Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is pyruvylated during 3-bromopyruvate mediated cancer cell death

Abstract

Background: The pyruvic acid analog 3-bromopyruvate (3BrPA) is an alkylating agent known to induce cancer cell death by blocking glycolysis. The anti-glycolytic effect of 3BrPA is considered to be the inactivation of glycolytic enzymes. Yet, there is a lack of experimental documentation on the direct interaction of 3BrPA with any of the suggested targets during its anticancer effect.

Methods and results: In the current study, using radiolabeled ((14)C) 3BrPA in multiple cancer cell lines, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the primary intracellular target of 3BrPA, based on two-dimensional (2D) gel electrophoretic autoradiography, mass spectrometry and immunoprecipitation. Furthermore, in vitro enzyme kinetic studies established that 3BrPA has marked affinity to GAPDH. Finally, Annexin V staining and active caspase-3 immunoblotting demonstrated that apoptosis was induced by 3BrPA.

Conclusion: GAPDH pyruvylation by 3BrPA affects its enzymatic function and is the primary intracellular target in 3BrPA mediated cancer cell death.

Figure 1

Selective binding of 3BrPA to intracellular proteins. (A) Coomassie blue-stained SDS-PAGE gels and their corresponding autoradiograms are shown for HepG2 and Vx-2 cell lines treated with ¹⁴C-3BrPA. Arrow head indicates the gel band excised and subjected to mass spectrometry characterization. (B) Silver-stained 2D gel of whole cell lysate from SK-Hep1 cells treated with ¹⁴C-3BrPA and its corresponding autoradiogram showing ¹⁴C-3BrPA incorporation. The circle indicates the intense gel spot excised and characterized by mass spectrometry.

Figure 2

GAPDH pyruvylation with 3BrPA treatment. (A) LCMS/MS chromatogram showing GAPDH as the peptide identified from autoradiogram spot of the 2D gel. (B) Immunoblot of a 2D gel showing GAPDH at the same molecular mass and pI value as the spot identified on 2D autoradiogram. (C) Autoradiogram of immunoprecipitates from the SK-Hep1 cells treated with ¹⁴C-3BrPA showing the ¹⁴C incorporation. Lanes indicate; W, Whole cell lysate; lanes L, P, G and H correspond to specific antibodies against the targets LDH, PDH, GAPDH and HK II, respectively. T, Target-specific antibody and C, Control IgG for the respective target.

Figure 3

Effect of pyruvylation on enzymatic function. (A) Dose-dependent decrease in the GAPDH activity in 3BrPA-treated cell lines. (B) HK II activity after 3BrPA treatment of various cell lines. (C) Activity of purified GAPDH and HK II. Equal quantities of enzymes (enzyme units) were used for these assays.

Figure 4

Lactate levels and LDH activity in 3BrPA-treated cell lines. (A) Intracellular lactate at different concentrations of 3BrPA treatment. (B) LDH activity in SK-Hep1 and Hep3B cells in the presence or absence of 3BrPA. (C) Activity of purified LDH in the presence or absence of 3BrPA (100 µM). Results are represented as mean ± standard error (n=3). C, Control; T, treated with 3BrPA.

Figure 5

Effect of 3BrPA on the cell viability based on ATP depletion in Hep3B, HepG2, SK-Hep1 and Vx-2 cell lines. Cells were treated for (A) 3 h, (B) 24 h or (C) 48 h. Results are represented as mean ± standard error (n=3). The results were also confirmed by Trypan blue staining independently.

Figure 6

Flow cytometry showing 3BrPA-induced dose-dependent apoptotic cell death. A minimum of 10⁴ cells treated with 0 µM 3BrPA (Control) or 200 µM 3BrPA (Treated) for 2 h were subjected to analysis with Annexin V-PE/7-AAD staining.

Figure 7

Fluorescent (lower panel) and light (upper panel) micrographs showing Annexin V staining in 3BrPA (200 µM) treated cell lines and controls (vehicle alone).

Figure 8

Immunoblot showing caspase-3 activation by 3BrPA in different cell lines. Protein samples from equal number of cells were loaded onto each lane. The blot is a representative of duplicate experiments.