CuB at 98% purity was purchased from Must Biotechnology Co., Ltd. (Chengdu, China). MTT, dimethyl sulfoxide (DMSO), PD98059, SP600125, SB203580 and interleukin (IL)-6 were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were acquired from HyClone Laboratories (Logan, UT, USA). Propidium iodide (PI), AG490 and bicinchoninic acid (BCA) Protein Assay kit were obtained from Beyotime (Shanghai, China). An Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit was purchased from MultiSciences Biotech Co., Ltd (Hangzhou, China). The primary antibodies specific to p53 (rabbit pAb), p21 (mouse mAb), B-cell lymphoma 2 (Bcl-2; rabbit pAb), Bcl2-associated X protein (Bax; rabbit pAb), phospho-JAK2 (Tyr 1007/1008; rabbit mAb), JAK2 (mouse mAb), phospho-STAT3 (Tyr705; mouse mAb), phospho-extracellular signal-regulated kinases (p-ERK; Thr202/Tyr204; rabbit pAb), phospho-p38 (Thr180/Tyr182; rabbit pAb) and β-actin (mouse mAb) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The phospho-c-Jun N-terminal kinase (p-JNK; Thr183/Tyr185; rabbit mAb) primary antibody was purchased from Cell Signaling Technology (Danvers, MA, USA), cyclin-dependent kinase 1 (CDK1; mouse mAb), Cyclin B1 (rabbit pAb), STAT3 (rabbit pAb), ERK (rabbit pAb), JNK1 and 2 (rabbit pAb), and p38 primary antibody (rabbit pAb) and secondary polyclonal antibodies goat anti-mouse immunoglobulin (Ig) G horseradish-peroxidase (HRP)-conjugate and goat anti-rabbit IgG HRP-conjugate were obtained from Boster Biological Technology., Ltd. (Wuhan, China).

The human NB cell line (SH-SY5Y) was obtained from the China Center for Type Culture Collection (Wuhan University, Wuhan, China). The cells were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO₂. All the experiments were performed one day after the cells were seeded.

The effect of CuB on the growth and proliferation on cancer cells was assessed by measuring the metabolic activity (MTT assay). The cells were seeded in 96-well plates at a density of 2×10⁴ cells/well with 200 μl culture medium per well for 24 h. On the next day, the medium was replaced with fresh medium containing different concentrations of CuB (0–128 μM). Subsequent to incubation for an additional 24 and 48 h, a total of 20 μl MTT [5 mg/ml in phosphate-buffered saline (PBS)] solution was added to each well and incubated at 37°C for 4 h to metabolize the MTT into formazan. Next, the supernatant was discarded and 100 μl DMSO was added to each well to terminate the reaction. The absorbance was measured at 490 nm using a microplate reader (Model 680; Bio-Rad, Richmond, VA, USA). Three independent experiments were performed in triplicates. The percentage of proliferation was normalized relative to the control.

The cell cycle parameters were analyzed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The SH-SY5Y cells were cultured with the indicated concentrations of CuB for 24 h. The cells were harvested by centrifugation, then washed with cold PBS (pH 7.4) and fixed with 70% ice-cold ethanol overnight at −20°C. Following fixation, the cells were harvested and rinsed once with PBS (pH 7.4) and then incubated with 500 μl PI staining solution (50 μg/ml PI, 100 μg/ml RNase A) for 1 h at room temperature. The relative numbers of cells in the G1, S and G2/M phases of the cell cycle were measured.

The SH-SY5Y cells were exposed to the indicated concentrations of CuB for 24 h. The samples of 1–5×10⁵ cells were harvested and rinsed twice with cold PBS (pH 7.4). The cells were resuspended in 500 μl binding buffer and stained with 5 μl Annexin-FITC and 10 μl of PI for 5 min in the dark at room temperature. The stained apoptotic cells were counted using a FACScan flow cytometer.

Following treatment with the indicated concentrations of CuB, the SH-SY5Y cells were lysed and the protein concentration was determined using a BCA protein assay kit. The lysate containing 40 μg of protein was subjected to SDS-PAGE. The protein was transferred to a nitrocellulose membrane, and the membrane was blocked overnight with 1X Tris-Buffered Saline containing 0.1% Tween-20 and 5% skimmed milk at 4°C. Following blocking, the membrane was washed three times and incubated with the respective primary antibodies for 2 h at room temperature. Next, the membrane was washed three times and incubated with the diluted HRP-conjugated secondary antibody (1:5,000) for 1.5 h at room temperature. Subsequent to the three washes, the membrane was detected using an enhanced chemiluminescence kit (Millipore, Bedford, MA, USA).

All the values are expressed as the mean ± standard deviation. Significant differences between the groups were determined using Student’s t-test and the statistical significance was expressed as ^*P<0.05 and ^**P<0.01. All the figures shown represent the results from at least three independent experiments.

In order to investigate the growth inhibition effects of CuB, the MTT assay and PI staining were performed to evaluate the cell viability and cell cycle distribution, respectively.

The viability of cancer cells treated with CuB was investigated by the MTT assay. CuB inhibited cell proliferation in SH-SY5Y cells in a time- and dose-dependent manner (Fig. 1A). The concentrations of 4, 8 and 12 μM CuB were used in subsequent experiments.

Cell cycle arrest caused by CuB for 24 h was investigated by flow cytometry following PI staining. Evident changes were found in the cell cycle distributions when treated with CuB (Fig. 1B). The percentage of cells in the G2/M phase was increased in a dose-dependent manner. These data indicated that CuB treatment suppressed cell proliferation through an increased accumulation of cells in G2/M phase of the cell cycle.

Apoptosis in SH-SY5Y cells was detected by flow cytometry following Annexin V-FITC/PI double staining. The results revealed that the early apoptotic rate (Annexin V-FITC-positive and PI-negative cells) was significantly increased following CuB treatment for 24 h and the rate increased in a dose-dependent manner (Fig. 2).

The phosphorylated forms of JAK2 and STAT3 were assessed by western blot analysis in SH-SY5Y cells treated with CuB for 24 h. Constitutive activation of JAK2 and STAT3 were suppressed by CuB in a concentration-dependent manner (Fig. 3A). Next, the role of JAK2/STAT3 in CuB-induced apoptosis by a JAK2 inhibitor (AG490) and an activator of JAK2/STAT3 (IL-6) was determined. Western blot analysis and flow cytometry were respectively employed to detect protein expression and apoptosis with or without AG490 or IL-6 pretreatment. As expected, the protein levels of p-JAK2 and p-STAT3 were regulated by AG490 and IL-6, while the total protein levels of JAK2 and STAT3 did not exhibit any evident change. Compared with CuB treatment alone, the protein levels of p-JAK2 and p-STAT3 were markedly decreased and the rate of apoptotic cells was markedly increased following the treatment of CuB and AG490. However, the alterations induced by CuB could be attenuated partially by IL-6 (Fig. 3B and C).

Western blot analysis was performed to determine whether CuB affects the activation of MAPK cascades, including ERK, JNK and p38 MAPK in NB cells. As a result, CuB upregulated p-JNK and p-p38 MAPK, and downregulated p-ERK in SH-SY5Y cells (Fig. 4A). Therefore, PD98059, SB203580 and SP600125, which are specific inhibitors of ERK, p38 MAPK and JNK, respectively, were used to examine the role of the MAPK signaling pathway in CuB-treated cells. Upon pretreatment with the inhibitors, the protein levels of p-ERK, p-JNK and p-p38 MAPK were all decreased, and the decrease in p-ERK expression was significant compared with CuB treatment alone. ERK, JNK and p38 MAPK did not reveal evident changes (Fig. 4B). Apoptosis analysis by flow cytometry revealed that the ERK inhibitor PD98059 increased the percentage of apoptotic cells induced by CuB, whereas the rates of apoptotic cells induced by CuB were significantly abrogated by the p38 MAPK and JNK inhibitors (Fig. 4C).

Bcl-2 and Bax have been implicated in apoptosis and mitochondrial dysfunction. For this reason, the effects of CuB on the expression of these two proteins were investigated. The data in Fig. 5 demonstrated that CuB downregulated the anti-apoptotic Bcl-2 and upregulated pro-apoptotic Bax in a concentration-dependent manner in SH-SY5Y cells. In addition, cell cycle proteins linked with the G2/M phase, including cyclin B1 and CDK1, were also downregulated by CuB in a dose-dependent manner. In multi-cellular organisms, p53 is involved in the prevention of cancer. It acts as a tumor suppressor and was reported to regulate the cell cycle through the control of the expression of cyclin-dependent kinase inhibitor p21 (20). The results indicated that p53 and p21 were also upregulated by CuB in a dose-dependent manner in SH-SY5Y cells (Fig. 5).