Resveratrol enhances the antitumor effects of temozolomide in glioblastoma via ROS-dependent AMPK-TSC-mTOR signaling pathway

Abstract

Background and purpose: Resveratrol has been regarded as a promising candidate for cancer prevention and treatment. The present study was to investigate the impact of resveratrol on the antitumor effects of temozolomide (TMZ), a standard treatment regiment of glioblastoma (GBM), in vitro and in vivo.

Methods and results: We found that the combination of resveratrol and TMZ significantly resulted in G(2)/M cell cycle arrest by flow cytometry, triggered a robust increase in expression of astrocyte differentiation marker glial fibrillary acid protein (GFAP), downregulated the expression of matrix metalloproteinase-9 (MMP-9) by immunohistochemistry and western blot analysis as well as inhibited cell migration by scratch wound assay. Further study revealed that TMZ in combination with resveratrol remarkably increased reactive oxygen species (ROS) production, which serves as an upstream signal for AMP-activated protein kinase (AMPK) activation. Subsequently, activated AMPK inhibited mTOR signaling and downregulated antiapoptosis protein Bcl-2, which was contributed to the additive antiproliferation effects of combination treatment. In an orthotopic xenograft model of GBM, TMZ plus resveratrol treatment significantly reduced the volume of tumor, which was confirmed by decreased expression of Ki-67, a marker of proliferation index.

Conclusions: Our findings demonstrate for the first time that resveratrol can enhance TMZ-mediated antitumor effects in GBM in vitro and in vivo, via ROS-dependent AMPK-TSC-mTOR signaling pathway.

Figure 1

TMZ alone or in combination with resveratrol (RES) induces cell death that is additive in effect. (A) and (B) MTT assay results showed that TMZ and RES decreased the cell viability in a concentration dependent manner after treatment for 72 h, respectively, and the vehicle of 500 μM TMZ (TMZ 500 V) had no effect on the viability of these cells. (C) MTT assay indicated that combination of TMZ and RES had an additive effect on inhibiting cell proliferation. (D) Fluorescence microscopy images of Hoechst 33342‐stained cells showed the appearance of apoptotic morphology in TMZ at 100 μM plus RES at 10 μM for 72 h. Lower histogram: quantitative analysis of condensed nucleus as in above fluorescence images. P represents statistical analysis. *P < 0.05 versus control group; ^# P < 0.05 versus TMZ.

Figure 2

Combination treatment with TMZ and resveratrol (RES) modulates the cell cycle in SHG44 cells. Representative flow‐cytometric histograms of DNA content of TMZ and RES individually and the combination. (A) The treatment of TMZ at 100 μM and RES at 10 μM arrest cell cycle in G₂/M phase and S‐phase, respectively. Combination the two agents significantly arrest cell cycle at G₂/M phase. (B) The percentage of sub‐G₁ phase was measured by flow cytometry. P represents statistical analysis. *P < 0.05 versus control group; ^# P < 0.05 versus TMZ.

Figure 3

Combination of TMZ and resveratrol (RES) prevented cell migration and inhibited cell metastasis. Representative pictures of SHG44 cells were at 0 h and 48 h after treatment (when a wound was made), and 24 h later, respectively. SHG44 cells subjected to in vitro scratch assay. The migration ability was estimated quantitatively by measuring the remaining area between the two edges of the wound. (A) At the beginning of 24 h, the gap distance was reduced to 1 μM in control groups and RES‐treated groups, and the gap distance of the TMZ group and the combined group was 83.2 ± 12.8 μM and 84.4 ± 26.4 μM, respectively (P= 0.8676). (B) During the last 24 h, the gap distance was reduced to 1 μM under control conditions, and after the treatment of RES and TMZ alone, the gap distance was reduced to 41.7 ± 8.8 and 87.3 ± 21.3 μM, indicating the partial inhibition of cell migration, and the most inhibitory effect after combination treatment was 304.1 ± 31.7 μM. The covered distance was estimated in five different areas of the culture, in three independent experiments. (C) Cells were incubated with indicated the treatment of TMZ at 100 μM and RES at 10 μM for 72 h, and then, cell proteins were obtained and analyzed with anti‐MMP‐9 antibodies by Western blot. The combination treatment significantly down regulated the expression of MMP‐9. *P < 0.05 versus control group (CON); ^#P < 0.05 versus TMZ.

Figure 4

Differentiation effects on the treatment of TMZ and resveratrol (RES). SHG44 cells were treated with either TMZ at 100 μM or RES at 10 μM and the combination for 72h, the expression of GFAP was detected with fluorescence microscopy images (A) and Western blot (B), respectively. (C) In nude model, the GFAP expression (arrow) was by immunohistochemisty after the combination of TMZ (68 mg/kg × 3 d) and RES (40 mg/kg, daily). (T: tumor; N: normal brain). *P < 0.05 versus control group (CON);^# P < 0.01 versus TMZ group.

Figure 5

TMZ combined with resveratrol (RES) in GBM orthotopic xenografts. (A) Mice with established orthotopic xenografts from SHG44 were randomized to therapy with the indicated placebo (0.5% carboxymethylcellulose), TMZ (68 mg/kg × 3 d), RES (40 mg/kg, daily) and the combination, change in body weight for mice from the experiment treated with placebo, RES, TMZ, or combination. (B) Representative tumor volumes of orthotopic nude mice were evaluated by magnetic resonance images (MRI). (C) Pathologic analysis of mouse brains. Low (2×), intermediate power images of the tumor area in four different animals. Intracranial tumors were identified in coronal brain sections by H&E staining. (D) Immunohistochemical staining for Ki‐67 (×400). Brown nuclear staining for Ki‐67 indicated proliferating cells. The percentage of Ki‐67‐positive cells in treated tumors. Combination treatment of TMZ and RES showed significant effect in vivo. P values are comparing an indicated treatment, change in body weight for mice from the experiment treated with placebo, RES, TMZ, or combination. *P < 0.005 versus placebo group (CON); ^# P < 0.001 versus TMZ.

Figure 6

Combination with TMZ and resveratrol (RES)‐induced production of ROS and mediated AMPK signaling in SHG44 cells. (A) DCFH‐DA fluorescence (green) imaging of ROS in SHG44 cells. Cells were labeled with DCFH‐DA 10 mM and then treated with indicated combination of TMZ and RES for 6 h. (B) Antioxidant (NAC) inhibited the antitumor effect of combination with TMZ and RES. MTT assay showed pretreatment with NAC (1 mM) for 1 h inhibited the combination therapy‐induced cell viability decline. (C) Cells were incubated with indicated the treatment of TMZ at 100 μM and RES at 10 μM for 72 h, and then, cell proteins were obtained and analyzed with anti‐phospho‐AMPK and anti‐AMPK antibodies by Western blot. The combination of TMZ and RES significantly increased the phosphorylation of AMPK. AMPK inhibitor Compoud C (CC) partly abolished the antitumor effect of TMZ plus RES. MTT assay showed pretreatment with CC 1 μM partly reduced the combinational therapy induced the antigrowth (D) and apoptosis effect (E). (F) mTOR signaling in SHG44 cells remained responsive to the combination of TMZ and RES. mTOR activity is phosphorylated at a lowest level following the combination treatment. TSC was involved in mTOR signaling pathway in response to the treatment. Wild‐type and TSC−/− MEF cells were treated with indicated treatment for 72 h and then cellular viability was detected with MTT assay. Notably, compared to TEC−/− MEF cells, the combination TMZ and RES markedly decreased the cellular viability in wild‐type MEF cells (G). Wild‐type and TSC−/− MEF cells were incubated with the combination for 72 h, and then, cell proteins were obtained and analyzed with the expression of mTOR by Western blot (H). (I) The combination of TMZ and RES significantly inhibited the expression of Bcl‐2 in SHG44. (J) Schematic model for the mechanisms of the combination with TMZ and RES exerted antitumor effect. The combinational treatment induced ROS generation, thereby activated the proapoptotic stress kinase AMPK via TSC, AMPK inhibited the mTOR activation, which subsequently mediated the antitumor effects, and eventually triggered apoptosis. The results are representative of more than three independent experiments. *P < 0.05 versus control group (CON); ^# P < 0.05 versus TMZ;^$ P < 0.05 versus RES+TMZ. ^& P < 0.05 TSC^+/+ versus TSC^−/−.