AtT20 cells (mouse corticotroph tumor cell line) were provided by Shanghai Institute of Materia Medica (Shanghai, China) (29). AtT20 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen Life Technologies, Grand Island, NY, USA) at 37°C in 5% CO₂. UA purchased from Sigma-Aldrich (St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) as a 100 mM stock solution and stored at −20°C. The cells were pretreated with JNK inhibitor SP600125 (Calbiochem, La Jolla, CA, USA), which was dissolved in DMSO 1 h prior to UA treatment.

Subsequent to being harvested and centrifuged, the AtT20 cells were resuspended in RPMI-1640 medium with 1% FBS and seeded in a 96-well plate at a density of 1 × 10⁵/well. The cells were treated with different concentrations of UA for 24, 48 and 72 h, respectively, while only DMSO was added to the control wells. A total of 10 μl CCK-8 (Dojindo, Kumamoto, Japan) was added to each well and the microplate was incubated for 6 h at 37°C with 5% CO₂. The optical density (OD) at 450 nm was read with a 96-well plate reader (Bio-Rad, Reinach, Switzerland). Experiments were conducted with five replicates.

AtT20 cells were collected, centrifuged and washed with phosphate-buffered saline following treatment with the indicated amount of UA for 24 h with or without pretreatment of SP600125 (JNK inhibitor; Sigma-Aldrich) for 1 h followed by staining with Annexin V-fluorscein (FLOUS) Staining kit and PI (Roche Diagnostics, Mannheim, Germany) for 15 min at room temperature. For each example, 20,000 cells were analyzed on a flow cytometer (FACStar, BD Biosciences, Franklin Lanes, NJ, USA).

The caspase-3/7, -8 and -9 activities were measured by Caspase-Glo luminescent-based assays (Caspase-Glo kit; Promega, Madison, WI, USA). The cells were seeded with a total of 10⁵ cells/well in a 96-well plate. Following treatment with UA (at various concentrations) in combination with SP600125 for 24 h, 100 μl of the Caspase-Glo-3/7, -8, -9 reagents were added to each well. The mixtures were incubated for 1 h and then transferred to a fluorescence microtiter plate. Quantification of luminescence was measured by a luminometer. To normalize the fluorescence intensity, the optical density values of the CCK-8 assay were measured by a Infinite F500 microplate reader (Tecan, Männedorf, Switzerland) as the viable cell number.

The total RNA was extracted from cells with indicated treatments. A volume of 5 μl cDNA (ReverTra Ace qPCR RT kit, Toyobo, Osaka, Japan) was amplified in a final volume of 0.4 μM of each primer and 25 μl SYBR Green Real-time PCR Master Mix (Toyobo) in a final volume of 50 μl for 40 cycles (ABI Prism 7500 Sequence Detection system; Applied Biosystems, Carlsbad, CA, USA). The primers were as follows: Pro-opio melanocortin (POMC) forward, 5′-AACCTGCTGGCTTGCATCCG-3′ and reverse, 5′-GGGC TGTTCATCTCCGTTGCCT-3′. β-actin, forward, 5′-TGGAATC CTGTGGCATCCATGAAAC-3′ and reverse, 5′-TAAAACGCAG CTCAGTAACAGTCC-3′.

The AtT20 cells were harvested, seeded in 24-well plates at a density of 2×10⁶/500 μl/well and then added to different concentrations of UA for 48 h. The cell culture supernatants were collected and centrifuged. ACTH was assayed using a mouse ACTH ELISA kit (Uscn Life Science Inc., Wuhan, China) following the manufacturer’s instructions.

Protein samples were extracted in radio-immunoprecipitation assay lysis buffer containing protease inhibitors and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). Mitochondria were extracted using a mitochondrial isolation kit (Thermo Fisher Scientific). Following incubation for 10–30 min at 4°C, the supernatant was collected by centrifugation (10,000 × g, 15 min, 4°C). The cell lysates (20–40 μg) which were determined using the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL, USA) proteins were separated by 12% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were washed twice in Tris-buffered saline and Tween 20 (TBST) and incubated with blocking buffer (5% skimmed milk in TBST) for 60 min at room temperature. Next, the membranes were washed three times and incubated overnight at 4°C with primary antibodies. The membranes were incubated with secondary antibodies (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. Following washing three times, the antibodies bound to the proteins were detected using enhanced chemiluminescence (Invitrogen Life Technologies). The following primary antibodies were used: anti-ACTH antibody (Ab) (1:1,000; Abcam); anti-phospho-p42/44 mitogen-activated protein kinase (MAPK) (Thr202/Tyr204) Ab, anti-p42/44MAPK Ab, anti-phospho-p38 (Thr180/Tyr182) Ab, anti-p38 Ab, anti-phospho-JNK (Thr183/Tyr185) Ab, anti-JNK Ab, anti-B cell lymphoma 2 (Bcl-2) Ab, anti-Bcl-2-associated X (Bax) Ab, anti-β-actin Ab (all 1:1,000; Cell Signaling Technology, Inc.); anti-phospho-Bcl-2 (Ser70) Ab (1:1,000; Sigma-Aldrich); anti-cytochrome c Ab (1:1,000; Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA). First, the phospho-specific MAPK were detected. Next, the membranes were stripped and re-probed with anti-JNK, anti-extracellular signal-regulated protein kinases (ERK) and anti-p38 antibodies. The densities of phosphorylated MAPK bands were normalized to that of the total MAPK bands. The expression levels of p-Bcl-2 and total Bcl-2 were assessed in the same manner. The secondary antibodies were Goat anti-rabbit IgG.

The results were expressed as the mean ± standard deviation of three separate experiments. Data were analyzed by one-way analysis of variance followed by Fisher’s least significant difference test or by Kruskal-Wallits test using SPSS 11 version software. P<0.05 was used to indicate a statistically significant difference.

In order to investigate the effect of UA on cytotoxicity, the AtT20 cells were exposed to UA (10–100 μM) for 24, 48 and 72 h, respectively. The CCK-8 results revealed that UA inhibited the viability of AtT20 cells in a dose- and time-dependent manner (Fig. 1). The IC₅₀ value for 24 h was 20.02 μM. Considering the IC₅₀ for 24 h, the concentration of 20 μM was used for further analysis.

The apoptosis of AtT20 cells induced by UA was further evaluated. AtT20 cells were treated with UA (10, 20 and 40 μM) for 24 h, respectively. Afterwards, Annexin V-FLOUS/PI staining was performed and assessed by flow cytometry. The percentage of apoptotic cells, including early apoptotic cells (Annexin V-FLOUS⁺/PI⁻) and late apoptotic cells (Annexin V-FLOUS⁺/PI⁺) was significantly increased in a dose-dependent manner (Fig. 3A). In addition, the activity of caspase-3/7, which is the executioner of apoptosis, was examined. Caspase-3/7 activity was gradually enhanced from 2.8- to 13.7-fold as the dose of UA increased (Fig. 3D). These results confirm that cell viability loss induced by UA in AtT20 cells is facilitated by the induction of apoptosis.

Next, it was investigated whether UA-induced apoptosis was accompanied with a decreased ACTH secretion. It has been reported that ACTH is derived from the POMC precursor peptide (30). In order to examine the effect on the intracellular levels of ACTH, UA (10, 20 and 40 μM) was added to AtT20 cells for 48 h, respectively, and the extracted total RNA was analyzed by qPCR. The results demonstrated that UA inhibited POMC mRNA (precursor of ACTH) in a dose-dependent manner. 40 μM UA achieved a maximum effect of inhibition (~0.3-fold compared with the control) (Fig. 2A). Western blot analysis also proved that UA decreased ACTH synthesis in a dose-dependent manner (Fig. 2B).

For determining the levels of ACTH, the conditioned medium was harvested and analyzed by ELISA. The data demonstrated that ACTH was reduced by UA in a dose-dependent manner. Compared with the control, ACTH levels were 0.62-, 0.48-, 0.3-fold, respectively (Fig. 2C). These results indicated that UA decreased the expression of POMC mRNA as well as ACTH synthesis and secretion in AtT20 cells in a dose-dependent manner.

Apoptosis is mediated by endogenous and exogenous signal transduction. To address whether the two pathways were involved in UA-induced apoptosis, caspase-8, -9, cytochrome c, Bcl-2 and Bax were assayed. AtT20 cells were treated with UA (10, 20 and 40 μM) for 24 h. Caspase-8 activity increased by 1.35-, 4.93-, 7.5-fold, respectively, as compared with the control (Fig. 3B). The levels of Bcl-2 and cytochrome c in mitochondria were reduced while cytochrome c in cytosol was increased along with increasing dose of UA (Fig. 3E and F). The protein levels of Bax were normal while the ratio of Bcl-2/Bax decreased (Fig. 3E and G). Caspase-9 was also significantly increased by 4.27-fold subsequent to treatment with 40 μM UA (Fig. 3C). The results revealed that endogenous and exogenous signaling pathways contributed to UA-induced apoptosis.

Cell proliferation and apoptosis, and the MAPK family are closely associated (31). UA has been shown to activate the MAPK signaling pathway in various cells (23,32). To further investigate the exact molecular mechanism of UA-induced apoptosis and identify whether the MAPK signaling pathway was also activated by UA in AtT20 cells, phosphorylation of JNK, ERK-1/2 and p38 was detected. The results revealed that 20 μM UA upregulated the expression of phosphorylation of JNK, which reached a maximum from 1 to 3 h, but had no effect on the phosphorylation of ERK-1/2 and p38 (Fig. 4A and B). To further confirm whether JNK activation mediated UA-induced apoptosis, AtT20 cells were pretreated with 10 μM SP600125 (JNK inhibitor) for 1 h followed by 20 μM UA for 24 h (Fig. 4C). The percentages of early and late apoptotic cells were 19.6 and 10.6% respectively, which were significantly less compared with that of cells in the group treated with 20 μM UA only (Fig. 3A).

It has been reported that JNK activation inactivated Bcl-2 by increasing phosphorylation of Bcl-2 (33,34). The results showed that 20 μM UA treatment for 3 h increased phosphorylated-Bcl-2 (Ser 70), while inhibition of JNK activation by SP600125 could downregulate the phosphorylation (Fig. 5A), demonstrating that JNK activation was involved in the upregulation of UA-induced Bcl-2 phosphorylation and resulting apoptosis.

To evaluate whether the activation of caspases was affected by Bcl-2, caspase-9 and -3/7 were assessed. The results revealed that caspase-3/7 and -9 activation were partly blocked in the UA group in the presence of SP600125 (Fig. 5A–C). Thus, the data indicated that JNK activation was involved in the endogenous signaling pathway in UA-induced apoptosis by increasing phosphorylated Bcl-2.