Anat Cell Biol.  2018 Dec;51(4):274-283. 10.5115/acb.2018.51.4.274.

Quercetin induces cell death in cervical cancer by reducing O-GlcNAcylation of adenosine monophosphate-activated protein kinase

• Ali A
• Kim MJ
• Kim MY
• Lee HJ
• Roh GS
• Kim HJ
• Cho GJ
• Choi WS

• KMID: 2430195
• DOI: http://doi.org/10.5115/acb.2018.51.4.274

Abstract

Hyper-O-GlcNAcylation is a general feature of cancer which contributes to various cancer phenotypes, including cell proliferation and cell growth. Quercetin, a naturally occurring dietary flavonoid, has been reported to reduce the proliferation and growth of cancer. Several reports of the anticancer effect of quercetin have been published, but there is no study regarding its effect on O-GlcNAcylation. The aim of this study was to investigate the anticancer effect of quercetin on HeLa cells and compare this with its effect on HaCaT cells. Cell viability and cell death were determined by MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assays. O-GlcNAcylation of AMP-activated protein kinase (AMPK) was examined by succinylated wheat germ agglutinin pulldown and immunoprecipitation. Immunofluorescence staining was used to detect the immunoreactivitiy of O-linked N-acetylglucosamine transferase (OGT) and sterol regulatory element binding protein 1 (SREBP-1). Quercetin decreased cell proliferation and induced cell death, but its effect on HaCaT cells was lower than that on HeLa cells. O-GlcNAcylation level was higher in HeLa cells than in HaCaT cells. Quercetin decreased the expression of global O-GlcNAcylation and increased AMPK activation by reducing the O-GlcNAcylation of AMPK. AMPK activation due to reduced O-GlcNAcylation of AMPK was confirmed by treatment with 6-diazo-5-oxo-L-norleucine. Our results also demonstrated that quercetin regulated SREBP-1 and its transcriptional targets. Furthermore, immunofluorescence staining showed that quercetin treatment decreased the immunoreactivities of OGT and SREBP-1 in HeLa cells. Our findings demonstrate that quercetin exhibited its anticancer effect by decreasing the O-GlcNAcylation of AMPK. Further studies are needed to explore how quercetin regulates O-GlcNAcylation in cancer.

Keyword

Quercetin; O-GlcNAcylation; AMPK; Sterol regulatory element binding protein 1; Uterine cervical neoplasms

MeSH Terms

Adenosine*
AMP-Activated Protein Kinases
Cell Death*
Cell Proliferation
Cell Survival
Deoxyuridine
Diazooxonorleucine
Fluorescent Antibody Technique
HeLa Cells
Humans
Immunoprecipitation
Phenotype
Protein Kinases*
Quercetin*
Sterol Regulatory Element Binding Protein 1
Transferases
Triticum
Uterine Cervical Neoplasms*
AMP-Activated Protein Kinases
Adenosine
Deoxyuridine
Diazooxonorleucine
Protein Kinases
Quercetin
Sterol Regulatory Element Binding Protein 1
Transferases

Figure

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  Fig. 1 Quercetin decreases cell viability and induces cell death in HaCaT and HeLa cells. (A–C) MTT assay of HaCaT and HeLa cells after treatment with quercetin (0–200 µM) for 24 hours. Bar graph representing the viability of HaCaT (A) and HeLa (B) cells. (C) Line graph representing the comparison of cell viability between HaCaT and HeLa cells. (D) Representative western blot analysis and relative bar graph quantification of PARP, cleaved PARP, caspase 3, and cleaved caspase 3 in HeLa and HaCaT cells after treatment with 50 µM quercetin for 24 hours. Band intensity was normalised to β-actin. Each experiment was performed three times. PARP, poly (ADP ribose) polymerase; SEM, standard error of mean. Data represent the mean±SEM of three independent experiments. **P<0.005, ***P<0.001.

• 

  Fig. 2 Quercetin decreases the expression of OGT, O-GlcNAc, and O-GlcNAcylated AMPK. Representative western blot analysis and relative bar graph quantification of OGT (A) and O-GlcNAc (B) in HeLa and HaCaT cells after treatment with 50 µM quercetin for 24 hours. (C) Upper panel: western blots using anti-O-GlcNAc and AMPK antibodies, of O-GlcNAcylated proteins precipitated by agarose-bound succinylated WGA beads, from HaCaT and HeLa cells treated with or without 50 µM quercetin for 24 hours. The specificity of WGA binding was confirmed by adding GlcNAc. Lower panel: relative bar graph quantification of relative O-GlcNAc levels. (D) Upper panel: western blots, using anti-O-GlcNAc and AMPK antibodies, of AMPK immunoprecipitated from cell lysates of HaCaT and HeLa cells treated with or without 50 µM quercetin for 24 hours. Lower panel: relative bar graph quantification of relative O-GlcNAc levels. Densitometry of immunoprecipitated AMPK to O-GlcNAc was normalised to IgG. OGT, O-linked N-acetylglucosamine transferase; O-GlcNAc, O-linked N-acetylglucosamine; AMPK, AMP-activated protein kinase; WGA, wheat germ agglutinin; SEM, standard error of mean; CTL, control; Quer, quercetin; IB, immunoblot. Each experiment was performed three times. Data represent the mean±SEM of three independent experiments. *P<0.05, **P<0.005, ***P<0.001.

• 

  Fig. 3 O-GlcNAcylation regulates activation of AMPK. (A) Representative western blot analysis and relative bar graph quantification of O-GlcNAc, AMPK, and p-AMPK in HeLa and HaCaT cells after treatment with 100 µM DON or 10 µM TMG for 24 hours. β-actin was used as a loading control. (B) Upper panel: western blots, using anti-O-GlcNAc and AMPK antibodies, of O-GlcNAcylated proteins precipitated by agarose-bound succinylated WGA beads, from cell lysates of HaCaT and HeLa cells after treatment with 100 µM DON or 10 µM TMG for 24 hours. Specificity of WGA binding was confirmed by adding GlcNAc. Lower panel: quantitative analysis of relative O-GlcNAc level. (C) Upper panel: western blots, using anti-O-GlcNAc and AMPK antibodies, of AMPK immunoprecipitated from the cell lysates of HaCaT and HeLa cells treated with 100 µM DON or 10 µM TMG for 24 hours. Lower panel: quantitative analysis of relative O-GlcNAc levels. Densitometry of immunoprecipitated AMPK to O-GlcNAc was normalised to IgG. AMPK, AMP-activated protein kinase; O-GlcNAc, O-linked N-acetylglucosamine; DON, 6-diazo-5-oxo-norleucineis; TMG, Thiamet G; WGA, wheat germ agglutinin; CTL, control. Each experiment was performed three times. Data represent the mean±SEM of three independent experiments. *P<0.05, **P<0.005, ***P<0.001.

• 

  Fig. 4 Quercetin regulates SREBP-1 and its target proteins by activating AMPK via regulating O-GlcNAcylation. (A) Quercetin treatment causes decreased O-GlcNAcylation, increased p-AMPK levels, and decreased levels of SREBP-1 and its target proteins. Representative western blot analysis and relative bar graph quantification of O-GlcNAc, AMPK, pAMPk, SREBP-1, ACC, and FAS in the cell lysates of HeLa and HaCaT cells treated with or without 50 µM quercetin. (B) O-GlcNAcylation regulates expression of p-AMPK, SREBP-1, and SREBP-1 target proteins. Representative western blot analysis and relative bar graph quantification of O-GlcNAc, AMPK, pAMPk, SREBP-1, ACC, and FAS in the cell lysates of HeLa and HaCaT cells treated with 100 µM DON or 10 µM TMG. Band intensity was normalised to β-actin. SREBP-1, sterol regulatory element binding protein 1; AMPK, AMP-activated protein kinase; O-GlcNAc, O-linked N-acetylglucosamine; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; DON, 6-diazo-5-oxo-norleucineis; SEM, standard error of mean; TMG, Thiamet G; CTL, control; Quer, quercetin. Each experiment was performed three times. Data represent the mean±SEM of three independent experiments. *P<0.05, **P<0.005, ***P<0.001.

• 

  Fig. 5 Quercetin decreases the immunoreactivity of OGT and SREBP-1, and increases the number of TUNEL-positive cells. Representative images of double immunofluorescence staining for OGT (green) and SREBP-1 (red) in HaCaT and HeLa cells after treatment with or without 50 µM quercetin for 24 hours. Merged images were generated by overlaying the images of OGT and SREBP-1 staining. The enlarged portion is indicated by the box. (B) Representative image of TUNEL (red) along with DAPI (blue) staining for nuclear localisation in HaCaT and HeLa cells after treatment with or without 50 µM quercetin for 24 hours. Merge images were generated by overlaying the images of TUNEL and DAPI staining. Arrows indicate TUNEL-positive cells. OGT, O-linked N-acetylglucosamine transferase; SREBP-1, sterol regulatory element binding protein 1; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling; CTL, control; Quer, quercetin. Scale bars=100 µm.

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