Tetrandrine was purchased from Shanghai Ronghe Medical (Shanghai, China). 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), was purchased from Invitrogen (Carlsbad, CA, USA). FuGene HD tranfection reagent was from Promega (Madison, WI, USA) and trypan blue dye was from Sigma (St. Louis, MO, USA). The ROS Assay kit and LY294002 were purchased from Beyotime (Nantong, China). Propidium iodide (PI) was purchased from MP Biomedicals (Solon, OH, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco LLC (Solon, OH, USA). Antibodies against β-tubulin, cyclin D1, cyclin D2, cyclin D3, cyclin E1, cyclin E2, CDK2, CDK4, GSK-3β, p53 and p27 were from Proteintech (Wuhan, China), Akt and phosphor-Akt were obtained from Cell Signaling Technology (Boston, MA, USA). Antibody against mouse CD31 was obtained from Becton-Dickinson (Franklin, NJ, USA).

EOMA cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Huh7 cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China). All the cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO₂ in high-glucose DMEM supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin. Cell culture dishes and plates were obtained from the NEST Biotechnology Co., Ltd., Jiangsu, China.

Empty vector (PUSE) and Akt overexpression vector (PUSE-CA-Akt) were obtained form Upstate Biotechnology (Lake Placid, NY, USA). Briefly, EOMA cells were seeded at a concentration of 5×10⁴ cells/well in a 6-well plate and grown at 37°C for 22 h before transfection. Then the cells were transiently transfected using FuGene HD transfection reagent with 4 μg of PUSE or PUSE-CA-Akt, respectively. After transfection for 16–24 h, cells were treated with 30 μM tetrandrine for 48 h.

EOMA cells were seeded at a concentration of 5×10⁴ cells/well in a 6-well plate and grown at 37°C for 22 h. The effects of tetrandrine on cell proliferation were characterized by cell counting. In brief, various concentrations of tetrandrine solution were added to different wells for 48 h. To assess cellular proliferation, the cells were counted daily using a hemocytometer.

EOMA cells were seeded in 60-mm plates at a concentration of 5×10⁵ cells/plate. After incubation for 22 h, various concentrations of tetrandrine solution were added, and the cells were incubated for 48 h. EOMA cells were harvested and supplemented with 70% ice-cold ethanol overnight. The cells were treated with RNase (50 μg/ml) at 37°C for 1 h, and treated with PI (20 μg/ml) for 30 min without light at 4°C. The DNA content was analysed by flow cytometry (Beckman Coulter).

EOMA cells were treated with different concentrations of tetrandrine for 48 h and harvested. Then, supernatant was collected, and the protein concentration was determined with a Bicinchoninic acid protein assay kit (Pierce). Protein samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. Following antibody incubation, enhanced chemiluminescence was used to detect the proteins.

EOMA cells were washed twice with PBS. 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was diluted to 10 μM with HG-DMEM and added to the cells. The plates were then stored in an incubator at 37°C for 1 h. ROS were detected in EOMA cells, and images were acquired with a fluorescence microscope.

EOMA cells were seeded in 6-well plates for 22 h and subsequently incubated with various concentrations of tetrandrine, NAC (20 mM) or pretreated with 20 mM NAC for 1 h followed by 50 μM tetrandrine. After incubation for 48 h, EOMA cells were harvested, and cells were centrifuged (150 × g for 10 min), supplemented with DCFH-DA solution (1 μM) and incubated in the cell incubator at 37°C for 1 h. The EOMA cells were centrifuged (250 × g for 10 min) and washed with ice-cold PBS prior to flow cytometry (Beckman Coulter).

Five-week-old male BALB/c nude mice were obtained from the Disease Prevention Centre of Hubei Province (Wuhan, China). The Experimental Animal Centre of Wuhan University approved the experimental protocols. The Huh7 cells were counted, and 2×10⁷ cells were implanted in the right flank of each mouse. When the tumor volume reached 150–300 mm³, the mice were randomly distributed into control and treatment groups (n=7) and gavaged. The control group received treatment with the vehicle, which consisted of 0.5% (w/v) methylcellulose and 0.1% (v/v) Tween-80 in sterile water. The treatment group was given tetrandrine at 50 mg/kg of body weight for 20 days, and the tumors were dissected and weighed.

CD31 panendothelial antigen (platelet/endothelial cell-adhesion molecule) was used for microvessel staining in frozen sections (25,26). Frozen sections were then blocked with 5% goat serum and stained with rat anti-mCD31 antibody (1:50 dilution; BD Biosciences-Pharmingen) at room temperature for 1 h. Sections were then washed with PBS and incubated for 1 h with biotinylated poly-clonal anti-rat IgG antibody. The sections were washed three times with PBS, reacted with the ABC peroxidase kit (Vector Laboratories) at room temperature for 45 min, and washed twice with PBS prior to mounting for light microscopy and photography.

The number of vessels was counted in a blinded manner in 18 sections (2 sections per slide, 3 slides per tissue) of the tissue using a light microscope at ×200 magnification. Five fields in each section were randomly selected, and the number of vessels in each field was averaged (27).

The results are expressed as the mean ± SE. The Tukey-Kramer multiple comparisons test was used to determine statistical significance. A P-value of <0.05 was considered to be statistically significant.

Tetrandrine, a compound isolated from a traditional Chinese medicine plant (Fig. 1A), has been found to be an antitumor agent (Fig. 1A) (10,28). After treatment with tetrandrine at indicated concentrations for 12, 24, 48 and 72 h, EOMA cells were analysed in proliferation assays. The proliferation of EOMA cells was inhibited, and the growth of EOMA cells was clearly suppressed after treatment with tetrandrine from 20 to 80 μM for 48 h (Fig. 1B and C).

Based on the above results, tetrandrine concentrations of 10, 20, 30, 40 and 50 μM and an incubation period of 48 h were selected as conditions for cell cycle analysis by flow cytometry. As shown in Fig. 2A, the proportion of EOMA cells in S phase was decreased from 20.8% in control cells to 19.0, 16.0, 16.2, 14.2 and 13.9% in cells treated with tetrandrine for 48 h at concentrations of 10, 20, 30, 40 and 50 μM, respectively, while the proportion of cells in G1/G0 was increased from 60.9 to 63.7, 67.1, 62.7, 63.9 and 67.5% under the same treatments. Thus, these results indicated that tetrandrine induced G1/S cell cycle arrest in EOMA cells in a dose-dependent manner.

To further investigate the observed G1/S cell cycle arrest, cell cycle regulation proteins, including cyclin D1, cyclin D2, cyclin D3, cyclin E1, cyclin E2, CDK2, CDK4 and CDK6 were determined by western blotting. The expression levels of cyclin D1, cyclin D2, cyclin E1, cyclin E2, CDK2 CDK4 and CDK6 were decreased in tetrandrine-treated cells compared to the control cells, but the expression of cyclin D3 was not affected by tetrandrine treatment (Fig. 2B). In addition, the expression levels of the proteins, including GSK-3β, p53 and p27, which were the inhibitors for the cell cycle regulators, were upregulated with tetrandrine treatment (Fig. 2C).

Since tetrandrine has been reported as an anticancer agent both in vitro and in vivo by inducing apoptosis through the formation of ROS (29), which can serve as important second messengers to regulate a variety of downstream signalling pathways (30–32), we determined the effect of tetrandrine on intracellular ROS levels in EOMA cells using DCFH-DA fluorescence. As shown in Fig. 3A, the number of fluorescent sites, representing the level of ROS, was higher in tetrandrine-treated cells than in the control cells. Flow cytometry results also showed that the ROS level was increased by treatments with 30 and 50 μM of tetrandrine as compared to the control, up to 2-fold in the 50 μM tetrandrine-treated cells (Fig. 3B). These results demonstrate that ROS was produced in EOMA cells in response to tetrandrine.

To further investigate the role of ROS in the upregulation of tetrandrine-treated EOMA cells, the ROS inhibitor was used. ROS accumulation was observed in EOMA cells after 48 h of tetrandrine treatment, but was markedly abrogated when cells were pretreated with the ROS scavenger NAC, demonstrating an effective elimination of tetrandrine-induced ROS production by NAC (Fig. 4A). Also in the cell cycle analysis, pretreatment with NAC inhibited the G1/S cell cycle arrest in tetrandrine-treated EOMA cells (Fig. 4B and C).

To determine the association between cell cycle arrest and the increased ROS in EOMA cells, the expression levels of cyclins and their upstream proteins were analysed by western blotting. The downregulation of cyclin D1, cyclin E1, CDK2, CDK4 and CDK6 in EOMA cells was inhibited by pretreatment with 20 mM of NAC for 1 h prior to treatment with 50 μM of tetrandrine for 48 h (Fig. 4D).

It is known that ROS generation is related with the PI3K-Akt signalling pathway. Akt is a critical kinase that regulates a variety of biological processes, including cell survival, proliferation, autophagy and apoptosis (33–36). To identify if Akt was involved in tetrandrine treated EOMA cells, western blots were performed as previously described. As shown in Fig. 5A, the level of phosphorylated Akt was decreased after tetrandrine treatment although the total Akt protein level remained unchanged. Then, to determine whether tetrandrine induced ROS upregulation by decreasing Akt activity, the EOMA cells were treated with tetrandrine in combination with LY294002 (a PI3K/Akt inhibitor) for 48 h and the ROS level was determined by flow cytometry. The results indicate that LY294002 did not inhibit tetrandrine-induced increase of ROS in EOMA cells (Fig. 5B).

Next, to determine whether the phosphorylation of Akt was regulated by the increased levels of ROS, the EOMA cells were pretreated with 20 mM of NAC for 1 h followed by treatment with 50 μM of tetrandrine for 48 h, and both total Akt and phosphorylated Akt were analysed by western blotting. As shown in Fig. 5C, the tetrandrine-induced decrease of Akt phosphorylation was blocked by NAC pretreatment. Taken together, these data suggest that ROS acted upstream of the Akt pathway to regulate the cell cycle.

To further establish that Akt was involved in tetrandrine-induced G1/S arrest, empty vector (PUSE) or PUSE-CA-Akt was transfected into EOMA cells. After tetrandrine treatment, the cell number of EOMA cells with Akt overexpression was nearly twice that of the control cells (Fig. 6A). It indicated that Akt overexpression could promote cell cycle transition and then had an effect on cell proliferation. Next, even with tetrandrine treatment, it showed that the proportion in S phase of EOMA cells with Akt overexpression had a significantly increase to 41.2%, compared with the control cells with 28.9% in S phase (Fig. 6B and C). Finally, the western blot analysis also checked the expression levels of cyclin D1, cyclin E1, CDK2, CDK4 and CDK6, which were important for G1/S arrest. As shown in Fig. 6D, when EOMA cells were transfected with the control vector, and the cells were treated with tetrandrine, cyclin D1, cyclin E1, CDK2, CDK4 and CDK6 were significantly downregulated. However, when the EOMA cells were transfected with Akt overexpressing vector, and treated with tetrandrine, the protein levels did not change much comparing with the control cells. This also suggested that tetrandrine-induced G1/S cell cycle arrest was rescued because of the overexpression of Akt. Collectively, considering the above, we concluded that the overexpression of Akt decreases tetrandrine-induced G1/S arrest.

Since the regulation of the endothelial cell cycle is critical to the function of the vascular system (14), it suggests that tetrandrine may be an effective angiogenesis inhibitor in cancer therapy. To evaluate the antitumor effect of tetrandrine in vivo, we investigated whether tetrandrine could inhibit tumor vessel growth in nude mice. As previously described, the mice bearing Huh7 tumor xenografts were gavaged with tetrandrine (50 mg/kg of body weight) or vehicle every other day, and the amount of vessels inside the tumor was observed in tumor sections after 20 days of treatment (9). Then we found the tumor vascular growth was visually inhibited by tetrandrine, and the data confirmed its anti-angiogenic activity (Fig. 7A). Vascular density was also measured in anti-CD31 stained vasculatures from both control tumor mice and tetrandrine-treated tumor mice. The results of immunohistochemical analysis showed a significant decrease of microvessel density in tetrandrine-treated group (Fig. 7B), further demonstrating the inhibition of liver cancer vascular growth in nude mice bearing liver cancer xenografts. Consistent with the reduction of tumor vascular growth by tetrandrine treatment in vivo, the average tumor weight in the animals treated with vehicle, tetrandrine therapy was 1.11 and 0.53 g (P<0.01), respectively (Fig. 7C). Therefore, tetrandrine also has antitumor effect and one of the important reasons is its anti-angiogenic function.