The mechanisms of malic enzyme 2 in the tumorigenesis of human gliomas

Abstract

The high level of resistance of glioblastoma multiforme (GBM) to currently used chemotherapies and other conventional therapies, its invasive characteristics and the presence of stem-like cells are the major factors that make the treatment of GBM difficult. Recent studies have demonstrated that the homeostasis of energy metabolism, glycolysis and mitochondrial oxidation of glucose are important for GBM cell growth and chemo-resistance. However, it is not clear which specific gene(s) are involved in the homeostasis of energy metabolism and invasiveness of GBM cells. We performed a preliminary analysis of data obtained from Gene Expression Omnibus profiles and determined that malic enzyme 2 (ME2) expression was positively associated with WHO grade in human primary gliomas. Hence, we evaluated the detailed working mechanisms of ME2 in human GBM cell processes, including proliferation, cell cycle, invasion, migration, ROS, and ATP production. Our data demonstrated that ME2 was involved in GBM growth, invasion and migration. ME2 has two cofactors, NAD+ or NADP+, which are used to produce NADH and NADPH for ATP production and ROS clearance, respectively. If the catalytic activity of ME2 is determined to be critical for its roles in GBM growth, invasion and migration, small molecule inhibitors of ME2 may be valuable drugs for GBM therapy. We hope that our current data provides a candidate treatment strategy for GBM.

Keywords: glioblastoma multiforme; malic enzyme 2; p53; reactive oxygen species; tumorigenesis.

Figure 1. Expression level of ME2 in human gliomas of various stages

Gene expression of ME2 in microarray datasets (GDS1962) of human gliomas: 26 cases of WHO grade II and III astrocytoma, including 7 cases of diffuse astrocytoma (WHO grade II), 19 cases of anaplastic astrocytoma (WHO grade III), and 81 cases of glioblastoma (WHO grade IV), compared with non-tumor controls. Two groups (benign and malignant) of data were analyzed using unpaired Student's t-test. (*** P < 0.001).

Figure 2. ME2 abundance in several human glioma cell lines

A. Cells lysates (GBM8401, U87MG, LN229 glioma cells and positive control HepG2 hepatoma cells); B. GBM8401/shME2 (286588 and 294005); C. LN229/shME2 (286588 and 294005) and shLuc control cells were subjected to Western blotting with antibody against ME2 and ACTN antibody as the loading control. SE represents short exposure time, and LE represents long exposure time. Results are representative data from two independent experiments.

Figure 3. The effects of ME2 on cell proliferation and cell cycle profile in human glioma cell lines

GBM8401 and LN229 shME2 cells were seeded (5×10⁴) in 12-well plates, and A. cell number were measured at the indicated time points; B. cell cycle analysis of GBM8401 and LN229 shME2 cells was determined by flow cytometry and C. BrdU incorporation by cells was performed using FITC BrdU Flow Kit. M1, BrdU-negative cells; M2, BrdU-positive cells. Cells untreated with BrdU was used as blank. The results of the statistical analysis are shown. Results are presented as the mean ± SD of triplicate samples from representative data of three independent experiments. (# p>0.05).

Figure 4. The effects of ME2 on soft-agar colony formation in human glioma cell lines

Soft agar assays of GBM8401 and LN229 shME2 (286588 and 294005) and shLuc control cells are shown in A. and the statistical analysis is shown in B. Colonies > 0.5 mm were counted using ImageJ software. Results were presented as the mean ± SD of triplicate samples from representative data of three independent experiments. (*** P < 0.001).

Figure 5. The effects of ME2 on invasion and migration in GBM8401 cells

Invasion assay A. and migration assay B. of GBM8401 shME2 (286588 and 294005) and shLuc control cells are shown, and the statistical analysis was performed. Results were presented as the mean ± SD of triplicate samples from representative data of three independent experiments. (*P < 0.05).

Figure 6. The effects of ME2 on ATP, ROS and lactate production in GBM8401 cells

A. ATP levels in GBM8401 shME2 (286588 and 294005) and shLuc control cells were measured and normalized to the respective protein concentration. (**P < 0.01) B. ROS formation in GBM8401 shME2 (286588 and 294005) and shLuc control cells was detected by DCFH-DA staining. Cells were stained with DCFH-DA and measured by flow cytometry. Cells untreated with DCFH-DA were used as blanks. Results were presented as the mean ± SD of triplicate samples from representative data of three independent experiments. (**P < 0.01) C. Lactate production level in GBM8401 shME2 (286588 and 294005) and shLuc control cells were measured and normalized to the respective protein concentrations. Results are presented as the mean ± SD of triplicate samples from representative data of three independent experiments. (*** P < 0.001).

Figure 7. The effects of ME2 on the AMPK signaling and EMT pathways in GBM8401 cells

Western blot analysis was utilized to determine the protein levels of AMPK signaling (p-ERK (Thr202/Tyr204), total ERK, p-Akt (Ser473), total Akt, p-AMPK (Thr172), total AMPK, p-ACC (Ser79) and total ACC) and EMT pathway markers (GFAP, vimentin, Snail, Twist and MMP-2) in GBM8401 shME2 (286588 and 294005) and shLuc control cells. ACTN was the loading control. Results are representative data of two independent experiments.

Figure 8. The effects of ME2 on the p53-dependent transcription functions in human glioma cell lines

A. RT-PCR analysis of p53, p21 and cyclin D1 gene expressions in GBM8401 and LN229 shME2 (286588 and 294005) and shLuc control cells. GAPDH was the loading control. B. Western blot analysis of p53, p21 and cyclin D1 protein levels in GBM8401 and LN229 shME2 (286588 and 294005) cells and shLuc control cells. GBM8401 C. and LN229 D. cells were transfected with indicated amounts of miR 17-5p for 72 hrs and Western blot analysis was utilized to determine the protein levels of p53, p21 and cyclin D1. ACTN was the loading control. Results are representative data of two independent experiments.