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

Ai G, Dachineni R, Kumar DR, Alfonso LF, Marimuthu S, Bhat GJ - Mol Med Rep (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, South Dakota State University College of Pharmacy, Avera Health and Sciences Center, Brookings, SD 57007, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Effects of aspirin on glucose-6-phosphate dehydrogenase (G6PD) activity in HCT 116 and HT-29 cells. Cells were cultured and left untreated or were treated with aspirin for 24 h. Protein (100 µg) was used to conduct a G6PD assay. The reaction mixture was incubated at 37°C for 30 min, and absorbance was measured at 450 nm. G6PD activity was expressed as a percentage of control. The experiments were repeated three times. Data are represented as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs. the control.
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f2-mmr-14-02-1726: Effects of aspirin on glucose-6-phosphate dehydrogenase (G6PD) activity in HCT 116 and HT-29 cells. Cells were cultured and left untreated or were treated with aspirin for 24 h. Protein (100 µg) was used to conduct a G6PD assay. The reaction mixture was incubated at 37°C for 30 min, and absorbance was measured at 450 nm. G6PD activity was expressed as a percentage of control. The experiments were repeated three times. Data are represented as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs. the control.

Mentions: To compare the levels of aspirin-mediated G6PD acetylation between HCT 116 and HT-29 cells, the cells were left untreated or were treated with two different concentrations of aspirin (0.5 and 2.5 mM) for 24 h. The lysates were then immunoprecipitated with anti-acetyl lysine antibody, and immunoblotted with anti-G6PD antibody. As shown in Fig. 1A, aspirin-induced acetylation of G6PD was detected at 0.5 mM in HCT 116 cells; however, markedly increased levels were detected at 2.5 mM. Conversely, in the HT-29 cells, G6PD acetylation was not observed at all at 0.5 mM, and was barely detected at 2.5 mM (Fig. 1B). As presented in Fig. 1C and D both isoforms of G6PD (isoforms a and b) were detected in HCT 116 and HT-29 cells. G6PD activity was then detected in lysates prepared from both HCT 116 and HT-29 cells. Aspirin progressively inhibited G6PD activity in HCT 116 cells beginning at 0.5 mM; however, inhibition was not so strong in HT-29 cells (Fig. 2). These results suggest that aspirin-mediated acetylation may lead to inhibition of G6PD activity within the cellular milieu.


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

Ai G, Dachineni R, Kumar DR, Alfonso LF, Marimuthu S, Bhat GJ - Mol Med Rep (2016)

Effects of aspirin on glucose-6-phosphate dehydrogenase (G6PD) activity in HCT 116 and HT-29 cells. Cells were cultured and left untreated or were treated with aspirin for 24 h. Protein (100 µg) was used to conduct a G6PD assay. The reaction mixture was incubated at 37°C for 30 min, and absorbance was measured at 450 nm. G6PD activity was expressed as a percentage of control. The experiments were repeated three times. Data are represented as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs. the control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940102&req=5

f2-mmr-14-02-1726: Effects of aspirin on glucose-6-phosphate dehydrogenase (G6PD) activity in HCT 116 and HT-29 cells. Cells were cultured and left untreated or were treated with aspirin for 24 h. Protein (100 µg) was used to conduct a G6PD assay. The reaction mixture was incubated at 37°C for 30 min, and absorbance was measured at 450 nm. G6PD activity was expressed as a percentage of control. The experiments were repeated three times. Data are represented as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs. the control.
Mentions: To compare the levels of aspirin-mediated G6PD acetylation between HCT 116 and HT-29 cells, the cells were left untreated or were treated with two different concentrations of aspirin (0.5 and 2.5 mM) for 24 h. The lysates were then immunoprecipitated with anti-acetyl lysine antibody, and immunoblotted with anti-G6PD antibody. As shown in Fig. 1A, aspirin-induced acetylation of G6PD was detected at 0.5 mM in HCT 116 cells; however, markedly increased levels were detected at 2.5 mM. Conversely, in the HT-29 cells, G6PD acetylation was not observed at all at 0.5 mM, and was barely detected at 2.5 mM (Fig. 1B). As presented in Fig. 1C and D both isoforms of G6PD (isoforms a and b) were detected in HCT 116 and HT-29 cells. G6PD activity was then detected in lysates prepared from both HCT 116 and HT-29 cells. Aspirin progressively inhibited G6PD activity in HCT 116 cells beginning at 0.5 mM; however, inhibition was not so strong in HT-29 cells (Fig. 2). These results suggest that aspirin-mediated acetylation may lead to inhibition of G6PD activity within the cellular milieu.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, South Dakota State University College of Pharmacy, Avera Health and Sciences Center, Brookings, SD 57007, USA.

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.

No MeSH data available.


Related in: MedlinePlus