, USA) and then transferred onto a PVDF membrane (Millipore, USA), which was incubated with 5% nonfat dry milk-TBST. The membranes were probed with primary antibodies (anti-JNK, anti-p-JNK, anti-ERK, anti-p-ERK, anti-AMPK, anti-p-AMPK, anti-PERK, anti-p-PERK, IRE1 and anti-GRP78, Cell Signaling Technology, USA). Then, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (Signalway Antibody, China) for 1 h, followed by visualization using LI-COR (Odyssey, USA) according to the manufacturer's instructions. An anti-GAPDH antibody (Signalway Antibody, China) was used as an internal control.

Nude mouse xenograft assay
Male BALB/c nude mice were obtained from the Chang Zhou Cavens Laboratory Animal Center. The animal protocol was approved by the Animal Ethics and Welfare Committee of Guangxi Medical University (201901007), and followed the Guiding Opinions on the Treatment of Laboratory Animals issued by the Ministry of Science and  Once the subcutaneous tumors were approximately 0.2×0.2 cm 2 in size, the mice were intraperitoneally injected with vehicle alone (0.5% methylcellulose) or TG (1 mg/kg) daily. We recorded the body weights and the tumors of the mice. The tumor volume (V) was calculated as follows: V =π/6 (1/2(A + B)). 3 The inhibition rate (IR) was calculated as follows: IR =1-mean tumor weight of the experimental group/mean tumor weight of the control group×100%. 27

Statistical analysis
Graphs were generated using GraphPad Prism 6.0. The results are expressed as the mean ± SD and were analyzed with SPSS 22.0 software. Student's t-test and one-way ANOVA were selected to analyze the differences among the groups. p<0.05 was considered statistically significant.

Cell lines identification
The identification for cell lines was analyzed using a crystal violet staining solution. As shown in Figure 1A and B, the typical morphological characteristics of the ACC cell lines SW-13 and NCI-H295R could be clearly observed.

TG inhibits the viability of human ACC cells
The CCK-8 results showed that the cell viability was decreased in both SW-13 and NCI-H295R following culture with TG at concentrations higher than 1 µM ( Figure 1C and D). The half maximal inhibition concentration of TG in the SW-13 and NCI-H295R cell lines was 150 SW-13 NCl-H295R

TG suppresses the migration and invasion ability of ACC cells
After incubation with different concentrations of TG (0, 2, 4, or 8 µM), the SW-13 and NCI-H295R cells were observed. As shown in Figure 2A and B, the migration and invasion abilities were dose-dependently inhibited by TG. The cells in both chambers were counted. TG, particularly at 8 µM, suppressed the motility of the ACC cells ( Figure 2C and D).

TG induces mitochondrial apoptosis in ACC cells
To determine whether TG inhibits cell viability by leading to cell apoptosis in ACC cell lines, the rate of apoptosis in the TG-treated cells was evaluated by flow cytometry. As shown in Figure 3A-C, after incubation with various concentrations of TG (0, 0.5, 1, 2, 4, 8, 10, or 16 µM) for 48 h, the apoptosis rate of the cells increased as the concentration of TG increased. These results suggest that the treatment with TG resulted in apoptosis in the ACC cells in vitro, which is consistent with the results of the CCK-8 assay showing that TG suppressed the viability of ACC cells ( Figure 1C and D).

TG inhibits cell viability and induces cell apoptosis by activating JNK signaling
To further clarify the molecular mechanism of the antiviability and apoptosis-inducing effects of TG in ACC cells, the apoptosis-related JNK signaling pathway and endoplasmic reticulum (ER) stress markers were investigated after the TG treatment. The expression levels of JNK signaling-related genes, including JNK, ATF6, PERK, LC3B, and HSAP, were obviously promoted by TG  ( Figure 4C and D). TG at a concentration of 8 µM exhibited the best performance with respect to the upregulation of the expression levels of the JNK, ATF6, PERK, LC3B and HSAP genes. The apoptosis-associated gene Bcl-2 was also detected by qPCR. Bcl-2 was increased in the TG8 groups compared with that in the TG0 group (P<0.05) ( Figure 4C and D). The expression of the JNK and p-JNK proteins was also significantly upregulated by TG ( Figure 5A-D).

TG induces cell apoptosis by activating JNK signaling
To further verify the molecular mechanism of JNK signaling in the TG-mediated ER stress-induced apoptosis in ACC cells, the JNK inhibitor SP600125 were used. The rate of apoptosis in the TG-treated cells with JNK inhibitor SP600125 was evaluated by flow cytometry. As shown in Figure 6A-E, the apoptosis rate of the cells and the expression of JNK had not significant difference in the treatment of SP600125. Suggesting the JNK signaling is the main molecular mechanism in the TG-mediated ER stress-induced apoptosis.

TG inhibited ACC tumor growth in nude mice
To investigate the effects of TG on ACC in vivo, SW-13 xenograft tumors were generated in nude mice. As shown in Figure 7A-F, the treatment with TG suppressed the growth of the SW-13 xenograft tumors in both the tumor volumes and weights. Furthermore, the mouse body weights in the TG group and control group were similar, suggesting that TG did not cause serious toxicity in the mice ( Figure 7D).

JNK signaling in ACC xenografts in nude mice treated with TG
The activation of JNK pathways plays a central role in the proliferation and apoptosis of cancer cells. We investigated whether JNK, ERK1/2, MAPK, PERK, IRE1 and GRP78 signaling pathway modulation accounted for the antitumor activity of TG. As shown in Figure 8, after 14 days of treatment with TG, increases in GRP78, IRE1 and the phosphorylation levels of JNK, ERK, PERK and MAPK were detected in the ACC xenograft tumors, whereas the total JNK, ERK, PERK and MAPK protein levels remained unchanged. These results suggest that the inhibition of tumors by TG is mediated through JNK, ERK and MAPK signaling pathway inactivation in ACC in vivo.

Discussion
ACC is rare, and typically, the tumor is characterized by invasive malignancy with a poor prognosis. To date, surgical treatment of the primary site has remained the mainstay of therapy, but the prognosis is poor. Recent studies investigating the treatment of ACC have mainly focused on exploring effective therapeutic agents. Thapsigargin (TG) is a natural product that has been reported to inhibit the function of the transmembrane portion of the sarcoplasmic/ER calcium adenosine triphosphatase pump, subsequently inducing apoptosis. 28,29 TG initiates ER stress via the UPR, which is initiated by 3 ER transmembrane proteins, namely, protein ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and ATF6. 30,31 TG has been considered a promising approach for the treatment of various cancers, such as breast cancer, pancreatic adenocarcinoma and melanoma. 30,32 Our results show that TG decreases the proliferation (Figure 1), migration and invasion (Figure 2) of SW-13 and NCI-H295R. TG also inhibited ACC xenograft tumor growth in vivo (Figure 7). In addition, TG promoted apoptosis in ACC cells (Figures 3, 4A and B). Wang et al 33 revealed that TG can induce apoptosis in human lung adenocarcinoma cells through cofilin-1 and paxillin, which is consistent with our present study.  A and B). The data is represented as means ± SD (n=5). *p<0.05 vs the control group.
The detailed mechanism studies revealed that TG promoted JNK signaling and ERS, thus promoting the downstream genes of pro-apoptosis regulators. JNK signaling-related markers, such as JNK, ATF6, PERK, LC3B, HSAP and Bcl-2, were also enhanced by TG ( Figure 4B). TG significantly increased the JNK and p-JNK protein levels in vitro ( Figure 5) and the JNK, p-JNK, ERK, p-ERK, MAPK, p-MAPK, PERK, p-PERK, IRE and GRP78 protein levels in vivo (Figure 8). When treated with JNK inhibitor SP600125, the apoptosis rate of the ACC cells had not significant difference ( Figure 6). It has been reported that apoptosis mediated by the ERS pathway plays a key role in many diseases. [34][35][36] To date, three important apoptotic pathways related to ER stress have been reported. First, the gene CHOP (C/EBP homologous protein)/GADD153, which regulates cellular bioenergetics and cell-death, is obviously induced by ER stress and plays an important role in ER stress-induced apoptosis. 37 The second pathway is the JNK pathway. 38 The third pathway involves caspase-12. 39,40 Mutations occurring in the JNK signaling pathway (eg, MAP3K1, MAP2K4, and MAP2K7) are implicated in the pathology of breast cancer. 41 Hiroaki Konishi et al 42 revealed that apoptosis is mediated by JNK activation. JNK, MAPK and P38 are famously known as stress-activated protein kinases because they can be activated by multiple stress stimuli. In addition, the activation of JNK, MAPK and p38 plays an important role in natural compound-induced apoptosis. It has been reported that many anticancer compounds activate MAPK signaling, ultimately leading to cancer cell apoptosis. 43 Consistent with these reports, TG can increase the phosphorylation levels of JNK, MAPK and ERK. Furthermore, the levels of the ERSrelated protein GRP78 were increased after the treatment with TG. These results suggest that TG regulates apoptosis in ACC by activating the JNK/MAPK/ERK signaling pathway.
In conclusion, this study indicates that TG could play a significant role in the treatment of ACC. Our research demonstrated that TG repressed proliferation and invasion in human ACC cells in vitro and inhibited ACC xenograft growth in vivo. Moreover, TG induced mitochondrial apoptosis and triggered the synergistic cytotoxicity of ERS and apoptosis, potentially through JNK signaling, to treat ACC.

Dovepress
Publish your work in this journal Drug Design, Development and Therapy is an international, peerreviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. Clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are a feature of the journal, which has also been accepted for indexing on PubMed Central. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www. dovepress.com/testimonials.php to read real quotes from published authors.