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Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers.

Sa-ngiamsuntorn K, Wongkajornsilp A, Kasetsinsombat K, Duangsa-ard S, Nuntakarn L, Borwornpinyo S, Akarasereenont P, Limsrichamrern S, Hongeng S - BMC Biotechnol. (2011)

Bottom Line: Their inducibility outperformed the classical HepG2 cells.The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes.The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkoknoi, Bangkok 10700, Thailand.

ABSTRACT

Background: The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies.

Results: The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells.

Conclusion: The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

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Related in: MedlinePlus

Basal CYP450 activity and its inducibility. The basal expression of CYP450 isotypes, CYP450 nuclear transcription factors, and UGT1A1 in MSCs, hepatocyte-like cells, HepG2 and the primary hepatocyte were analyzed using real-time qPCR (A). They were analyzed as fold-changes over that of untreated MSCs. The induction of CYP1A1 (B), CYP1A2 (C), CYP3A4 (D) or CYP2C9 (E) activities after adding the corresponding enzyme inducers for 72 h were analyzed using P450Glo™ assay kit (Promega) with different luciferin substrates. After 3-h incubation with specific substrate, luciferase activities were measured. Results are expressed as luciferase activities in relative luminescence unit (RLU) and mean ± SD of 3 independent experiments.
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Figure 4: Basal CYP450 activity and its inducibility. The basal expression of CYP450 isotypes, CYP450 nuclear transcription factors, and UGT1A1 in MSCs, hepatocyte-like cells, HepG2 and the primary hepatocyte were analyzed using real-time qPCR (A). They were analyzed as fold-changes over that of untreated MSCs. The induction of CYP1A1 (B), CYP1A2 (C), CYP3A4 (D) or CYP2C9 (E) activities after adding the corresponding enzyme inducers for 72 h were analyzed using P450Glo™ assay kit (Promega) with different luciferin substrates. After 3-h incubation with specific substrate, luciferase activities were measured. Results are expressed as luciferase activities in relative luminescence unit (RLU) and mean ± SD of 3 independent experiments.

Mentions: After maturation induction, hepatocyte-like cell culture was continued in IMDM with 1% FBS, 2% DMSO for 2 weeks at confluence [26]. Cells were harvested and determined for phase I and phase II enzyme expressions (Figure 4A). Specific transcription factors such as AHR, PXR and CAR in hepatocyte-like cells were increased by about 10 times those of MSCs. These genes are involved in the transcription of CYP450 isotypes [27,28]. Genes that were highly expressed in hepatocyte-like cells included one phase II enzyme UGT1A1 and 6 CYP450 isotypes (CYP2B6, CYP2D6, CYP2C9, CYP2C19, CYP3A4, and CYP1A1). In particular, the expression level of CYP2B6 in hepatocyte-like cells was even higher than that of HepG2 while other isotypes achieved comparable expression levels to those of HepG2. However, all CYP450 isotype expressions in hepatocyte-like cells were only 10-20% that of normal hepatocytes. The authenticity of the real-time RT-PCR products of hepatocyte markers and CYP450 were confirmed through the analysis for melting curve using Sequence Detection Software version 2.01 (Applied Biosystems, CA).


Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers.

Sa-ngiamsuntorn K, Wongkajornsilp A, Kasetsinsombat K, Duangsa-ard S, Nuntakarn L, Borwornpinyo S, Akarasereenont P, Limsrichamrern S, Hongeng S - BMC Biotechnol. (2011)

Basal CYP450 activity and its inducibility. The basal expression of CYP450 isotypes, CYP450 nuclear transcription factors, and UGT1A1 in MSCs, hepatocyte-like cells, HepG2 and the primary hepatocyte were analyzed using real-time qPCR (A). They were analyzed as fold-changes over that of untreated MSCs. The induction of CYP1A1 (B), CYP1A2 (C), CYP3A4 (D) or CYP2C9 (E) activities after adding the corresponding enzyme inducers for 72 h were analyzed using P450Glo™ assay kit (Promega) with different luciferin substrates. After 3-h incubation with specific substrate, luciferase activities were measured. Results are expressed as luciferase activities in relative luminescence unit (RLU) and mean ± SD of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198927&req=5

Figure 4: Basal CYP450 activity and its inducibility. The basal expression of CYP450 isotypes, CYP450 nuclear transcription factors, and UGT1A1 in MSCs, hepatocyte-like cells, HepG2 and the primary hepatocyte were analyzed using real-time qPCR (A). They were analyzed as fold-changes over that of untreated MSCs. The induction of CYP1A1 (B), CYP1A2 (C), CYP3A4 (D) or CYP2C9 (E) activities after adding the corresponding enzyme inducers for 72 h were analyzed using P450Glo™ assay kit (Promega) with different luciferin substrates. After 3-h incubation with specific substrate, luciferase activities were measured. Results are expressed as luciferase activities in relative luminescence unit (RLU) and mean ± SD of 3 independent experiments.
Mentions: After maturation induction, hepatocyte-like cell culture was continued in IMDM with 1% FBS, 2% DMSO for 2 weeks at confluence [26]. Cells were harvested and determined for phase I and phase II enzyme expressions (Figure 4A). Specific transcription factors such as AHR, PXR and CAR in hepatocyte-like cells were increased by about 10 times those of MSCs. These genes are involved in the transcription of CYP450 isotypes [27,28]. Genes that were highly expressed in hepatocyte-like cells included one phase II enzyme UGT1A1 and 6 CYP450 isotypes (CYP2B6, CYP2D6, CYP2C9, CYP2C19, CYP3A4, and CYP1A1). In particular, the expression level of CYP2B6 in hepatocyte-like cells was even higher than that of HepG2 while other isotypes achieved comparable expression levels to those of HepG2. However, all CYP450 isotype expressions in hepatocyte-like cells were only 10-20% that of normal hepatocytes. The authenticity of the real-time RT-PCR products of hepatocyte markers and CYP450 were confirmed through the analysis for melting curve using Sequence Detection Software version 2.01 (Applied Biosystems, CA).

Bottom Line: Their inducibility outperformed the classical HepG2 cells.The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes.The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkoknoi, Bangkok 10700, Thailand.

ABSTRACT

Background: The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies.

Results: The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells.

Conclusion: The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

Show MeSH
Related in: MedlinePlus