Triptolide inhibits cell proliferation and tumorigenicity of human neuroblastoma cells

Triptolide is a diterpene triepoxide, extracted from the Chinese herb Tripterygium wilfordii Hook F, which has been shown to have antitumor activity in a number of cancers. Neuroblastoma is an aggressive extracranial pediatric solid tumor, with significant chemotherapeutic resistance. In this study, triptolide was hypothesized to be a potential therapeutic agent for neuroblastoma. The effects of triptolide on neuroblastoma cell growth and tumor development were investigated. Cell growth and proliferation were evaluated using a cell counting kit-8 assay and a 5-bromo-2-deoxyuridine staining assay. Cell cycle and apoptosis were detected by flow cytometry. Reverse transcription-quantitative polymerase chain reaction was conducted to detect the expression levels of the apoptosis-associated proteins, caspase-3 and caspase-9. The tumorigenicity of neuroblastoma cells was assessed by a soft agar clonogenic assay and an in vivo tumorigenic assay. The results demonstrated that exposure of BE(2)-C human neuroblastoma cells to triptolide resulted in a reduction in cell growth and proliferation, and the induction of cell death and apoptosis, together with cell cycle arrest in the S phase. A soft agar assay indicated that triptolide inhibited the colony-forming ability of BE(2)-C neuroblastoma cells. The xenograft experiment showed that triptolide significantly reduced tumor growth and development in vivo. The data suggested that this Chinese herb may be a potential novel chemotherapeutic agent for neuroblastoma.


Introduction
Neuroblastoma is a common extracranial pediatric solid tumor, accounting for up to 10% of pediatric cancers and ultimately resulting in 15% of cancer-related mortality in children (1,2). Histologically, neuroblastoma is a heterogeneous group of tumors ranging from benign changes in sympathetic neurons to tumors that cause critical illness as a result of extensive invasion and metastasis (3)(4)(5). The clinical presentation of neuroblastoma is variable and advanced cases are often found to be highly resistant to conventional treatment modalities based on surgery, chemotherapy and radiotherapy (6). Therefore, recent studies have primarily focused on these particularly aggressive cases, with the goal of identifying additional therapeutic options (7).
Natural compounds extracted from herbs, such as Taxol, have been widely used in cancer therapy (8). A number of active compounds isolated from Chinese herbs have been shown to have antitumor properties (9,10). Thus, traditional Chinese medicine provides an important additional option for the development of novel cancer treatments. Triptolide is a diterpene triepoxide and is extracted from the Chinese herb Tripterygium wilfordii Hook F, which has been used to treat inflammation and autoimmune diseases in Chinese medicine (11,12). Recently, evidence has shown that triptolide has a potent immunosuppressive effect and antineoplastic activity in certain types of cancer, including breast cancer, pancreatic cancer, melanoma and prostate cancer (13)(14)(15)(16). In addition, triptolide has been shown to exert its antitumor properties through induction of apoptosis and inhibition of cell proliferation, angiogenesis, cell invasion and metastasis (17)(18)(19)(20). This study investigated the effects of triptolide on malignant neuroblastoma cell growth and cell proliferation with the aim of providing evidence that may support the use of triptolide as a novel drug for the treatment of neuroblastoma.

Materials and methods
Cell culture. BE(2)-C neuroblastoma cells were obtained from the American Type Culture Collection (Manassus, VA, USA), and was cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 (DMEM/F-12; Invitrogen, Carlsbad, CA, USA) plus 10% fetal Triptolide inhibits cell proliferation and tumorigenicity of human neuroblastoma cells bovine serum (Invitrogen) and 1% penicillin and streptomycin (Invitrogen), and was incubated at 37˚C in a 5% CO 2 humidified incubator. Purified triptolide (>98%) was purchased from Sigma-Aldrich (T3652; St. Louis, MO, USA), which was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at a stock concentration of 50 mM and stored at 4˚C.
Cell growth and viability assays. BE(2)-C cells grown in 96-well culture plates were treated either with various doses of triptolide (5, 10, 25, 50 or 100 nM), or DMSO. The cell growth rate was analyzed with the cell counting kit-8 (CCK-8) growth assay after 24 h culture. Briefly, cells in each well were incubated with 10 µl CCK-8 reagent at 37˚C for 2 h. The optical density was measured at a wavelength of 450 nm using a microplate reader (Model 550, Bio-Rad, Hercules, CA, USA). In addition, BE(2)-C cells were treated with 25 or 50 nM for 24 h, photographed using an Olympus 1X71 (Olympus Corporation, Tokyo, Japan) and counted with a TC10 ™ Automated Cell Counter (Bio-Rad).
Cell cycle assay. After treatment with triptolide (25 nM) for 24 h, cells were collected by centrifugation at 211 x g for 5 min, washed with ice-cold PBS, fixed with 70% ethanol, stained with 20 µg/ml propidium iodide (Invitrogen) and analyzed by flow cytometry (BD FACSVerse™, BD BioSciences, Franklin Lakes, NJ, USA). The data were analyzed with CellQuest Pro software, version 5.0 (BD BioSciences).
Cell death and apoptosis assays. Cells were either untreated or treated with triptolide. DMSO was used as control. After 24 h treatment, adherent and floating cells were pooled, collected by centrifugation at 211 x g for 5 min, and washed once with ice-cold PBS. The cell death rate was detected with 0.2% trypan blue dye (Bio-Rad). Apoptotic cells were determined by the Annexin V-fluorescein isothiocyanate (FITC) kit (Sigma-Aldrich), using flow cytometry according to the manufacturer's instructions.
Soft agar clonogenic assay. Cells (1x10 3 ) were mixed in 0.3% Noble agar in a growth medium containing vehicle or triptolide, and plated into six-well plates containing a solidified bottom layer (0.6% Noble agar in the same growth medium). Colonies were photographed after 14 days (Olympus 1X71) and cell numbers were counted from at least five randomly selected fields.
In vivo tumorigenic assay. Six female non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (4 weeks old) were used and maintained under specific pathogen-free conditions. Cells (1x10 6 ) were suspended in 200 µl serum-free DMEM/F12, and injected subcutaneously into both flanks. After tumor formation, mice were divided randomly into treatment and control groups. Mice in the treatment group were injected with triptolide at 0.4 mg/kg daily for seven days (21), while control mice were injected with DMSO. Tumor size was measured using calipers and tumor volume was calculated using the formula 4/3πr 3 , where r is the radius of the tumor. Mouse body weight was monitored daily. Three weeks after tumor growth, xenograft tumors were removed and weighed immediately after the mice were sacrificed by cervical dislocation. All animal experiments were approved by the Institutional Animal Care and Use Committee of Southwest University (Chonqing, China).

Statistical analysis.
All observations were confirmed by at least three independent experiments. Quantitative data are expressed as the mean ± standard deviation. Two-tailed Student's t-test was performed for paired samples using GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results
Triptolide inhibits neuroblastoma cell growth and viability. BE(2)-C cells were treated with increasing doses of triptolide for 24 h. A concentration-dependent response to triptolide in the BE (2)-C cells was observed. As shown in Fig. 1A, triptolide inhibited cell growth even at a low dose of 5 nM. The cell viability was significantly reduced to 50% at 50 nM of triptolide. Triptolide also inhibited cell growth in a time dependent manner ( Fig. 1B and C). Moreover, immunofluorescent staining using a BrdU label confirmed that triptolide markedly inhibited cell proliferation ( Fig. 2A and B).
Triptolide induces neuroblastoma cell cycle arrest and apoptosis. The effect of triptolide on cell cycle was investigated. It was found that the percentage of cells in S phase increased from 36.06 to 58.16% (Fig. 3A and B). This result suggests that triptolide induces cell cycle arrest in the S phase, which may contribute to inhibition of cell proliferation.
It was also observed that exposure of BE(2)-C cells to triptolide could induce cell death and apoptosis. Triptolide    Fig. 4A). The apoptosis rate was also increased after treatment with 25 nM triptolide for 24 h (Fig. 4B). As shown in Fig. 4C and D, following triptolide treatment mRNA expression levels of caspase-9 and caspase-3 were increased 70.9 and 9.7 fold, respectively, compared with control. These results indicate that triptolide induces cell death and apoptosis through caspase-9 and caspase-3 activation.
Triptolide suppresses neuroblastoma cell colony formation in vitro and tumorigenicity in vivo. The role of triptolide in neuroblastoma tumorigenesis was examined. BE(2)-C cells treated with 25 nM triptolide gave rise to smaller and and sparser colonies in soft agar, compared with cells treated with DMSO ( Fig. 5A and B). The xenograft study in NOD/SCID mice showed that the volume and weight of xenograft tumors in the triptolide treatment group were lower than those in the DMSO group (Fig. 6). These data indicate that triptolide may inhibit neuroblastoma cell self-renewal and tumorigenesis. In addition, there was no significant difference in mouse body weight after triptolide treatment (Fig. 6D), which suggests that the administered dose of triptolide may have minimal toxic side effects.

Discussion
Recently, Chinese herbs have attracted attention from researchers worldwide due to their potential efficacy in the treatment of a number of diseases (22,23). A large number of active compounds have been extracted from Chinese herbs. Tripterygium wilfordii Hook F has been used in traditional Chinese medicine for centuries for the treatment of fever, chills, carbuncles and edema (24,25). The diterpenoid epoxide triptolide is one of the two main bioactive components of Tripterygium wilfordii Hook F, which exhibits antitumor activity (26,27). However, there is little data regarding the efficacy of triptolide against neuroblastoma cells. This study aimed to investigate the effect of triptolide on neuroblastoma cell growth and tumor development, with the aim of providing more information for the development of novel neuroblastoma treatments.
The current study demonstrated that triptolide not only induced neuroblastoma cell death and apoptosis via caspase-9/caspase-3 pathway activation, but also inhibited cell growth and viability by inducing cell cycle arrest at the S phase. Furthermore, the results showed that triptolide inhibited neuroblastoma cell colony-forming capability in vitro and tumor progression in vivo. In conclusion, triptolide may be a potent natural candidate for neuroblastoma treatment.