Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT-29 colorectal cancer cells, in order to compare aspirin-mediated acetylation of G6PD and its activity between HCT 116 and HT-29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT-29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin-acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH.

Aspirin increases mitochondrial fatty acid oxidation

“…This acetyl group has been shown by others to acetylate cytosolic proteins in cultured cells [24–26], but the effects on mitochondrial proteins have not been investigated. First, we measured aspirin-induced acetylation in whole cell extracts.…”

Aspirin increases metabolism through germline signalling to extend the lifespan of Caenorhabditis elegans

“…The primary metabolite of aspirin (acetyl SA) SA can bind to plant and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which plays a central role in glycolysis and suppresses nuclear translocation [ 35 ]. Similar results showed that aspirin could acetylate and inhibit glucose-6-phosphate dehydrogenase (G6PD), which catalyses the first reaction in the pentose phosphate pathway and is important for the regulation of oxidative stress [ 36 ]. For the mechanism of action in anti-cancer and chemoprevention activities, aspirin and salicylic acid can directly bind to CDK2 and down-regulate cyclin A2/CDK2 proteins [ 37 ] and inhibit CBP and p300 lysine acetyltransferase activity in vitro through direct competition with acetyl-Coenzyme A at the catalytic site [ 38 ].…”

Disrupting G6PD-mediated Redox homeostasis enhances chemosensitivity in colorectal cancer

“…Beside, recent study showed that aspirin inhibits G6PD activity in CRC cells through acetylation. 32 Additionally, a novel clinical-stage radiosensitizer and chemosensitizer RRx-001 exerts the anti-proliferative effects through inhibiting G6PD activity in human tumor cells. 33 Future study should focus on the development of selective inhibitors for G6PD and the combinatorial effects with clinical chemotherapy drugs.…”