Ethyl pyruvate inhibits glioblastoma cells migration and invasion through modulation of NF-κB and ERK-mediated EMT

Abstract

Background: Glioblastoma is a grade IV glioma with the highest degree of malignancy and extremely high incidence. Because of the poor therapeutic effect of surgery and radiochemotherapy, glioblastoma has a high recurrence rate and lethality, and is one of the most challenging tumors in the field of oncology. Ethyl pyruvate (EP), a stable lipophilic pyruvic acid derivative, has anti-inflammatory, antioxidant, immunomodulatory and other cellular protective effects. It has been reported that EP has potent anti-tumor effects on many types of tumors, including pancreatic cancer, prostate cancer, liver cancer, gastric cancer. However, whether EP has anti-tumor effect on glioblastoma or not is still unclear.

Methods: Glioblastoma U87 and U251 cells were treated with different concentrations of EP for 24 h or 48 h. CCK8 assay and Colony-Formation assay were performed to test the viability and proliferation. Wound-healing assay and Transwell assay were carried out to measure cell invasion and migration. Western blot was not only used to detect the protein expression of epithelial-mesenchymal transition (EMT)-related molecules, but also to detect the expression and activation levels of NF-κB (p65) and Extracellular Signal Regulated Kinase (ERK).

Results: In glioblastoma U87 and U251 cells treated with EP, the viability, proliferation, migration, invasion abilities were inhibited in a dose-dependent manner. EP inhibited EMT and the activation of NF-κB (p65) and ERK. With NF-κB (p65) and ERK activated, EMT, migration and invasion of U87 and U251 cells were promoted. However the activation of NF-κB (p65) and ERK were decreased, EMT, migration and invasion abilities were inhibited in U87 and U251 cells treated with EP.

Conclusion: EP inhibits glioblastoma cells migration and invasion by blocking NF-κB and ERK-mediated EMT.

Keywords: EMT (epithelial–mesenchymal transition); ERK; Ethyl pyruvate; Glioblastoma; Invasion; Migration; NF-κB.

Figure 1. EP inhibits cell viability and colony formation in U251 and U87 cells.

(A and B) Effect of EP on cell viability in U251 and U87 cells. CCK8 assay was carried out to detect the cell viability 48 h after cells were treated with increasing doses of EP. EP significantly decreased cell viability in a dose dependent manner. (C–L) Effect of EP on colony formation in U251 and U87 cells. EP significantly decreased the number of colonies in a dose-dependent manner. Cell clones were stained with crystal violet, then the stained clones were dissolved with 1% SDS and the absorbance of the solution at 450 nm wavelength was detected after 10–14 days. Data are represented as mean ± SD of three independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Ordinary one-way ANOVA Multiple comparisons).

Figure 2. EP attenuate the migration and invasion of U251 and U87 cells.

(A–Z) Wound healing assay of U251 and U87 cells after treatment with 0, 10, 20, 30 mM EP for 0, 12 and 24 h. (AA–JJ) Transwell migration assay of U251 and U87 cells after treatment with 0, 10, 20, 30 mM EP for 24 h. (KK–TT) Transwell invasion assay of U251 and U87 cells after treatment with 0, 10, 20, 30 mM EP for 24 h. Data are represented as mean ± SD of three independent experiments. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Two-way ANOVA Multiple comparisons).

Figure 3. EP inhibits epithelial-mesenchymal transition and inhibits NF-κB (p65) and ERK phosphorylation in U251 and U87 cells.

(A–C) EP promotes epithelial marker E-cadherin expression and inhibits the expression of mesenchymal marker Vimentin and EMT related transcription factor ZEB1, Snail, Twist1 in U251 and U87 cells with a dose-dependent manner. All protein levels were evaluated in EP treated cells after 48 h. (D–F) EP inhibits the expression and activation of NF-κB (p65) and ERK in U251 and U87 cells with a dose-dependent manner. All protein levels were evaluated in EP treated cells after 48 h. Data are represented as mean ± SD of three independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Two-way ANOVA Multiple comparisons).

Figure 4. Effects of NF-κB and ERK on U251 and U87 cells migration and invasion ability.

(A–Z) Wound healing assay of U251 and U87 cells after treated with control group, TNF-α (10 μM) group, TNF-α coupled with U0126 (10 pg/L) group and TNF-α coupled with EP (20 mM) group for 0, 12 and 24 h. (AA–TT) Transwell migration assay and Transwell invasion assay of U251 and U87 cells after treated with control group, TNF-α (10 μM) group, TNF-α coupled with U0126 (10 pg/L) group and TNF-α coupled with EP (20 mM) group for 24 h. Data are represented as mean ± SD of three independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Two-way ANOVA Multiple comparisons).

Figure 5. EP inhibitis EMT induced by NF-κB and ERK pathways in U251 and U87 cells.

(A–C) The expression level of NF-κB (p65), ERK and their phosphorylated proteins in U251 and U87 cells treated with different conditions including control group, TNF-α (10 μM) group, TNF-α coupled with U0126 (10 pg/L) group, TNF-α coupled with EP (20 mM) group. (D–F) The protein expression levels of E-cadherin and mesenchymal associated genes/activators in U251 and U87 cells treated with different conditions including control group, TNF-α (10 μM) group, TNF-α coupled with U0126 (10 pg/L) group, TNF-α coupled with EP (20 mM) group. Data are represented as mean ± SD of three independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (Two-way ANOVA Multiple comparisons).