Triptolide suppresses growth and hormone secretion in murine pituitary corticotroph tumor cells via NF-kappaB signaling pathway

Biomedicine & Pharmacotherapy 95 (2017) 771–779

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Original article

Triptolide suppresses growth and hormone secretion in murine pituitary corticotroph tumor cells via NF-kappaB signaling pathway

MARK

Ran Lia, Zhuo Zhanga, Junwen Wanga, Yiming Huanga, Wei Suna, Ruifan Xiea,b, Feng Hua, ⁎ Ting Leia, a Department of Neurosurgery, Sino-German Neuro-Oncology Molecular Laboratory, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Avenue 1095, Wuhan 430030, China b Research Group Experimental Neurooncology, Clinical Cooperation Unit Neurooncology, German Cancer Research Center (DKFZ), Heidelberg 69120, Germany

A R T I C L E I N F O

A B S T R A C T

Keywords: Triptolide Cushing’s disease Apoptosis NF-κB ACTH

Triptolide is a principal diterpene triepoxide from the Chinese medical plant Tripterygium wilfordii Hook. f., whose extracts have been utilized in dealing with diverse diseases in traditional Chinese medicine for centuries. Recently, the antitumor effect of triptolide has been found in several pre-clinical neoplasm models, but its effect on pituitary corticotroph adenomas has not been investigated so far. In this study, we are aiming to figure out the antitumor effect of triptolide and address the underlying molecular mechanism in AtT20 murine corticotroph cell line. Our results demonstrated that triptolide inhibited cell viability and colony number of AtT20 cells in a dose- and time-dependent pattern. Triptolide also suppressed proopiomelanocortin (Pomc) mRNA expression and extracellular adrenocorticotropic hormone (ACTH) secretion in AtT20 cells. Flow cytometry prompted that triptolide leaded to G2/M phase arrest, apoptosis program and mitochondrial membrane depolarization in AtT20 cells. Moreover, dose-dependent activation of caspase-3 and decreased Bcl2/Bax proportion were observed after triptolide treatment. By western blot analysis we found that triptolide impeded phosphorylation of NF-κB p65 subunit and extracellular signal-regulated kinase (ERK), along with reduction of cyclin D1, without any impact on other NF-κB related protein expression like total p65, p50, IκB-α, p-IκB-α. Furthermore, the mouse xenograft model revealed the inhibition of tumor growth and hormone secretion after triptolide administration. Altogether this compound might be a potential pharmaceutical choice in managing Cushing’s disease.

1. Introduction Cushing’s disease, characterized by a superfluous secretion of adrenocorticotropic hormone (ACTH) from the pituitary and chronic endogenous hypercortisolism, results from the lack of feedback regulation mechanism of the hypothalamic-pituitary-adrenal axis and leads to numerous morbidities such as central fatness, diabetes mellitus, hypertension, disturbed mood, predisposition to infection and osteoporosis [1,2]. It is defined as a severe and potentially fatal disease, accounting for 10–15% of pituitary tumors as well as 80% of endogenous Cushing’s syndrome [3]. In virtue of absence of standard reliable medical therapy and the high occurrence of long-term hypopituitarism caused by radiation therapy, the current first-line treatment for this disorder is recommended to receive transsphenoidal microsurgery with a high recurrent possibility to 20–25% in long-term follow-up [4–6]. Novel therapeutic approaches for Cushing’s disease are urgently needed in this challenging condition.

⁎

Corresponding author. E-mail address: [email protected] (T. Lei).

http://dx.doi.org/10.1016/j.biopha.2017.08.127 Received 27 July 2017; Received in revised form 24 August 2017; Accepted 29 August 2017 0753-3322/ © 2017 Published by Elsevier Masson SAS.

Tripterygium wilfordii Hook. f (TWHf), namely “Thunder God Vine”, was exploited to medicate immune-inflammatory disorders including rheumatoid arthritis, lupus erythematosus, chronic nephritis owning to its potent anti-infalmmatory and immunosuppressive features in traditional Chinese medicine [7,8]. Triptolide, a principal diterpene terpenoids isolated from this natural herb, has been uncovered to restrain tumor proliferation and motivate apoptosis in various malignant tumor forms both in vitro and in vivo models, such as pancreatic cancer, hepatocellular carcinoma, breast cancer, adrenal cancer, and neuroblastoma [9–15]. The underlying mechanisms of its antitumor effect have been reported to be related with reticence of SP-1 and HSP70 transcriptional activity, kindling TRAIL-induced death receptor mediated apoptosis pathway, blockage of NF-κB activity. NF-κB, a key transcription factor that consists of a heterodimer of p65 and p50 subunits, modulates through its transcriptional activation to cell growth, differentiation and survival in cellular responses to cytokines and growth factors [16,17]. Under physiological status, NF-κB

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activity is impeded by the interaction with inhibitory κB proteins (I-κB proteins) in the cytoplasm. Phosphorylation of I-κB provokes degradation of I-κB by the proteasome, and then NF-κB can translocate to the nucleus for regulating the expression of target genes, including apoptotic and anti-apoptotic genes like Bax and Bcl-2 [18,19]. Recently, several natural compounds such as curcumin and ursolic acid, have been found to have anti-tumorigenic and hormone-suppressive effects on both murine corticotroph cells and primary cultured cells from human ACTH-secreting pituitary adenoma by lowering constitutive NFκB activity, followed by a caspase-dependent apoptotic cell death [20,21]. Interestingly, inhibition of NF-κB activity also led to a distinct reduction of cellular viability in a well characterized rat pituitary adenoma cell line [22,23]. Triptolide has been proved to be a potent inhibitior of NF-κB pathway, leading to the down-regulation of its downstream genes involving in inflammatory response or cell growth [24,25]. Therefore, it’s reasonable to propose that triptolide can suppress growth and hormone secretion in corticotroph tumor cells by blocking NF-κB activation. To test this hypothesis, we investigated for the first time the effect of triptolide on Cushing disease by using the mouse pituitary corticotroph AtT20 cells in vitro and mouse xenograft model in vivo. We demonstrated the suppressive effect of triptolide on the gowth of AtT20 cells and xenograft model. Down-regulation of NF-κB by triptolide decreased expression of anti-apoptotic protein Bcl-2 and cyclin D1, resulting in caspase dependent mitochondrial apoptosis and cell cycling arrest respectively. In addition, triptolide also exerted inhibitory effect on Pomc gene transcription and ACTH secretion. This study provides as a novel prospective that triptolide could be ultilized as a therapeutic medication in the treatment of Cushing disease.

triptolide. Cells were maintained for two weeks, the medium was renewed twice per week. The colonies were visualized by crystal violet staining after fixation with 4% paraformaldehyde. 2.4. Evaluation of ACTH secretion and Pomc gene expression ACTH secretion was evaluated by detecting mouse ACTH immunoreactivity in the supernatants of AtT20 cells culture medium after indicated treatment for 24 h with commercial mouse ACTH ELISA kit (Uscn Life Science Inc., China) according to the manufacturer’s instruction. The results were quantified according to standard curve and normalized with the cell number measured by CCK8 assay. Total cell mRNAs were isolated with TRIzol reagent (Invitrogen, USA) following the manufacturer’s protocol. The ultraviolet (UV) spectrophotometry (Bio-Rad, USA) was used to determine the concentrations of total RNA. cDNA was obtained from reverse transcription of 1 μg of total RNA as template by ReverTra Ace-A kit (Toyobo, Japan) according to the manufacturer’s instruction. Pomc expression evaluation was performed by quantitative real-time PCR using SYBRGreen PCR Master Mix (Toyobo, Japan) following the manufacturer’s introduction. The primers were adopted as follows: Pomc forward, 5′AACCTGCTGGCTTGCATCCG-3′ and reverse, 5′-GGGCTGTTCATCTCCGTTG CCT-3′. β-actin, forward, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and reverse, 5′-TAAAACGCAGCTCAGTAACAGTCC-3′. PCR procedures were applied as follows: 50 °C for 2 min, 95 °C for 2 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 30 s, 72 °C for 45 s and 72 °C for 10 min. 2.5. Cell cycle determination by flow cytometry Propidium iodide (PI) (Beyotime Biotech., China) staining was performed to confirm the changes of DNA content in triptolide treated cells. After triptolide treatment at indicated concentrations (0, 50, 100 nM) for 96 h, AtT20 cells were collected after trypsinization and fixed in 70% cold ethanol at 4 °C overnight, and then incubated with 10 μg/μl PI and 100 μg/ml RNase A (Beyotime Biotech., China) for 1 h at room temperature in the dark. The stained cells were subjected to FACScaliber II sorter machine (BD Biosciences). Cell Quest Research Software (BD Biosciences) was operated for further statistical analysis.

2. Materials and methods 2.1. Chemicals and reagents A commercial triptolide was purchased from Selleck chemicals (PG490, Catalog No. S3604). Briefly, the extract products from root of tripterygium wilfordii was concentrated, then absorbed by macroporous, followed by silica gel chromatography and preparation of liquid phase separation, finally purified. Triptolide was reconstituted in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to achieve a 1 mM stock solution, aliquots were frozen at −20 °C.

2.6. Cell apoptosis analysis and mitochondrial membrane depolarization assay by flow cytometry Triptolide-induced apoptosis of AtT20 cells was assessed by Annexin V-FITC/PI double staining. AtT20 cells were stimulated to indicated dose of triptolide for two days. The adherent and non-adherent cells of different groups were harvested after treatment and incubated with FITC-labeled Annexin V and PI as the manufacturer's protocols (KeyGEN Biotech, China), consequently cells were subjected to FACScaliber II sorter machine (BD Biosciences) and then analyzed as described above. To further determine the mitochondrial membrane potential collapse in triptolide treated AtT20 cells, JC-1 staining was used. Following the manufacturer’s procedure (Beyotime Biotech., China), cells were harvested and then stained using JC-1 dye under different demand, finally scrutinized by flow cytometry as previous description.

2.2. Cell line and cell culture The mouse ACTH-secreting pituitary adenoma cell line AtT-20 (AtT20/D16v-F2) was purchased from American Tissue and Culture Collection (ATCC, USA). AtT20 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, USA) containing 10% (V/ V) fetal bovine serum (FBS) (Hyclone, USA), 5 U/ml Penicillin, 5 mg/ ml Streptomycin under humidified atmosphere of 5% CO2 at 37 °C. 2.3. Cell viability and colony formation assay AtT20 cells were seeded into 96-well plates at a density of 5 × 103 cells with complete medium (10% FBS) per well. After 24 h, medium was changed with 2% FBS with the indicated concentrations of triptolide. Cell viability was assessed by Cell Counting Kit 8 (CCK8, Dojindo Laboratories, Kumamoto, Japan). CCK8 assay was performed following manufacturer recommendations. Spectrophotometer was used to quantify the optical density (OD) value at the wavelength of 450 nm. In addition, the effect of triptolide on AtT20 cell growth was also validated by automatic cell counting with an adapted Coulter counter. Furthermore, we employed colony formation assay to determine the growth suppresive effect of triptolide in vitro. AtT20 cells were seeded into 6-well plates (1000 cells/well). After 24 h, the medium was changed to fresh complete medium including the indicated dosage of

2.7. Caspase-3 activity analysis Caspase-3 activity assay kit (Beyotime Biotech., China) was performed to detect the caspase-3 activation in AtT20 cells exposure to triptolide. Following the manufacturer’s protocol, 10 μl protein (adjusted to same concentration), 10 μl Ac-DEVD-pNA and 80 μl detecting buffer were added in 96-well plate per well, and incubated for 2 h at room temperature in the dark. The OD value was measured by spectrophotometer at 405 nm wavelength. According to pNA standard curve, the caspase 3 activity in each sample was quantified combining with the concentration of each sample. 772

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Fig. 1. Triptolide inhibited the growth, viability and ACTH secretion of AtT20 cells. (A) AtT20 cells were treated with increasing doses of triptolide. Cell growth was determined by CCK8 assay after 96 h. The data were quantified as% of vehicle (0.01% DMSO). (B) AtT20 cells were treated with increasing doses of triptolide for 96 h. Cells were trypsinized and counted. The data were calculated as% of vehicle control. (C) AtT20 cells were treated with indicated doses of triptolide for 14–21 days. The colonies were visualized by crystal violet staining and quantified in 4 random fields each well. The average number of colonies were calculated and presented as% of vehicle control. (D) AtT20 cells were treated with 100 nM triptolide for 24 h, 48 h, 72 h and 96 h respectively. Cell growth was determined by CCK8 assay. The data were calculated as% of corresponding vehicle control. (E) Cells were treated with indicated doses of triptolide for 24 h. Effects of triptolide on Pomc mRNA expression were assessed by real-time PCR, using β-actin as a control. (F) Cells were treated with indicated doses of triptolide for 24 h. Equal amount of the cell culture supernatants were used for hormone measurement by competitive-ELISA, ACTH secreting levels were calculated according to the standard reference provided by the company. Data were normalized with the number of cells. All Data were presented as means ± SD of three independent experiments each performed with at least quadruplicates. *P < 0.05; **P < 0.01. vs control.

size measurements were carried every 48 h using caliper. Tumor volume was counted with formula (width)2 × (length)/2. When the tumour volume had reached around about 100 mm3, triptolide administration was initiated. Triptolide was dissolved in DMSO and a total amount of 0.15 mg/kg triptolide per mouse was administered three times a week by i.p. injections. Vehicle-treated animals with equal volume of DMSO served as controls. At the end of the experiment, blood samples were collected by cardiac puncture from anesthetized mice, then ACTH ELISA kit (Uscn Life Science Inc., China) was used to measure plasma ACTH according to the manufacturer’s introductions, as well as serum corticosterone (Uscn Life Science Inc., China). Xenografts were totally removed, measured, and fixed in phosphatebuffered 4% paraformaldehyde solution for further morphological analysis. All animal experiments in this study were implemented according to the Institutional Animal Care and Use Committee guidelines and approved by Ethical Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technlogy. Full exertions were made to lessen animal suffering and to minimize the number of experimental animals.

2.8. Western blot analysis Whole-cell protein lysate was extracted from AtT20 cells using cold RIPA buffer containing protease inhibitor cocktail and PMSF (Beyotime Biotech., China) and quantified with the BCA assay kit (Beyotime Biotech., China). 20 μg protein samples were run in 10% or 15% SDSPAGE gel. Proteins were then transferred onto polyvinylidenedifluoride membranes (Bio-Rad, USA) using standard procedures. The blotting membranes were blocked with 5% nonfat milk in TBST (Tris buffered saline–Tween 20) at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies, followed by incubation with corresponding anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (1:3000, Proteintech., USA) for 1 h at room temperature. Peroxidase activities were measured using ECL (Bio-Rad). The signal was detected using Quantity One software. The following primary antibodies were used: caspase-3, P50, IκB-α, p- IκB-α, β-actin(1:1000, Santa Curz Biotech., USA), P65, p-P65, Bcl-2, Bax, cyclin D1, ERK1/2, p-ERK1/2 (1:1000, Cell Signaling Technology, USA), respectively. 2.9. In vivo xenograft growth study

2.10. Immunohistochemical staining Female 4–6 weeks old athymic mice (n = 12) (Wuhan Laboratory Animal Center) were feeded in a specific pathogen-free circumstances. After harvested and resuspended in PBS mixed with equal amount Matrigel (BD, USA), 5 × 106 cells (in 50 μl of PBS-Matrigel mixture) were inoculated subcutaneously into unilateral groin of each mouse (n = 6 per group using number-based simple randomization). Tumor

The mouse xenografts were processed into paraffin blocks according to the standard protocols. Hematoxylin-eosin staining was performed using standard method. Paraffin sections (4 μm) were immunostained for ACTH (1:1 000, Dako, Germany), Ki-67 (1:1 000, Dako, Germany), cleaved-caspase 3 (1:500, Cell Signaling Technology, USA). 773

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was strongly enlarged in triptolide treated group comparing to the control group (Fig. 2D and E; 5.02% ± 1.66%, 11.23% ± 1.42%, 29.7% ± 4.82% respectively for control, 50 nM and 100 nM group; 50 nM group vs. control, P = 0.01; 100 nM group vs. control, P < 0.01), which suggested that triptolide induced mitochondrial membrane potential collapse in AtT20 cells. In addition, the activity of the pro-apoptotic downstream protein caspace-3 was detected through the absorbance of its substrate, Ac-DEVD-pNA. As shown in Fig. 2F, triptolide distinctly induced caspase-3 activation (50 nM group vs. control, P < 0.01; 100 nM group vs. control, P < 0.01), which was also confirmed by cleaved caspase-3 expression by western blot (Fig. 2G). Besides caspase family proteins, the Bcl-2 family members also contributed to mitochondria-mediated apoptosis process. To further investigate if Bcl-2 family proteins were also involved in the triptolide caused apoptosis of AtT20 cells, the expression levels of Bcl-2 and Bax were measured by western blot. The pro-survival protein Bcl-2 was down-regulated after exposure to triptolide, but no effect on the levels of pro-apoptotic protein Bax was observed (Fig. 2G). Notably, the proportion of Bcl-2/Bax also declined (Fig. 2H; 0.62 ± 0.08 at 50 nM group vs. control, P = 0.02; 0.12 ± 0.05 at 100 nM group vs. control, P < 0.01), which was consistent with the shift of caspase-3 activity and apoptosis sclae after triptolide treatment.

Diaminobenzidine (DAB) was used as substrate for the HRP conjugated secondary antibody (Vector Laboratories). Nuclei were counterstained by Harris hematoxylin. Immunoreactivity was evaluated by the percentage of positive cells and their intensity. 2.11. Data analysis All values were presented as the mean ± SD (standard deviation) of at least three indipendent experiments and statistically analyzed by SPSS 18.0 software (SPSS Inc., USA). Two-tail unpaired Student's t-test and one way Anova were used for group comparison. The P value less than 0.05 was considered to be statistically significant. 3. Results 3.1. Triptolide inhibited cell viability and clonogenic ability of AtT20 cells The anti-proliferative effect of triptolide on AtT20 cells was determined by CCK-8 assay and direct cell counting. Triptolide suppressed AtT20 cell viability in a concentration-dependent manner, even at a low dose of 50 nM compared with control group (Fig. 1A; 80.67% ± 6.66% vs. control, P = 0.02) (Fig. 1B; 83.67% ± 7.51% vs. control, P = 0.03). In addition, the growth inhibitory effect of triptolide in a dose dependent pattern was also verified by a colony formation model compared with control group (Fig. 1C; 45% ± 6.24% in 50 nM group vs. control, P < 0.01). Furthermore, a lower viability of corticotroph tumor cells upon longer exposure to triptolide of the same dose was also observed (Fig. 1D).

3.4. Triptolide exerted impact on NF-κB and ERK signaling To confirm whether triptolide induced suppressive effect and apoptosis procedure was correlated with inhibition of NF-κB activity, western blot were conducted after the stimulation of triptolide. Notably, a prominent reduction of the phosphorylated p65 was observed after 100 nM triptolide treatment (Fig. 3A, 50 nM group vs. control, P = 0.01; 100 nM group vs. control, P < 0.01). However, no significant difference was observed in the expression levels of other NFκB associated proteins, such as p50, IκB-α, and p- IκB-α by comparing different concentration groups (Fig. 3A, P > 0.05). In addition, we also found a robust reduction of the phosphorylated ERK in AtT20 cells after triptolide treatment (Fig. 3B; 50 nM group vs. control, P < 0.01; 100 nM group vs. control, P < 0.01), suggesting that ERK pathway was also affeted in the triptolide induced suppressive effect accompanied apoptosis program. Furthermore, the expression of cyclin D1 also decreased after exposure to triptolide (Fig. 3B), consistent with the cell cycle arrest.

3.2. Triptolide reduced Pomc transcriptional activity and ACTH secretion in AtT20 cells The main characteristic of corticotroph adenomas is excessive ACTH secrestion. To investigate the hormone suppressive activity of triptolide in AtT20 cells, the total RNA was extracted after 24 h exposure to triptolide (0, 50, 100 nM), relative gene expression was analyzed by qPCR. As the precursor peptide of ACTH, Pomc gene expression determined the intracellular levels of ACTH. Pomc mRNA expression was significantly reduced after triptolide treatment (Fig. 1E; 0.61 ± 0.1 in 50 nM group vs. control, P < 0.01; 0.43 ± 0.11 in 100 nM group vs. control, P < 0.01). Furthermore, in order to determine the extracellular secretion levels of ACTH, the supernatants of AtT20 cells were collected and analyzed by ELISA after stimulation with triptolide for 24 h. The data showed that triptolide significantly decreased the ACTH secretion as well (Fig. 1F; 80.0% ± 10.58% in 50 nM group vs. control, P = 0.04; 45.33% ± 7.57% in 100 nM group vs. control, P < 0.01).

3.5. Triptolide treatment reduced tumor growth and hormone secretion in vivo

3.3. Triptolide induced cell cycle arrest, mitochondrial apoptosis of AtT20 cells

To further confirm the obtained results in vitro, we investigated the role of triptolide in a xenograft model. We implanted AtT20 cells subcutaneously with or without triptolide treatment. Tumor size, weight, plasma ACTH and corticosterone levels were analyzed. The tumor growth was significantly reduced in triptolide treated group compared to the vehicle group (P ≤ 0.05; Fig. 4A and B). The final weight of xenograft in triptolide treated group was significantly lower than the vehicle group (Fig. 4C; 127.67 ± 21.94 mg in treated group vs. 83.33 ± 14.44 mg in vehicle group; P = 0.04). The plasma ACTH and corticosterone levels of the mice under triptolide treatment were also significantly lower than those of vehicle group (P < 0.05; Fig. 4D and E). Moreover, immunohistochemical analysis of tumor specimens showed decrease in ACTH protein expression and Ki-67 proliferation index, but increase in cleaved caspase 3 expression in triptolide treatment group (Fig. 4F). These results suggested the triptolide-induced inhibitory effect on tumor proliferation and hormone secretion, in accordance with previous in vitro studies.

Having demonstrated the suppressive effect of triptolide on AtT20 cells growth, flow cytometry was explored to investigate the possible effect of triptolide on cell cycle distribution. After triptolide treatment for 2 days, we observed a decreased cell population in G0/G1 phase, accompanied by a dose-dependent increase in G2/M phase, suggesting a cell cycle arrest induced by triptolide (Fig. 2A). Moreover, the apoptotic rate of cells exposed to different concentrations of triptolide (0, 50, 100 nM) was 5.16% ± 1.8%, 14.82% ± 4.14% and 31.79% ± 3.51%, respectively (Fig. 2B and C), indicating a significant increase of apoptosis induced by triptolide (50 nM group vs. control, P = 0.02; 100 nM group vs. control, P < 0.01). In order to identify whether the triptolide-induced apoptosis was due to mitochondrial membrane depolarization, AtT20 cells were incubated with JC-1 dye after triptolide treatment. The results from flow cytometry demonstrated that the population of JC-1 green stained cells 774

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Fig. 2. Triptolide induced cell cycle arrest and mitochondria mediated apoptosis of AtT20 cells. AtT20 cells were stimulated at the various doses of triptolide for 48 h. (A) Cell cycle distribution was determined using PI staining. The cell proportions in G0/G1 phase were both significantly reduced in two experimental groups companying with a rising cell ratios in G2/ M phase. (B) Cell apoptosis was detected by flow cytometry using Annexin V-FITC and PI double staining. Flow cytogram was presented representatively. (C) Quantification of FACS analysis. The total apoptosis rate (early apoptosis rate plus late apoptosis rate) of each group is averaged with SD from three independent experiments. (D) Mitochondrial membrane depolarization was measured by flow cytometry using JC-1 staining. Flow cytogram was presented representatively and data were shown as% of gated cells. (E) Quantification of FACS analysis. The disrupted mitchondria rate of each group is averaged with SD from three independent experiments. (F) Unit content of caspase-3 activity was determined by colorimetric analysis and calculated according to the standard substance provided by the company. (G) Activation of cleaved caspase-3 protein and reduction of Bcl-2 protein expression after triptolide treatment. (H) The ratio of Bcl-2 and Bax are quantified. All data were shown as means ± SD of three independent experiments. *P < 0.05; **P < 0.01. vs control group.

4. Discussion

subsequent activation of caspase-3 and decreased Bcl-2/Bax proportion suggested that triptolide harbored a pro-apoptotic effect on murine ACTH-secreting pituitary tumor cells. Furthermore, we successfully further verified those inhibitory effect of triptolide by a mouse xenograft model of Cushing disease with triptolide administration in vivo. The inhibition of NF-κB activity and inactive ERK1/2 might be responsible for those encouraging results by intensive analysis of potential signaling pathways. The anti-tumor activity of chemotherapeutic agents is mainly mediated by initiation of DNA damage and cell death after apoptosis, a process which requires NF-κB [30]. Accumulating studies have proved the prominent importance of the transcription factor NF-κB in regulating the expression of genes involved in tumor development [17]. As an anti-apoptotic transcription factor, NF-κB customarily shelters cells by motivating prosurvival proteins expression. Its activation is regulated by an adverse feedback pathway depending on the transcriptional level of IκB family members [18]. Recent studies demonstrated that the NF-κB signaling pathway is clearly down-regulated by triptolide in immune cells, fibroblast, epithelial cells and a quantity of cancer cells [25,31–37]. To investigate a possible effect of triptolide on the NF-κB pathway in AtT20 cells, western blot assay was conducted. We found that triptolide treatment resulted in a significant reduction of phosphorylated p65 subunit of NF-κB in protein level, but not other NF-κB

Recently, Chinese herbs have been accentuated increasingly in research by reason of their potential curative efficacy and lower side effects in the treatment of plentiful diseases. TWHf, which was first documented in Shennong Bencao Jing, is broadly distributed in Asian areas and utilized to manage immune-inflammatory diseases for centuries [26]. Since triptolide was successfully extracted and purified from TWHf in 1970s, its clinical pharmacological effects were largely studied. Thereinto, its anti-tumor effect was revealed in plentiful novel researches [27]. This natural agent revealed a great antitumor potential in the treatment of numerous solid tumors [28]. However, the role of triptolide on pituitary tumors has not been deeply investigated. To our knowledge, only one in vivo study mentioned the promotion effect of TWHf on the synthesis and secretion of ACTH in rat normal pituitary gland [29]. In the current study we demonstrated that nanomolar dosage of triptolide was able to reduce the viability and colony formation ability of AtT20 cells in a dose- and time- dependent pattern. We next elucidated that triptolide remarkably decreased Pomc mRNA levels and extracellular secretion of ACTH from AtT20 cells in a dose-dependent manner. Triptolide also induced G2/M phase arrest and apoptosis procedure of AtT20 cells. Mitochondrial membrane depolarization,

Fig. 3. Triptolide exerted impact on NF-κB and ERK signaling. (A) P65, p-p65, p50, IκB-α, p- IκB-α were detected by Western blot. Data presented is a representative result of 3 independent experiments. Relative expression ratio of p-p65/t-p65 was presented as the fold-change of the control. (B) ERK, pERK, cyclin D1 were detected by Western blot. Data presented is a representative result of 3 independent experiments. Relative expression ratio of p-ERK/total ERK was presented as the fold-change of the control. All data were shown as means ± SD of three independent experiments. *P < 0.05; **P < 0.01. vs control group.

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Fig. 4. Effects of triptolide on AtT20 cell tumor growth and hormone secreation in vivo. Athymic mice bearing corticotroph tumour cells were treated with triptolide for 2 weeks. (A)The development of mean tumor size during the treatment period was documented. (B) Representative photographs of xenograft tumors were presented. (C) The mean tumor weight after the treatment was shown. (D, E) Plasma concentration of ACTH and corticosterone after last treatment were measured calculated according to the standard substance provided by the company. (F) H & E and Immunohistochemical staining for Xenograft tumors. Representative images of each group were presented (Original magnification 40×). All data were shown as means ± SD. *P < 0.05; **P < 0.01. vs vehicle group.

play a key role in regulating the mitochondria-mediated (intrinsic) apoptosis pathway [42]. As a pro-apoptotic protein, Bax evokes cytochrome C move to mitochondrial external membrane, while Bcl-2 harbors an anti-apoptotic properties through stabilizing mitochondria membrane and restraining cytochrome C release. The balance between the two members is crucial in the progression of apoptosis [43,44]. Caspase-3 activation eventually results in cell apoptosis. Triptolide has been reported to prompt Bcl-2 cleavage and mitochondria mediated

associated protein expression, such as p65, p50, IκB-α, p- IκB-α. Phosphorylation of p65 was reported to be related with nuclear translocation of NF-κB and activation of several downstream genes by positive effect on co-activators [38,39]. Inactive p65, namely decreased phosphorylation level, suppressed NF-κB nuclear translocation, potentiated the inhibition of NF-κB activity and influenced the expression of multiple target genes [40,41]. The Bcl-2 family proteins, whose expression is regulated by NF-κB, 777

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Acknowledgments

apoptosis process, and then to lessen the expression of pro-survival genes and cell cycle related proteins such as Bcl-2 and cyclin D1 [25]. Cyclin D1 is a proto-oncogene involved in G1-G2/M progression of the cell cycle, its expression is higher in pituitary adenomas than in the normal pituitary cells [45], and associated with recurrence [46]. In our study, we also demonstrate that, as NF-κB target genes, Bcl-2 and cyclin D1 were both down regulated after exposure to triptolide, resulting in cell cycle arrest and mitochondria mediated apoptosis initiation. Excess serum ACTH levels and subsequent hypercortisolemia are correlated with the furtherance of Cushing disease, its clinical therapeutic goal was to normalize serum ACTH level. In corticotroph cells, the POMC peptide is cleaved into ACTH, so Pomc gene was the crucial therapeutic target for Cushing disease. It was reported that, the transcriptional activation of the Pomc gene was negatively regulated by a NF-κB-dependent pathway in AtT20 corticotroph cells [47]. Under proinflammatory cytokine stimuli, upregulation of Pomc gene was related with NF-κB activation in pituitary corticotroph cells [48,49]. Consistent with these studies, our results showed that, downregulation of NF-κB was associated with suppressive effect on Pomc gene expression and ACTH secretion in AtT20 cells after treatment of triptolide. In addition, the MAPK (mitogen-activated protein kinases)/ERK signaling pathway also harbored the ability to influence tumorigenesis by regulation of cell growth, differentiation and survival [50]. However, recent studies have suggested a more complicated role of ERK, regarding cell proliferation and cell death. Triptolide was found to reduce the expression of MAPK phosphatase-1 (MKP-1), which dephosphorylates the ERK-1/2 and the p38 MAPK, to execute its growth inhibitory and apoptosis promoting effects [28]. Triptolide inhibited the proliferation of the HT22 hippocampal cells by persistent ERK-1/2 activation [51]. Elevated ERK also mediated triptolide-induced caspasedependent apoptosis in breast cancer cells [52]. However, as a classic pro-survival pathway, MAPK-ERK activation was typically associated with unrestricted cellular proliferation and decreased sensitivity to apoptotic-inducing agents [53]. The inhibitors of the ERK1/2 cascade (for example, trametinib) were promising to serve as antitumor drugs [54,55]. ERK activation protected cells from apoptosis by inhibiting caspase cascade [56]. It has been reported that ERK1/2 was overphosphorylated in all types of pituitary adenomas, comparing to normal pituitary gland [57]. Increased phosphorylation of ERK1/2 was associated with proliferation in AtT20 cells [58]. In our study, we also observed a remarkable inhibition of ERK1/2 phosphorylation in AtT20 cells, which could be a possible mechanism for contributing to induction of cell death after triptolide treatment. Lately, the detection of the ubiquitin-specific protease8 (USP8) mutation in more than 60% of ACTH-secreting corticotrophin adenomas provided exciting advances in the understanding of the pathogenesis and new therapeutic target of Cushing’s disease [59–61]. USP8 mutants disminished epidermal growth factor receptor (EGFR) ubiquitination, subsequently potentiated EGFR-MAP kinase signaling, triggering Pomc transcription and ACTH synthesis. As the downstream of EGFR, MAPK/ERK signaling cascade plays an important role in activity of the Pomc promoter [62]. After incubation with triptolide, the reduction of the phosphorylated ERK1/2 would suppress the activation of the Pomc gene promoter, accompanied by the hypoproduction of ACTH in AtT20 cells, which hints at the potential of triptolide to be effective in the management of ACTH-producing pituitary adenoma.

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5. Conclusion Triptolide inhibited pituitary corticotroph tumor cell growth in vitro and in vivo by induction of mitochondria-mediated apoptosis, cell cycle arrest and decreased cellular ACTH secretion as well. Reduction of NFκB activity and phosphorylated ERK1/2 levels were, at least partly, responsible for these effects. Therefore, triptolide is a potential therapeutic agent in the treatment of Cushing disease with effective suppression of ACTH level. 778

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