20(S)-ginsenoside-Rg3 reverses temozolomide resistance and restrains epithelial-mesenchymal transition progression in glioblastoma

Abstract

Glioblastoma multiforme (GBM) is one of the most malignant human intracranial tumors. Temozolomide (TMZ) is the primary alkylating agent for GBM patients. However, many GBM patients are resistant to TMZ. Therefore, patients with GBM urgently need more effective therapeutic options. 20(S)-ginsenoside-Rg3 (20(S)-Rg3) is a natural chemical with anti-tumor effects, but at present there is little understanding of its functional mechanism. Several research reports have demonstrated that O⁶ -methylguanine DNA-methyltransferase (MGMT) repairs damaged DNA and contributes to TMZ resistance in gliomas. In addition, recent studies have shown that MGMT gene expression could be regulated by the Wnt/β-catenin pathway. However, whether 20(S)-Rg3 inhibits MGMT expression and augments chemosensitivity to Temozolomide (TMZ) in glioma cells remains unclear. In this study, we explored the modulating effects of 20(S)-Rg3 on MGMT. We used glioma cell lines, primary cell strain (including T98G, U118 and GBM-XX; all of them are MGMT-positive glioma cell lines) and xenograft glioma models to examine whether 20(S)-Rg3 increased the sensitivity to TMZ and to reveal the underlying mechanisms. We found that the MGMT expression was effectively downregulated by 20(S)-Rg3 via the Wnt/β-catenin pathway in glioma cell lines, and TMZ resistance was significantly reversed by 20(S)-Rg3. Meanwhile, 20(S)-Rg3 shows no obvious cytotoxicity at its effective dose and is well tolerated in vivo. In addition, we found that 20(S)-Rg3 significantly restrains the epithelial-mesenchymal transition (EMT) progression of glioma cells. Taken together, these results indicate that 20(S)-Rg3 may be a novel agent to use in treatment of GBM, especially in TMZ-resistant GBM with high MGMT expression.

Keywords: 20(S)-ginsenoside-Rg3; O6-methylguanine DNA-methyltransferase; Wnt/β-catenin pathway; glioblastoma multiforme; temozolomide.

Figure 1

20(S)‐Rg3 inhibits the expression of O⁶‐methylguanine DNA‐methyltransferase (MGMT) in glioma cell lines. T98G, U118 and GBM‐XX cells were seeded in 96‐well flat‐bottom plates at 5000 cells/well, cultured in DMEM supplemented with 10% FBS, and then treated with increasing concentrations of 20(S)‐Rg3 or temozolomide (TMZ), or DMSO as a control; 72 h later, 10 μL of cell counting kit‐8 mix reagent was added to 100 μL of media per well, and the cells were incubated at 37°C for 2 h. The optical density (OD) was measured at 450 nm with a spectrophotometer. A,B, The half maximal inhibitory concentration of 20(S)‐Rg3 and TMZ on glioma cells is approximately 200 and 250 μmol/L. C, T98G, U118 and GBM‐XX were treated with 20(S)‐Rg3 (100 μmol/L) for 72 h, and the total RNA was extracted with TRIzol; then the expression of MGMT mRNA level was determined by quantitative real‐time PCR (n = 4). ***P < .001 compared with the control group. D,E, T98G, U118 and GBM‐XX were treated with 20(S)‐Rg3 (100 μmol/L); 72 h later, expression of MGMT was determined by western blot (n = 4). ***P < .001 compared with control group. F,G, T98G, U118 and GBM‐XX cells were cultured on coverslips. After the cells attached to the slips over 24 h, 20(S)‐Rg3 (100 μmol/L) was added to the culture media for 72 h. Then expression of MGMT in glioma cells was determined by immunocytochemistry assay (n = 4). *P < .05, **P < .01 compared with control group

Figure 2

20(S)‐Rg3 inhibits Wnt/β‐catenin pathways activity. After treatment of 20(S)‐Rg3 (100 μmol/L), Wnt/β‐catenin pathway‐related proteins were determined by western blot. The expression of β‐catenin was repressed in all of T98G, U118 and GBM‐XX cell lines, 2 important transcriptional factors of Wnt/β‐catenin signaling, LEF1 and TCF1/TCF7, were both depressed by 20(S)‐Rg3. Meanwhile, the target genes CD44, C‐Jun, C‐Myc, cyclinD1, MMP7 and survivin were decreased, and the changes were similar to those of MGMT

Figure 3

20(S)‐Rg3 augments temozolomide (TMZ)‐mediated chemotherapy. A,B,C, T98G, U118 and GBM‐XX cells were seeded in 96‐well plates at a density of 5 × 10³ cells/well. After 12 h of cell attachment, we treated them with TMZ (100 μmol/L) and/or 20(S)‐Rg3 (100 μmol/L), or DMSO as a control for 72 h. Then cell viability was determined by cell counting kit‐8 mix reagent at indicated time points (24, 48, 72 h). As shown in cell viability assays, 20(S)‐Rg3 (100 μmol/L) had no cytotoxicity by itself. However, 20(S)‐Rg3 significantly enhanced the cytotoxicity of TMZ (n = 6), *P < .05, ***P < .001 compared with control group, n.s., not statistically significant. D,E, The combination of 20(S)‐Rg3 and TMZ significantly increase the apoptosis of glioma cells. T98G, U118 and GBM‐XX cells were treated with 20(S)‐Rg3 (100 μmol/L) or/and TMZ (100 μmol/L) for 72 h, and then the apoptosis rates of glioma cells were determined by flow cytometry (n = 3). ***P < .001 compared with control group; n.s., not statistically significant. F, After treatment of 20(S)‐Rg3 (100 μmol/L) and/or TMZ (100 μmol/L) on T98G for 48 h, the expression of apoptosis‐related proteins including BAX, Bcl‐2, caspase‐3, cleaved‐caspase‐3, PARP and cleaved‐PARP, were determined by western blot. As shown in the figure, 20(S)‐Rg3+ TMZ increased the expression of BAX, cleaved‐caspase‐3 and cleaved‐PARP, and conversely decreased Bcl‐2, caspase‐3 and PARP expression in glioma cell line T98G

Figure 4

20(S)‐Rg3 displays synergistic activity with temozolomide (TMZ) in vivo. A,B, Tumor xenografts were established by subcutaneous inoculation of U118 cells into the armpit of the right upper limb of nude mice. Seven days later, tumors reached approximately 100 mm³; mice were treated every day for 4 wks with 20 mg/kg TMZ alone or 20 mg/kg 20(S)‐Rg3+ 20 mg/kg TMZ. The size of tumors in nude mice was monitored weekly with a vernier caliper; 4 wks later, mice were killed and tumors were excised and weighed (n = 6). *P < .05, ***P < .001. C, O⁶‐methylguanine DNA‐methyltransferase (MGMT) expression level of tumor tissue was determined by western blot (n = 3). *P < .05 compared with control group

Figure 5

20(S)‐Rg3 inhibits migration and invasion of glioma cells. A, T98G, U118 and GBM‐XX cells were seeded into 6‐well plates and incubated at 37°C until cells reached a confluence of at least 90%, then scratched and stimulated with 20(S)‐Rg3 (60, 120 μmol/L) or without 20(S)‐Rg3, and migration was monitored by cells within the wound area at 0 and 24 h. B, Migrated cells were quantitated using the ImageJ software program (n = 4). *P < .05, **P < .01, ***P < .001. C, 1 × 10⁵ T98G, U118 and GBM‐XX cells were seeded in the upper chamber with serum‐free medium in 24‐well Transwell plates that were pre‐coated with Matrigel; 300 μL complete culture medium containing 10% FBS and 20(S)‐Rg3 (0, 60, 120 μmol/L) was added to the lower chamber as a chemo‐attractant. We dyed them with crystal violet 24 h later. D, Invasive cells were quantitated using the ImageJ software program (n = 3). *P < .05, **P < .01, ***P < .001 compared with control group

Figure 6

20(S)‐Rg3 increased the expression level of E‐cadherin and decreased the expression levels of N‐cadherin and vimentin. A, After treatment of 20(S)‐Rg3(120 μmol/L for 48 h), epithelial‐mesenchymal transformation‐related proteins, including E‐cadherin, N‐cadherin and vimentin, were determined by western blot. The expression of E‐cadherin in T98G, U118 and GBM‐XX increased; meanwhile, N‐cadherin and vimentin decreased uniformly. B, Immunofluorescence and assay showed the same result as with western blots in T98G and GBM‐XX cells. C,D Effect of 20(S)‐Rg3 on peritoneal spreading and metastasis in vivo. GBM‐XX cells (100 μL, 2 × 10⁶) were injected i.p. into each 4‐wk‐old male nude mouse. Seven days later, mice received PBS or 20 mg/kg of 20(S)‐Rg3 every day by i.p. injection for 20 d. After 20 d, mice were killed and peritoneal metastatic nodules were calculated. n = 4, *P < .05 compared with the control group