J Korean Neurosurg Soc.  2017 Feb;60(2):130-137. 10.3340/jkns.2016.0101.006.

Memantine Induces NMDAR1-Mediated Autophagic Cell Death in Malignant Glioma Cells

• Yoon WS
• Yeom MY
• Kang ES
• Chung YA
• Chung DS
• Jeun SS

• KMID: 2374873
• DOI: http://doi.org/10.3340/jkns.2016.0101.006

Abstract

OBJECTIVE
Autophagy is one of the key responses of cells to programmed cell death. Memantine, an approved anti-dementia drug, has an antiproliferative effect on cancer cells but the mechanism is poorly understood. The aim of the present study was to test the possibility of induction of autophagic cell death by memantine in glioma cell lines.
METHODS
Glioma cell lines (T-98 G and U-251 MG) were used for this study.
RESULTS
The antiproliferative effect of memantine was shown on T-98 G cells, which expressed N-methyl-D-aspartate 1 receptor (NMDAR1). Memantine increased the autophagic-related proteins as the conversion ratio of light chain protein 3-II (LC3-II)-/LC3-I and the expression of beclin-1. Memantine also increased formation of autophagic vacuoles observed under a transmission electron microscope. Transfection of small interfering RNA (siRNA) to knock down NMDAR1 in the glioma cells induced resistance to memantine and decreased the LC3-II/LC3-I ratio in T-98 G cells.
CONCLUSION
Our study demonstrates that in glioma cells, memantine inhibits proliferation and induces autophagy mediated by NMDAR1.

Keyword

Memantine; Autophagy; N-methyl-D-aspartate; Glioma

MeSH Terms

Autophagy*
Cell Death
Cell Line
Gastrin-Secreting Cells
Glioma*
Memantine*
N-Methylaspartate
RNA, Small Interfering
Transfection
Vacuoles
Memantine
N-Methylaspartate
RNA, Small Interfering

Figure

• 

  Fig. 1 Memantine-induced cytotoxicity in glioma cell lines. After incubation with memantine (0–600 μM), malignant glioma cells were grown for 48 h starting at a density of 104/well. The number of viable cells was counted and expressed as a percentage of the untreated control. The results are representative of 3 independent experiments. *p<0.05, Student’s t-test.

• 

  Fig. 2 Expression levels of NMDAR1 and NMDAR2B in U-251 MG and T-98 G cells. The cell lines were analyzed using western blotting (A) and RT-PCR (B). NMDAR1 was present only in T-98 G cells, but NMDAR2B was detectable in both glioma cell lines. β-actin and GAPDH were used as internal controls. NMDAR: N-methyl-D-aspartate receptor, RT-PCR: reverse transcription polymerase chain reaction, GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 3 Memantine upregulated LC3-II and beclin-1 in T-98 G cells. A: Protein levels of LC3-I and LC3-II were analyzed in T-98 G cells treated with 0 and 400 μM memantine for 48 h. The graph shows the results of densitometric quantitation of western blots, presented as the LC3-II/LC3-I ratio relative to the control (0 μM of memantine). LC3-II was significantly increased by 400 μM memantine. B: Protein expression levels of AMPKα, beclin-1, APG5L, and ULK1 were evaluated using western blotting after incubation with memantine (0 and 400 μM) for 48 hr in T-98 G cells. The results of densitometric quantitation of immunoblots are presented as the ratio relative to the control (0 μM of memantine). Beclin-1 was significantly upregulated after the treatment with 400 μM of memantine. *p<0.05, Student’s t-test. AMPK: AMP-activated protein kinase.

• 

  Fig. 4 Memantine induced autophagic ultrastructural features in T-98 G cells according to TEM. A: The control cells showed the normal cytoplasm including mitochondria, ribosomes, and endoplasmic reticulum. B: After incubation with 400 μM memantine for 48 h, the cells developed autophagic vacuoles with lamellar structure in the cytoplasm. TEM: transmission electron microscopy.

• 

  Fig. 5 A knockdown of NMDAR1 using siRNA transfection in T-98 G cells. T-98 cells incubated in EMEM was used as the control value. A: After the transfection with anti-NMDAR1 siRNA (10 nM) for 48 hr, the expression of NMDAR1 was evaluated using western bloting. B: Cell viability of control and siRNA-transfected T-98 G cells was analyzed after incubation with various concentration of memantine (0–600 μM). Significant differences in cell viability was observed at 400, and 600 μM of memantine. C: Protein level of LC3-I and LC3-II was evaluated in control and NMDAR1 knockdown cells using western blotting after incubation with 400 μM memantine. The results of densitometric quantitation of immunoblots were represented as the LC3-II/LC3-I ratio relative to control cells. *p<0.05, Student’s t-test. NMDAR: N-methyl-D-aspartate receptor, siRNA: small interfering RNA, EMEM: Eagle’s minimum essential medium.

Reference

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