Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 May 31;14(5):e0217695.
doi: 10.1371/journal.pone.0217695. eCollection 2019.

Mechanism of the efflux transport of demethoxycurcumin-O-glucuronides in HeLa cells stably transfected with UDP-glucuronosyltransferase 1A1

Beibei Zhang  1 , Jing Yang  1 , Zifei Qin  1   2 , Shishi Li  2 , Jinjin Xu  2 , Zhihong Yao  2   3 , Xiaojian Zhang  1 , Frank J Gonzalez  4 , Xinsheng Yao  2   3
Affiliations

Mechanism of the efflux transport of demethoxycurcumin-O-glucuronides in HeLa cells stably transfected with UDP-glucuronosyltransferase 1A1

Beibei Zhang et al. PLoS One. .

Abstract

Demethoxycurcumin (DMC) is a safe and natural food-coloring additive, as well as an agent with several therapeutic properties. However, extensive glucuronidation in vivo has resulted in its poor bioavailability. In this study, we aimed to investigate the formation of DMC-O-glucuronides by uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) and its transport by breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in HeLa cells stably transfected with UGT1A1 (named HeLa1A1 cells). The chemical inhibitors Ko143 (a selective BCRP inhibitor) and MK571 (a pan-MRP inhibitor) both induced an obvious decrease in the excretion rate of DMC-O-glucuronides and a significant increase in intracellular DMC-O-glucuronide concentrations. Furthermore, BCRP knock-down resulted in a marked reduction in the level of excreted DMC-O-glucuronides (maximal 55.6%), whereas MRP1 and MRP4 silencing significantly decreased the levels of excreted DMC-O-glucuronides (a maximum of 42.9% for MRP1 and a maximum of 29.9% for MRP3), respectively. In contrast, neither the levels of excreted DMC-O-glucuronides nor the accumulation of DMC-O-glucuronides were significantly altered in the MRP4 knock-down HeLa cells. The BCRP, MRP1 and MRP3 transporters were identified as the most important contributors to the excretion of DMC-O-glucuronides. These results may significantly contribute to improving our understanding of mechanisms underlying the cellular disposition of DMC via UGT-mediated metabolism.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Liquid chromatography tandem mass spectrometry analysis of demethoxycurcumin and demethoxycurcumin-O-glucuronides.
(A) Ultra high-performance liquid chromatography chromatograms of demethoxycurcumin and demethoxycurcumin-O-glucuronides (G1 and G2) generated in wild-type HeLa cells and HeLa1A1 cells; (B) Extracted ion chromatograms of demethoxycurcumin and demethoxycurcumin-O-glucuronides (G1 and G2); (C) ESI-MS/MS spectra of demethoxycurcumin and demethoxycurcumin-O-glucuronides (G1 and G2) in positive ion mode; (D) Glucuronidation pathways of demethoxycurcumin by HeLa1A1 cells.
Fig 2
Fig 2. UGT1A1 catalyzed the demethoxycurcumin-O-glucuronidation, and expression of UGT1A1 and efflux transporters in HeLa1A1 cells.
(A) Kinetic profiles for demethoxycurcumin-O-glucuronides (G1 and G2) (0.5–40 μM) by UGT1A1; (B) Kinetic profiles for demethoxycurcumin-O-glucuronides (G1 and G2) (0.5–40 μM) by HeLa1A1 cell lysate; In each panel, the insert figure showed the corresponding Eadie-Hofstee plot. (C) Protein expression of UGT1A1, BCRP and four MRP family transporters in HeLa and HeLa1A1 cells. All experiments were performed in triplicate (n = 3). Data were expressed as mean ± SD (n = 3).
Fig 3
Fig 3. Validation of UGT1A1 in the demethoxycurcumin-O-glucuronidation by inhibitory assays and activity correlation analysis assays.
(A) Inhibitory effects of nilotinib (10 μM), glycyrrhetinic acid (20 μM) and protopanaxatriol (500 μM) on the glucuronidation activities of G1 and G2 at 4 μM of demethoxycurcumin; (B) Correlation analysis between β-estradiol-3-O-glucuronidation and demethoxycurcumin-O-glucuronidation (G1) in a bank of individual human liver microsomes (n = 12); (C) Correlation analysis between β-estradiol-3-O-glucuronidation and demethoxycurcumin-O-glucuronidation (G2) in twelve individual HLMs; All experiments were performed in triplicate (n = 3). Data were expressed as mean ± SD (n = 3). *, # p < 0.05, **, ## p < 0.01 or ***, ### p < 0.001 compared with that of control group of G1 and G2, respectively.
Fig 4
Fig 4. Concentration dependent excretions of demethoxycurcumin-O-glucuronides (G1 and G2) in HeLa1A1 cells.
(A) Excreted demethoxycurcumin-O-glucuronides (G1) when treated with different concentrations of demethoxycurcumin (2, 4 and 8 μM); (B) Excreted demethoxycurcumin-O-glucuronides (G1) in extracellular solution at different concentrations of demethoxycurcumin (2, 4 and 8 μM); (C) Excreted rates of G1 and G2 at different concentrations (2, 4 and 8 μM); All experiments were performed in triplicate (n = 3). Data were expressed as mean ± SD (n = 3). *, # p < 0.05, **, ## p < 0.01 or ***, ### p < 0.001 compared with that of demethoxycurcumin at 2 or 4 μM, respectively.
Fig 5
Fig 5. Effects of Ko143 on the formation and efflux excretion of demethoxycurcumin-O-glucuronides in HeLa1A1 cells.
(A) Effects of Ko143 (5 and 20 μM) on the accumulation of demethoxycurcumin-O-glucuronides (G1) in extracellular medium; (B) Effects of Ko143 (5 and 20 μM) on demethoxycurcumin-O-glucuronides (G2) excretion in extracellular solutions; (C) Effects of Ko143 (5 and 20 μM) on the efflux excretion rates of G1 and G2; (D) Effects of Ko143 (5 and 20 μM) on the intracellular G1 and G2 levels; (E) Effects of Ko143 (5 and 20 μM) on the fraction metabolized (fmet) of demethoxycurcumin; All experiments were performed in triplicate (n = 3). Data were presented as mean ± SD (n = 3). *, # p<0.05, **, ## p<0.01 and ***, ### p<0.001 compared with that of control group of G1 or G2, respectively.
Fig 6
Fig 6. Transporter inhibitor MK571 inhibited the generation and excretion of demethoxycurcumin-O-glucuronides in HeLa1A1 cells.
(A) MK571 (5 and 20 μM) inhibited the excretion of demethoxycurcumin-O-glucuronides (G1) in extracellular medium; (B) Inhibitory effects of MK571 (5 and 20 μM) on the excretion of demethoxycurcumin-O-glucuronides (G2) in extracellular solution; (C) MK571 (5 and 20 μM) decreased the efflux excretion rates of G1 and G2; (D) MK571 (5 and 20 μM) evaluated the intracellular levels of G1 and G2; (E) MK571 (5 and 20 μM) reduced the overall formation (fmet) of G1 and G2 in HeLa1A1 cell; The concentration of demethoxycurcumin was 4 μM. All experiments were performed in triplicate (n = 3). Data were presented as mean ± SD (n = 3). *, # p < 0.05, **, ## p < 0.01 and ***, ### p < 0.001 compared with that of control group of G1 or G2, respectively.
Fig 7
Fig 7. Inhibitory effects of chemical inhibitors Ko143 and MK571 on demethoxycurcumin-O-glucuronidation activity.
(A) Inhibitory effects of chemical inhibitors on glucuronidation activity by UGT1A1; (B) Inhibitory effects of chemical inhibitors on glucuronidation activity by HeLa1A1 cell lysate. The concentration of demethoxycurcumin was 4 μM. All experiments were performed in triplicate (n = 3). Data were expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with that of control group of G1 or G2, respectively. # p < 0.05, ## p < 0.01 and ### p < 0.001 compared with that of Ko143 (5 μM) or MK571 (5 μM), respectively.
Fig 8
Fig 8. Effects of BCRP silencing on excretion rates and intracellular levels of demethoxycurcumin-O-glucuronides in HeLa1A1 cells.
(A) Effects of excreted demethoxycurcumin-O-glucuronides (G1) in extracellular medium; (B) Effects of excreted demethoxycurcumin-O-glucuronides (G2) in extracellular medium; (C) Effects of the efflux excretion rates of G1 and G2; (D) Effects of the intracellular G1 and G2 levels; (E) Effects of the fraction metabolized (fmet) of demethoxycurcumin; (F) Effects of gene silencing on the protein level of BCRP. The concentration of demethoxycurcumin was 4 μM. All experiments were performed in triplicate (n = 3). Data were presented as mean ± SD (n = 3). *, # p < 0.05, **, ## p < 0.01 and ***, ### p < 0.001 compared with that of control group of G1 or G2, respectively.
Fig 9
Fig 9. Effects of knock-down of MRP1 transporter on demethoxycurcumin-O-glucuronides excretion and cellular glucuronidation in HeLa1A1 cells.
(A) Effects of accumlated demethoxycurcumin-O-glucuronides (G1) in extracellular medium; (B) Effects of excreted demethoxycurcumin-O-glucuronides (G2) in extracellular medium; (C) Effects of the excretion rates of G1 and G2; (D) Effects of the intracellular G1 and G2 levels; (E) Effects of fmet values of demethoxycurcumin; (F) Effects of gene silencing on the protein level of MRP1. The concentration of demethoxycurcumin was 4 μM. All experiments were performed in triplicate (n = 3). Data were expressed as mean ± SD (n = 3). *, # p < 0.05, **, ## p < 0.01 and ***, ### p < 0.001 compared with that of control group of G1 or G2, respectively.
Fig 10
Fig 10. MRP3 silencing led to reduce excretion rates and increase intracellular demethoxycurcumin-O-glucuronides in HeLa1A1 cells.
(A) MRP3 transporter silencing inhibited the accumlated demethoxycurcumin-O-glucuronides (G1) in extracellular medium; (B) MRP3 transporter silencing decreased the excreted demethoxycurcumin-O-glucuronides (G2) in extracellular solution; (C) MRP3 silencing significantly reduced the excretion rates of G1 and G2; (D) MRP3 silencing resulted in evaluation of intracellular G1 and G2 levels; (E) MRP3 silencing decreased total cellular glucuronidation (G1 ang G2) of demethoxycurcumin; (F) Effects of gene silencing on the protein level of MRP3. The concentration of demethoxycurcumin was 4 μM. All experiments were performed in triplicate (n = 3). Data were presented as mean ± SD. *, # p < 0.05, **, ## p < 0.01 and ***, ### p < 0.001 compared with that of control group of G1 or G2, respectively.
Fig 11
Fig 11. Effects of shRNA-mediated MRP4 transporter knock-down on demethoxycurcumin-O-glucuronides excretion and cellular glucuronidation in HeLa1A1 cells.
(A) MRP4 knock-down decreased the excreted demethoxycurcumin-O-glucuronides (G1); (B) MRP4 knock-down reduced the accumlated demethoxycurcumin-O-glucuronides (G2) in extracellular medium; (C) MRP4 knock-down inhibited the excretion rates of G1 and G2; (D) The intracellular G1 and G2 levels were evaluated when MRP4 transporter was knock-down; (E) MRP4 knock-down resulted in decrease of overall cellular glucuronidation (G1 and G2) of demethoxycurcumin; (F) Effects of gene silencing on the protein level of MRP4. The concentration of demethoxycurcumin was 4 μM. All experiments were performed in triplicate (n = 3). Data were expressed as mean ± SD (n = 3). *, # p < 0.05, **, ## p < 0.01 and ***, ### p < 0.001 compared with that of control group of G1 or G2, respectively.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Li R, Xiang C, Ye M, Li HF, Zhang X, Guo DA. Qualitative and quantitative analysis of curcuminoids in herbal medicines derived from Curcuma species. Food Chem. 2011;126: 1890–1895. 10.1016/j.foodchem.2010.12.014 - DOI - PubMed
    1. Hsiao YT, Kuo CL, Lin JJ, Huang WW, Peng SF, Chueh FS, et al. Curcuminoids combined with gefitinib mediated apoptosis and autophagy of human oral cancer SAS cells in vitro and reduced tumor of SAS cell xenograft mice in vivo. Environ Toxicol. 2018. May 2 10.1002/tox.22568 - DOI - PubMed
    1. Arshad L, Haque MA, Bukhari SNA, Jantan I. An overview of structure-activity relationship studies of curcumin analogs as antioxidant and anti-inflammatory agents. Future Med Chem. 2017;9: 605–626. 10.4155/fmc-2016-0223 - DOI - PubMed
    1. Ahmed T, Gilani AH. Therapeutic Potential of Turmeric in Alzheimer's Disease: Curcumin or Curcuminoids? Phytother Res. 2014;28: 517–525. 10.1002/ptr.5030 - DOI - PubMed
    1. Teymouri M, Barati N, Pirro M, Sahebkar A. Biological and pharmacological evaluation of dimethoxycurcumin: A metabolically stable curcumin analogue with a promising therapeutic potential. J Cell Physiol. 2018;233: 124–140. 10.1002/jcp.25749 - DOI - PubMed

Publication types

MeSH terms

Substances

Grants and funding

This work was supported by National Major Scientific and Program of Introducing Talents of Discipline to Universities (B13038) to XY, State Key Program of National Natural Science Foundation of China (81630097) to XY, National Natural Science Foundation of Guangdong Province (2017A03031387) to ZY, National Natural Science Foundation of Guangdong Province (2018A030310639) to ZQ and Hospital Youth Foundation of the First Affiliated Hospital of Zhengzhou University (YNQN2017200) to BZ.
-