Co-Cultured Continuously Bioluminescent Human Cells as Bioreporters for the Detection of Prodrug Therapeutic Impact Pre- and Post-Metabolism
<p>Autobioluminescent cells successfully detected both pre- and post-metabolized prodrug metabolic impacts from (<b>a</b>) the known cytotoxic compounds Mitomycin C and (<b>b</b>) Doxorubicin hydrochloride. (<b>c</b>) Treatment with Cyclophosphamide, which is known to become metabolically active only after liver cell metabolism, showed metabolic effects in the downstream human breast cancer (T47D) cell line, but not the upstream liver cancer (HepG2) cell line. Values are the averages of triplicate tests, with error represented as the standard error of the mean (SEM).</p> "> Figure 2
<p>Detection of metabolic impact using autobioluminescence correlated with ATP content for both (<b>a</b>) Cytarabine and (<b>b</b>) Methotrexate, and was more sensitive for the detection of (<b>c</b>) Oligomycin B. Values are the averages of triplicate tests, with error represented as SEM.</p> "> Figure 3
<p>The autobioluminescence assay correlated with the fluorescent dichlorofluorescin diacetate (DCFH-DA) assay for the detection of reactive oxygen species as mediators of metabolic impact for both (<b>a</b>) Doxorubicin and (<b>b</b>) tert-butyl H<sub>2</sub>O<sub>2</sub> (TBHP). Values are the averages of triplicate tests, with error represented as SEM.</p> "> Figure 4
<p>No correlation was observed between the results of autobioluminescent assays and glutathione depletion assays for following treatment with (<b>a</b>) Digoxin or (<b>b</b>) Rotenone. Values are the averages of triplicate tests, with error represented as SEM.</p> "> Figure 4 Cont.
<p>No correlation was observed between the results of autobioluminescent assays and glutathione depletion assays for following treatment with (<b>a</b>) Digoxin or (<b>b</b>) Rotenone. Values are the averages of triplicate tests, with error represented as SEM.</p> "> Figure 5
<p>CYP1A1 was the most upregulated enzyme for both the (<b>a</b>) HepG2 and (<b>b</b>) T47D cell lines at the time autobioluminescent metabolic impact was reported. No significant upregulation of any Phase II detoxification enzyme was observed in the (<b>b</b>) T47D cell line, while SULT1A1, SULT1A2, and mGST1 were upregulated in the (<b>a</b>) HepG2 cell line. For each detoxification enzyme, significant induction in treated cells relative to their untreated controls (<span class="html-italic">p</span> ≤ 0.05) is denoted by a single asterisk (*). Under a given compound treatment condition, significant differences in the level of upregulation between different detoxification enzymes is indicated by a double-asterisk (**).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Development of Autobioluminescent Cell Lines
2.2. ATP Content Screening
2.3. Reactive Oxygen Species Screening
2.4. Glutathione Depletion Screening
2.5. Detoxification Pathway Biomarker Activation Screening
2.6. Autobioluminescent Metabolic Activity Screening
2.7. Statistical Analysis
3. Results
3.1. Development of Autobioluminescent Cell Lines
3.2. Detection of Pre- and Post-Biotransformed Compound Effects Using Autobioluminescent Cell Lines
3.3. Identification of the Mechanisms of Action Underlying Autobioluminescent Dynamics
3.4. Correlation between Autobioluminescet Dynamics Detoxification Pathway Activation
4. Discussion
Acknowledgments
Author Contributions
Conflicts of Interest
References
- Tralau, T.; Luch, A. Drug-mediated toxicity: Illuminating the ‘bad’ in the test tube by means of cellular assays? Trends Pharmacol. Sci. 2012, 33, 353–364. [Google Scholar] [CrossRef] [PubMed]
- Astashkina, A.; Mann, B.; Grainger, D.W. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity. Pharmacol. Ther. 2012, 134, 82–106. [Google Scholar] [CrossRef] [PubMed]
- Knight, A. Non-animal methodologies within biomedical research and toxicity testing. ALTEX 2008, 25, 213–231. [Google Scholar] [CrossRef] [PubMed]
- Phillips, B.W.; Crook, J.M. Pluripotent human stem cells: A novel tool in drug discovery. Biodrugs 2010, 24, 99–108. [Google Scholar] [CrossRef] [PubMed]
- Breslin, S.; O’Driscoll, L. Three-dimensional cell culture: The missing link in drug discovery. Drug Discov. Today 2013, 18, 240–249. [Google Scholar] [CrossRef] [PubMed]
- Close, D.M.; Patterson, S.S.; Ripp, S.; Baek, S.J.; Sanseverino, J.; Sayler, G.S. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line. PLoS ONE 2010, 5, e12441. [Google Scholar] [CrossRef] [PubMed]
- Xu, T.; Ripp, S.; Sayler, G.; Close, D. Expression of a humanized viral 2A-mediated lux operon efficiently generates autonomous bioluminescence in human cells. PLoS ONE 2014, 9, e96347. [Google Scholar] [CrossRef] [PubMed]
- Close, D.; Xu, T.; Smartt, A.; Rogers, A.; Crossley, R.; Price, S.; Ripp, S.; Sayler, G. The evolution of the bacterial luciferase gene cassette (lux) as a real-time bioreporter. Sensors 2012, 12, 732–752. [Google Scholar] [CrossRef] [PubMed]
- Tinikul, R.; Chaiyen, P. Structure, Mechanism, and Mutation of Bacterial Luciferase. Adv. Biochem. Eng. Biotechnol. 2016, 154, 47–74. [Google Scholar] [PubMed]
- Westerink, W.; Schoonen, W.G. Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol. In Vitro 2007, 21, 1581–1591. [Google Scholar] [CrossRef] [PubMed]
- Westerink, W.; Schoonen, W.G. Phase II enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol. In Vitro 2007, 21, 1592–1602. [Google Scholar] [CrossRef] [PubMed]
- Roy, P.; Li, J.Y.; Crespi, C.L.; Waxman, D.J. Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles. Drug Metab. Dispos. 1999, 27, 655–666. [Google Scholar] [PubMed]
- Muus, P.; Haanen, C.; Pennings, A.; Ruitenbeek, W.; Van den Bogert, C. Influence of cytarabine on mitochondrial function and mitochondrial biogenesis. Semin. Oncol. 1987, 14, 245–250. [Google Scholar] [PubMed]
- Beckers, A.; Organe, S.; Timmermans, L.; Vanderhoydonc, F.; Deboel, L.; Derua, R.; Waelkens, E.; Brusselmans, K.; Verhoeven, G.; Swinnen, J.V. Methotrexate enhances the antianabolic and antiproliferative effects of 5-aminoimidazole-4-carboxamide riboside. Mol. Cancer Ther. 2006, 5, 2211–2217. [Google Scholar] [CrossRef] [PubMed]
- Murphy, M.P.; Holmgren, A.; Larsson, N.G.; Halliwell, B.; Chang, C.J.; Kalyanaraman, B.; Rhee, S.G.; Thornalley, P.J.; Partridge, L.; Gems, D. Unraveling the biological roles of reactive oxygen species. Cell Metab. 2011, 13, 361–366. [Google Scholar] [CrossRef] [PubMed]
- Winterbourn, C.C. Reconciling the chemistry and biology of reactive oxygen species. Nat. Chem. Biol. 2008, 4, 278–286. [Google Scholar] [CrossRef] [PubMed]
- Close, D.; Hahn, R.; Ripp, S.; Sayler, G.S. Determining toxicant bioavailability using a constitutively bioluminescent human cell line. In Proceedings of the 3rd Annual Biomedical Science and Engineering Conference, Oak Ridge, TN, USA, 15–17 March 2011; pp. 1–4. [Google Scholar]
- Close, D.; Webb, J.; Ripp, S.; Patterson, S.; Sayler, G. Remote detection of human toxicants in real-time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter. In Proceedings of the 2012 SPIE Defense, Security, and Sensing Conference, Baltimore, MD, USA, 23–27 April 2012; p. 837117. [Google Scholar]
- Xu, T.; Close, D.; Smartt, A.; Ripp, S.; Sayler, G. Detection of organic compounds with whole-cell bioluminescent bioassays. In Bioluminescence: Fundamentals and Applications in Biotechnology–Volume 1, Advances in Biochemical Engineering/Biotechnology; Thouand, G., Marks, R., Eds.; Springer: Berlin/Heidelberg, Germany, 2014; Volume 1, pp. 111–151. [Google Scholar]
- Xu, T.; Marr, E.; Lam, H.; Ripp, S.; Sayler, G.; Close, D. Real-time toxicity and metabolic activity tracking of human cells exposed to Escherichia coli O157:H7 in a mixed consortia. Ecotoxicology 2015, 24, 2133–2140. [Google Scholar] [CrossRef] [PubMed]
- Class, B.; Thorne, N.; Aguisanda, F.; Southall, N.; Mckew, J.; Zheng, W. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial lux reporter. J. Lab. Autom. 2015, 20, 164–174. [Google Scholar] [CrossRef] [PubMed]
- John, B.A.; Xu, T.; Ripp, S.; Wang, H.C.R. A real-time non-invasive auto-bioluminescent urinary bladder cancer xenograft model. Mol. Imaging Biol. 2017, 19, 10–14. [Google Scholar] [CrossRef] [PubMed]
- Riss, T. Is Your MTT Assay Really the Best Choice? Available online: http://www.promega.com/resources/pubhub/is-your-mtt-assay-really-the-best-choice/ (accessed on 25 February 2017).
- D’Autréaux, B.; Toledano, M.B. ROS as signalling molecules: Mechanisms that generate specificity in ROS homeostasis. Nat. Rev. Mol. Cell Biol. 2007, 8, 813–824. [Google Scholar] [CrossRef] [PubMed]
- Halliwell, B.; Gutteridge, J.M. Free Radicals in Biology and Medicine; Oxford University Press: New York, NY, USA, 2015. [Google Scholar]
- Close, D.M.; Ripp, S.; Sayler, G.S. Reporter proteins in whole-cell optical bioreporter detection systems, biosensor integrations, and biosensing applications. Sensors 2009, 9, 9147–9174. [Google Scholar] [CrossRef] [PubMed]
© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Xu, T.; Conway, M.; Frank, A.; Ripp, S.; Sayler, G.; Close, D. Co-Cultured Continuously Bioluminescent Human Cells as Bioreporters for the Detection of Prodrug Therapeutic Impact Pre- and Post-Metabolism. Sensors 2017, 17, 2827. https://doi.org/10.3390/s17122827
Xu T, Conway M, Frank A, Ripp S, Sayler G, Close D. Co-Cultured Continuously Bioluminescent Human Cells as Bioreporters for the Detection of Prodrug Therapeutic Impact Pre- and Post-Metabolism. Sensors. 2017; 17(12):2827. https://doi.org/10.3390/s17122827
Chicago/Turabian StyleXu, Tingting, Michael Conway, Ashley Frank, Steven Ripp, Gary Sayler, and Dan Close. 2017. "Co-Cultured Continuously Bioluminescent Human Cells as Bioreporters for the Detection of Prodrug Therapeutic Impact Pre- and Post-Metabolism" Sensors 17, no. 12: 2827. https://doi.org/10.3390/s17122827