Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid, has been reported to have a radiosensitization effect on tumors. However, its effects on human glioma and the specific molecular mechanisms of these effects remain unknown. In this study, we demonstrated that Tet has a radiosensitization effect on human glioma cells. It has been hypothesized that Tet has a radiosensitization effect on glioma cells by affecting the glioma cell cycle and DNA repair mechanism and that ERK mediates these activities. Therefore, we conducted detailed analyses of the effects of Tet on the cell cycle by performing flow cytometric analysis and on DNA repair by detecting the expression of phosphorylated H2AX by immunofluorescence. We used western blot analysis to investigate the role of ERK in the effect of Tet on the cell cycle and DNA repair. The results revealed that Tet exerts its radiosensitization effect on glioma cells by inhibiting proliferation and decreasing the expression of phosphorylated ERK and its downstream proteins. In summary, our data indicate that ERK is involved in Tet-induced radiosensitization of glioma cells via inhibition of glioma cell proliferation or of the cell cycle at G0/G1 phase.
The most common primary brain tumor is glioma, which constitutes approximately 80% of all malignant brain tumors (Ostrom
Tetrandrine (Tet) is a bisbenzylisoquinoline alkaloid extracted from the root of the Chinese traditional medicine
Extracellular signal-regulated kinases (ERKs) belong to the Ras/Raf/MEK/ERK pathway, which plays an important role in the survival of cells, including cancer cells (Sun
In this study, we investigated the radiosensitive effects of Tet on glioma and further examined the effect of ERK on Tet-induced radiosensitization.
The human glioma cell lines U251 and U87 were obtained from Shanghai Institute of Cell Biology, the Chinese Academy of Sciences, Shanghai, China. These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Paisley, Scotland, UK) with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin and 100 units/ml penicillin (Biyuntian Biotechnology, Suzhou, China). All cell lines were maintained in a humidified incubator (Thermo Fisher Scientific, MA, USA) at 37°C and 5% CO2.
Cells were plated onto 96-well plates in triplicate at a density of 3×103 cells per well and allowed to adhere overnight in DMEM medium. Cells were incubated with Tet (Sigma-Aldrich Bio, CA, USA) for 24 h. After this incubation period, 20 μl/well of MTT solution (5 mg/ml phosphate-buffered saline [PBS]) was added, and the cells were incubated for 5 h. Then, the medium was aspirated and replaced with 150 μl/well of acidic isopropanol (0.04 N HCl in isopropanol) to dissolve the formazan salt that had formed. The absorbance (OD) of the formazan solution, which reflects the cell growth conditions, was measured at 570 nm using a microplate spectrophotometer (ELx800 BioTek Instruments, LA, USA). The results are represented as the OD ratio of the 20% inhibitory concentration (IC20) of Tet at 24 h, which was calculated and chosen for the following experiments (Ma
The cells were lysed in SDS buffer containing protease inhibitors and phosphorylated protease inhibitors (F. Hoffmann-La Roche Ltd., Basel, Switzerland). The obtained protein samples were subjected to 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes with a pore size of 0.22 μm, blocked in 5% fat-free milk and incubated overnight at 4°C with antibodies directed against phosphorylated ERK (p-ERK), total ERK (t-ERK), CCND1, PCNA and GAPDH (Cell Signaling Technology, NY, USA). All of these antibodies were diluted 1:1000. Protein expression was detected by horse-radish peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL, USA, diluted 1:10,000). Immunoreactive bands were visualized using an enhanced chemiluminescence assay (Pierce).
Cells were cultured in DMEM complete medium for 24 h and then treated with 20 μM U0126 (Cell Signaling Technology) for 24 h.
Cells were exposed to radiation at a dose rate of 1.21 Gy/min with 160 kV photons at room temperature using a RS2000 Biological Research Irradiator that contained a linear accelerator (Rad Source Technologies Asia Limited, LA, USA).
Glioma cells were cultured on cover glasses slips and treated with or without Tet at IC20 for 24 h. Then, the cells were exposed to radiotherapy, fixed in 4% formaldehyde for 10 min, permeabilized with 0.1% Triton X-100 in PBS, and incubated with 5% bovine serum albumin (BSA) and rabbit anti-human monoclonal phosphorylated H2AX (p-H2AX, 1:400; Cell Signaling Technology) for 60 min at room temperature and overnight at 4°C, respectively. Cultured cell slides were then incubated with Alexa Fluor 594 goat anti-rabbit IgG secondary antibody (1:1000; Cell Signaling Technology) for 1 h and with 5 μg/ml of DAPI for 15 min at 37°C. The slides were then washed with PBS and imaged under a fluorescence microscope (Olympus IX51, Fukushima, Japan). The number of p-H2AX foci per cell was determined using stored images of 50 cells (Xiao
U87 and U251 cells (2×106 cells/flask) cultured in flasks (Corning, USA) were treated with Tet at the respective IC20 concentrations or with the same volume of DMSO (control) for 24 h. Then, 100~1×105 cells were seeded in T75 flasks. After 8 h, the control and Tet-treated cells were exposed to radiation (0, 2, 4, 6, or 8 Gy) and then cultured with medium for 14–20 days. When colonies had formed, the cells were fixed in 4% formaldehyde and stained with gentian violet. The numbers of clones were then scored manually. Specifically, a colony was defined as more than 50 cells in a group. Survival fractions (SFs) were calculated as follows: SF=[the mean plating efficiency of radiation (± Tet)-treated cells divided by the mean plating efficiency of control (± Tet) cells], expressed as a %. SFs of combination-treated cells were corrected for the cytotoxicity of Tet. Radiation treatment (RT) survival curves were fitted according to the linear-quadratic model using GraphPad Prism 5.0 software (GraphPad, San Diego, CA, USA): Survival= e–(αD+βD2) (Xiao
Cells were seeded in six-well plates, treated with Tet at IC20 for 24 h and then exposed to radiation. Before the cells were analyzed, 100 μl of binding buffer containing 1 μl of 100 μg/ml propidium iodide (PI) was added to these cells, and the cells were incubated for 30 min in the dark. Analyses were performed using a FACScan flow cytometer (Beckman Coulter, CA, USA). Cell cycle distribution was calculated based on DNA plots using MultiCycle software (Phoenix Flow Systems, San Diego, CA, USA).
All experiments were performed at least three times (n=3). The data are presented as χ̄ ± SEM. The experimental data were statistically analyzed using SPSS 13.0 for Windows (Chicago, IL, USA). Two-way ANOVA was used to study the influences of RT dose and Tet treatment on cell survival as determined by clonogenic assay. A two-sample
As shown in Fig. 1A, cell viability significantly decreased with increased Tet concentrations in a dose-dependent manner. The IC20 values of Tet for U87 and U251 cells were 3.91 ± 1.09 and 4.36 ± 1.32, respectively. Thus, 4 μM Tet was chosen for the following experiments. We treated two glioma cell lines (U251 and U87) with 4 μM Tet for 24 h followed by exposure to different doses of radiotherapy. Next, we investigated the SFs of the two glioma cell lines by clonogenic assay. As shown in Fig. 1B and 1C, the SFs of Tet treatment groups decreased after received radiotherapy compared with control cell lines (U251, U87) at the same dose. Finally, we calculated the parameters for RT and RT+Tet. As shown in Table 1, the SF2 of RT+Tet was lower than that of RT alone, and the SERs of U251 and U87 cells were 1.716 and 2.884. Drugs are considered to cause radiation sensitivity when the SER is above 1 (Xiao
Initially, we observed the cell cycle distribution of U251 and U251+Tet cells after radiotherapy by flow cytometry (FCM). As shown in Fig. 2A and 2B, the proportions of cells in G0/G1 phase in the Tet treatment group were higher than were those in the control group. We also noticed that the proportions of cells in G2/M phase increased gradually after radiotherapy until 12 h in both U251 and U251+Tet groups in Fig. 2C; however, we did not observe that Tet could increase the G2/M proportions after the cells received radiotherapy. Next, we detected the repair of radiation-induced DNA double-strand breaks (DSBs) by calculating the number of p-H2AX foci. As shown in Fig. 2D and 2E, the average p-H2AX foci. Numbers increased, reached peaks at 4 h after 2 Gy radiotherapy and then decreased gradually in both U251 and U251+Tet groups. More importantly, no differences in the p-H2AX foci numbers were observed between the U251 and U251+Tet groups. These data indicated that Tet exerted a radiosensitization effect on glioma through inhibiting proliferation and blocking cells at G0/G1 phase, not by enhancing DNA damage.
As we observed that Tet could enhance radiotherapy by inhibiting proliferation, we detected the proliferation-related protein p-ERK and its downstream proteins. As shown in Fig. 3A, p-ERK expression increased at 4 and 6 h after radiotherapy. Simultaneously, p-ERK expression could not be stimulated when U251 cells were treated with Tet, even after receiving radiotherapy at the same time point. We also investigated the expression of the proliferation-related proteins CCND1 and PCNA, which are also downstream proteins of p-ERK. The expression of CCND1 and PCNA decreased after the cells were treated with Tet. The relative expression levels of these proteins are shown in Fig. 3B–3E. These data indicated that Tet could inhibit p-ERK and its downstream proteins even after receiving radiotherapy.
We used U0126 to inhibit p-ERK expression (Stepanenko
The anti-tumor mechanism of Tet, isolated from the root of
The effects of Tet inhibition of tumor cell proliferation are variable. In hepatoma, Tet was shown to induce cell cycle arrest at G2/M phase (Ng
In conclusion, we demonstrated that Tet exerts its radiosensitization effect on glioma cells by inhibiting proliferation, which is mediated by decreasing the expression of p-ERK and its downstream proteins (Fig. 5). In this context, Tet could potentially be used as a radiosensitizer when patients with glioma receive radiation.
This research was supported by a grant from the Science and Technology Project of Guangzhou (No. 2014J4100103).