Corilagin induces apoptosis and autophagy in NRF2‑addicted U251 glioma cell line

Abstract

Corilagin, extracted from the Euphorbiaceae and Phyllanthus plants, inhibits the growth of a number of types of tumors. Compared with temozolomide, the traditional chemotherapy drug, corilagin has demonstrated stronger antitumor activity. However, the pharmaceutical mechanism of corilagin in glioma remains unclear. Nuclear factor erythroid 2 like 2 (NFE2L2 or NRF2) is positively associated with several types of tumor including glioma. In the present study, NRF2 expression was higher in glioma tissues compared with non‑glioma specimens. Therefore, it was hypothesized that corilagin targets NRF2 regulation of U251 cell apoptosis. The present study used Hoechst 33258 staining to demonstrate that corilagin induced glioma cell apoptosis and observed that the expression of the apoptosis‑related gene Bcl‑2 was reduced. In addition, corilagin induced autophagy and promoted the conversion of light chain 3 (LC3) protein from LC3Ⅰ to LC3II. NRF2 expression was downregulated by corilagin stimulation. Furthermore, the gene expression pattern following knockdown of NRF2 in U251 cells using siRNA was consistent with corilagin stimulation. Therefore, it was preliminarily concluded that corilagin induces apoptosis and autophagy by reducing NRF2 expression.

Keywords: corilagin; nuclear factor erythroid 2 like 2; glioma; autophagy; apoptosis.

Figure 1.

NRF2 is overexpressed in glioma tissues and negatively correlates with the survival rates of patients. (A) NRF2 was overexpressed in the 15 glioma tissues compared with the 9 control samples by western blotting. (B) The relative protein expression level of NRF2 was calculated using β-actin as the loading control (*P<0.05). (C) The NRF2 mRNA expression profiles in control brain tissue (n=207), GBM (T=163) and LGG (T=518) from a data set from GEPIA; *P<0.05. (D) The difference between the low and high NRF2 expression groups. There were 337 patients with low expression of NRF2 and there were 338 patients with high expression of NRF2 (GEPIA). HR demonstrated that the risk of mortality in the NRF2 high expression group was 1.9 times higher compared with that of the NRF2 low expression group. NRF2, nuclear factor erythroid 2 like 2; T, glioma tissues; C, control samples; GBM, glioblastoma multiforme; LGG, low grade glioma; GEPIA, Gene Expression Profiling Interactive Analysis; HR, Hazard Ratio.

Figure 2.

Corilagin downregulates the expression of NRF2. (A) Following stimulation of U251 cells by 100 µg/ml corilagin for 48 h, the protein expression of NRF2 was detected by western blotting and (B) the relative protein expression level of NRF2 was calculated using β-actin as the loading control (*P<0.05). (C) Following stimulation of U251 cells by 100 µg/ml corilagin for 48 h, the protein expression of NRF2 was detected by immunofluorescence. Scale bar, 20 µm. NRF2, nuclear factor erythroid 2 like 2; con, control.

Figure 3.

Corilagin induces the apoptosis of U251 glioma cell line. (A) Hoechst 33258 staining showed that the level of cell apoptosis following adding different concentrations of corilagin (0, 25, 50 and 100 µg/ml) for 48 h to U251 cells. Scale bar, 50 µm. (B) Western blotting identified the protein expression level of Bcl-2 following adding different concentrations of corilagin (0, 25, 50 and 100 µg/ml) for 48 h to U251 cells. (C) The mRNA expression of Bcl-2 was detected in corilagin-stimulated cells by reverse transcription-quantitative PCR normalized to β-actin (*P<0.05; ***P<0.001). con, control.

Figure 4.

Corilagin induces the autophagy of U251 glioma cell line. (A) Following stimulation of U251 cells by EBSS and 100 µg/ml corilagin, cell autophagy-related protein LC3B were detected by immunofluorescence. Scale bar, 10 µm. (B) Western blotting demonstrated that the protein level of LC3II in the EBSS and corilagin treatment group were increased and the expression of P62 was significantly decreased. (C) The mRNA expression level of P62 was significantly reduced in the corilagin-treated (100 µg/ml) group as determined by reverse transcription-quantitative PCR normalized to β-actin (**P<0.01). (D) The relative protein ratio of LC3II/LC3I and P62 were calculated using β-actin as the loading control (**P<0.01; *P<0.05). EBSS, Earle's Balanced Salt Solution; LC3, light chain 3; con, control;

Figure 5.

NRF2 regulates apoptosis and autophagy. (A) The U251 cells were transfected with siRNAs by Lipofectamine^® 2000. The protein expression of NRF2, P62, Bcl-2 and LC3 were detected by western blotting. (B) The relative protein quantitative analysis of the ratio LC3II/LC3I, P62, Bcl-2 and NRF2 were calculated using β-actin as the loading control (*P<0.05). NRF2, nuclear factor erythroid 2 like 2; con, control; si, short interfering; NC, negative control; siN, siRNA of NRF2.