Using acetaminophen's toxicity mechanism to enhance cisplatin efficacy in hepatocarcinoma and hepatoblastoma cell lines

Alexander J. Neuwelt; Y. Jeffrey Wu; Narcyz Knap; Marcin Losin; Edward A. Neuwelt; Michael A. Pagel; Steven Warmann; Joerg Fuchs; Piotr Czauderna; Michal Wozniak

doi:10.1593/neo.09688

Using acetaminophen's toxicity mechanism to enhance cisplatin efficacy in hepatocarcinoma and hepatoblastoma cell lines

Alexander J. Neuwelt
, Y. Jeffrey Wu
, Narcyz Knap
, Marcin Losin
, Edward A. Neuwelt
, Michael A. Pagel
, Steven Warmann
, Joerg Fuchs
, Piotr Czauderna
, Michal Wozniak

Neurology

Research output: Contribution to journal › Article › peer-review

32 Scopus citations

Abstract

BACKGROUND/AIMS: Acetaminophen overdose causes hepatotoxicity mediated by toxic metabolites generated through the cytochrome P450 enzyme. The objective of this study was to investigate whether acetaminophen (AAP) can enhance cisplatin (CDDP) cytotoxicity against human hepatocarcinoma and hepatoblastoma cells in vitro and whether this effect can be prevented by N-acetylcysteine (NAC). METHODS: In vitro studies (glutathione [GSH] level, cell viability, and immunoblot assays) were performed using human hepatocarcinoma and hepatoblastoma cells cultured in AAP, CDDP, and the combination of both with or without delayed NAC administration. The pharmacology and toxicology of high-dose AAP in rats were also examined. RESULTS: Acetaminophen decreased GSH levels in liver cancer cells in a dose- and time-dependent manner. Acetaminophen combined with CDDP had enhanced cytotoxicity over CDDP alone. The cytotoxicity caused by AAP plus CDDP was decreased by NAC, with the effectiveness being time-dependent. The GSH level was lowered in the liver but not in the blood or the brain in rats treated with a high dose of AAP (1000 mg/kg). The expression of CYP2E1 protein, a key cytochrome P450 enzyme, varies among species but is not correlated to AAP sensitivity in liver cancer cells. CONCLUSIONS: Our results suggest that a chemotherapeutic regimen containing both AAP and CDDP with delayed NAC rescue has the potential to enhance chemotherapeutic efficacy while decreasing adverse effects. This would be a promising approach particularly for hepatoblastomas regardless of cellular CYP2E1 protein level but could also be beneficial in other malignancies.

Original language	English (US)
Pages (from-to)	1003-1011
Number of pages	9
Journal	Neoplasia
Volume	11
Issue number	10
DOIs	https://doi.org/10.1593/neo.09688
State	Published - Oct 2009

Funding

Abbreviations: AAP, acetaminophen; CDDP, cisplatin; CYP2E1, cytochrome P450 2E1; HB, hepatoblastoma; GSH, glutathione; NAC, N-acetylcysteine; NAPQI, N-acetyl-p-benzoquinonimine; PARP, poly ADP ribose polymerase Address all correspondence to: Prof. Michal Wozniak, MD, PhD, Chair, Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211 Gdansk, Poland. E-mail: [email protected] 1This work was supported by the Biogen Idec Corporation and Fulbright Foundation to A.J.N. and by a grant from the National Institutes of Health (R37-NS044687) to E.A.N. E.A.N., Oregon Health & Science University, Portland Veterans Affairs Medical Center, and the Department of Veterans Affairs have a significant financial interest in Adherex, a company that may have a commercial interest in the results of this research and technology. This potential conflict of interest was reviewed and managed by the Oregon Health & Science University Integrity Program Oversight Council and the Portland Veterans Affairs Medical Center’s Conflict of Interest in Research Committee. E.A.N. has divested his financial interests in Adherex. 2A.J.N. and Y.J.W. contributed equally to this publication. Received 27 April 2009; Revised 2 July 2009; Accepted 6 July 2009 Copyright © 2009 Neoplasia Press, Inc. All rights reserved 1522-8002/09/$25.00 DOI 10.1593/neo.09688 Human HepG2 hepatocarcinoma and HUH6 and HepT1 HB cell lines were obtained from Dr. Steven Warmann (Tuebingen, Germany) and were cultured in Dulbecco’s modified Eagle medium in 10% FBS and 1% streptomycin/penicillin cocktail (Sigma-Aldrich, Poznan, Poland). Cells were maintained at 37°C and in an atmosphere containing 5% CO2. Normal human hepatocytes were obtained through Liver Tissue Cell Distribution System (Pittsburgh, PA), which was funded by the National Institutes of Health contract no. N01-DK-7-0004/HHSN26700700004C. Normal rat and mouse hepatocytes were obtained from the Department of Comparative Medicine of Oregon Health & Science University.

Funders	Funder number
Biogen Idec Corporation
Fulbright Foundation
Author National Institutes of Health National Institutes of Health National Institutes of Health National Institutes of Health The Bev Hartig Huntington's Disease Foundation National Institutes of Health	N01-DK-7-0004/HHSN26700700004C
National Institute of Neurological Disorders and Stroke	R37NS044687
U.S. Department of Veterans Affairs

ASJC Scopus subject areas

Cancer Research

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10.1593/neo.09688

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@article{5b924d37d6a947c593bda817e79f76c1,

title = "Using acetaminophen's toxicity mechanism to enhance cisplatin efficacy in hepatocarcinoma and hepatoblastoma cell lines",

abstract = "BACKGROUND/AIMS: Acetaminophen overdose causes hepatotoxicity mediated by toxic metabolites generated through the cytochrome P450 enzyme. The objective of this study was to investigate whether acetaminophen (AAP) can enhance cisplatin (CDDP) cytotoxicity against human hepatocarcinoma and hepatoblastoma cells in vitro and whether this effect can be prevented by N-acetylcysteine (NAC). METHODS: In vitro studies (glutathione [GSH] level, cell viability, and immunoblot assays) were performed using human hepatocarcinoma and hepatoblastoma cells cultured in AAP, CDDP, and the combination of both with or without delayed NAC administration. The pharmacology and toxicology of high-dose AAP in rats were also examined. RESULTS: Acetaminophen decreased GSH levels in liver cancer cells in a dose- and time-dependent manner. Acetaminophen combined with CDDP had enhanced cytotoxicity over CDDP alone. The cytotoxicity caused by AAP plus CDDP was decreased by NAC, with the effectiveness being time-dependent. The GSH level was lowered in the liver but not in the blood or the brain in rats treated with a high dose of AAP (1000 mg/kg). The expression of CYP2E1 protein, a key cytochrome P450 enzyme, varies among species but is not correlated to AAP sensitivity in liver cancer cells. CONCLUSIONS: Our results suggest that a chemotherapeutic regimen containing both AAP and CDDP with delayed NAC rescue has the potential to enhance chemotherapeutic efficacy while decreasing adverse effects. This would be a promising approach particularly for hepatoblastomas regardless of cellular CYP2E1 protein level but could also be beneficial in other malignancies.",

author = "Neuwelt, \{Alexander J.\} and Wu, \{Y. Jeffrey\} and Narcyz Knap and Marcin Losin and Neuwelt, \{Edward A.\} and Pagel, \{Michael A.\} and Steven Warmann and Joerg Fuchs and Piotr Czauderna and Michal Wozniak",

note = "Funding Information: Abbreviations: AAP, acetaminophen; CDDP, cisplatin; CYP2E1, cytochrome P450 2E1; HB, hepatoblastoma; GSH, glutathione; NAC, N-acetylcysteine; NAPQI, N-acetyl-p-benzoquinonimine; PARP, poly ADP ribose polymerase Address all correspondence to: Prof. Michal Wozniak, MD, PhD, Chair, Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211 Gdansk, Poland. E-mail: [email protected] 1This work was supported by the Biogen Idec Corporation and Fulbright Foundation to A.J.N. and by a grant from the National Institutes of Health (R37-NS044687) to E.A.N. E.A.N., Oregon Health \& Science University, Portland Veterans Affairs Medical Center, and the Department of Veterans Affairs have a significant financial interest in Adherex, a company that may have a commercial interest in the results of this research and technology. This potential conflict of interest was reviewed and managed by the Oregon Health \& Science University Integrity Program Oversight Council and the Portland Veterans Affairs Medical Center{\textquoteright}s Conflict of Interest in Research Committee. E.A.N. has divested his financial interests in Adherex. 2A.J.N. and Y.J.W. contributed equally to this publication. Received 27 April 2009; Revised 2 July 2009; Accepted 6 July 2009 Copyright {\textcopyright} 2009 Neoplasia Press, Inc. All rights reserved 1522-8002/09/\$25.00 DOI 10.1593/neo.09688 Funding Information: Human HepG2 hepatocarcinoma and HUH6 and HepT1 HB cell lines were obtained from Dr. Steven Warmann (Tuebingen, Germany) and were cultured in Dulbecco{\textquoteright}s modified Eagle medium in 10\% FBS and 1\% streptomycin/penicillin cocktail (Sigma-Aldrich, Poznan, Poland). Cells were maintained at 37°C and in an atmosphere containing 5\% CO2. Normal human hepatocytes were obtained through Liver Tissue Cell Distribution System (Pittsburgh, PA), which was funded by the National Institutes of Health contract no. N01-DK-7-0004/HHSN26700700004C. Normal rat and mouse hepatocytes were obtained from the Department of Comparative Medicine of Oregon Health \& Science University.",

year = "2009",

month = oct,

doi = "10.1593/neo.09688",

language = "English (US)",

volume = "11",

pages = "1003--1011",

journal = "Neoplasia",

issn = "1522-8002",

publisher = "Elsevier Inc.",

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T1 - Using acetaminophen's toxicity mechanism to enhance cisplatin efficacy in hepatocarcinoma and hepatoblastoma cell lines

AU - Neuwelt, Alexander J.

AU - Wu, Y. Jeffrey

AU - Knap, Narcyz

AU - Losin, Marcin

AU - Neuwelt, Edward A.

AU - Pagel, Michael A.

AU - Warmann, Steven

AU - Fuchs, Joerg

AU - Czauderna, Piotr

AU - Wozniak, Michal

N1 - Funding Information: Abbreviations: AAP, acetaminophen; CDDP, cisplatin; CYP2E1, cytochrome P450 2E1; HB, hepatoblastoma; GSH, glutathione; NAC, N-acetylcysteine; NAPQI, N-acetyl-p-benzoquinonimine; PARP, poly ADP ribose polymerase Address all correspondence to: Prof. Michal Wozniak, MD, PhD, Chair, Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki St, 80-211 Gdansk, Poland. E-mail: [email protected] 1This work was supported by the Biogen Idec Corporation and Fulbright Foundation to A.J.N. and by a grant from the National Institutes of Health (R37-NS044687) to E.A.N. E.A.N., Oregon Health & Science University, Portland Veterans Affairs Medical Center, and the Department of Veterans Affairs have a significant financial interest in Adherex, a company that may have a commercial interest in the results of this research and technology. This potential conflict of interest was reviewed and managed by the Oregon Health & Science University Integrity Program Oversight Council and the Portland Veterans Affairs Medical Center’s Conflict of Interest in Research Committee. E.A.N. has divested his financial interests in Adherex. 2A.J.N. and Y.J.W. contributed equally to this publication. Received 27 April 2009; Revised 2 July 2009; Accepted 6 July 2009 Copyright © 2009 Neoplasia Press, Inc. All rights reserved 1522-8002/09/$25.00 DOI 10.1593/neo.09688 Funding Information: Human HepG2 hepatocarcinoma and HUH6 and HepT1 HB cell lines were obtained from Dr. Steven Warmann (Tuebingen, Germany) and were cultured in Dulbecco’s modified Eagle medium in 10% FBS and 1% streptomycin/penicillin cocktail (Sigma-Aldrich, Poznan, Poland). Cells were maintained at 37°C and in an atmosphere containing 5% CO2. Normal human hepatocytes were obtained through Liver Tissue Cell Distribution System (Pittsburgh, PA), which was funded by the National Institutes of Health contract no. N01-DK-7-0004/HHSN26700700004C. Normal rat and mouse hepatocytes were obtained from the Department of Comparative Medicine of Oregon Health & Science University.

PY - 2009/10

Y1 - 2009/10

N2 - BACKGROUND/AIMS: Acetaminophen overdose causes hepatotoxicity mediated by toxic metabolites generated through the cytochrome P450 enzyme. The objective of this study was to investigate whether acetaminophen (AAP) can enhance cisplatin (CDDP) cytotoxicity against human hepatocarcinoma and hepatoblastoma cells in vitro and whether this effect can be prevented by N-acetylcysteine (NAC). METHODS: In vitro studies (glutathione [GSH] level, cell viability, and immunoblot assays) were performed using human hepatocarcinoma and hepatoblastoma cells cultured in AAP, CDDP, and the combination of both with or without delayed NAC administration. The pharmacology and toxicology of high-dose AAP in rats were also examined. RESULTS: Acetaminophen decreased GSH levels in liver cancer cells in a dose- and time-dependent manner. Acetaminophen combined with CDDP had enhanced cytotoxicity over CDDP alone. The cytotoxicity caused by AAP plus CDDP was decreased by NAC, with the effectiveness being time-dependent. The GSH level was lowered in the liver but not in the blood or the brain in rats treated with a high dose of AAP (1000 mg/kg). The expression of CYP2E1 protein, a key cytochrome P450 enzyme, varies among species but is not correlated to AAP sensitivity in liver cancer cells. CONCLUSIONS: Our results suggest that a chemotherapeutic regimen containing both AAP and CDDP with delayed NAC rescue has the potential to enhance chemotherapeutic efficacy while decreasing adverse effects. This would be a promising approach particularly for hepatoblastomas regardless of cellular CYP2E1 protein level but could also be beneficial in other malignancies.

AB - BACKGROUND/AIMS: Acetaminophen overdose causes hepatotoxicity mediated by toxic metabolites generated through the cytochrome P450 enzyme. The objective of this study was to investigate whether acetaminophen (AAP) can enhance cisplatin (CDDP) cytotoxicity against human hepatocarcinoma and hepatoblastoma cells in vitro and whether this effect can be prevented by N-acetylcysteine (NAC). METHODS: In vitro studies (glutathione [GSH] level, cell viability, and immunoblot assays) were performed using human hepatocarcinoma and hepatoblastoma cells cultured in AAP, CDDP, and the combination of both with or without delayed NAC administration. The pharmacology and toxicology of high-dose AAP in rats were also examined. RESULTS: Acetaminophen decreased GSH levels in liver cancer cells in a dose- and time-dependent manner. Acetaminophen combined with CDDP had enhanced cytotoxicity over CDDP alone. The cytotoxicity caused by AAP plus CDDP was decreased by NAC, with the effectiveness being time-dependent. The GSH level was lowered in the liver but not in the blood or the brain in rats treated with a high dose of AAP (1000 mg/kg). The expression of CYP2E1 protein, a key cytochrome P450 enzyme, varies among species but is not correlated to AAP sensitivity in liver cancer cells. CONCLUSIONS: Our results suggest that a chemotherapeutic regimen containing both AAP and CDDP with delayed NAC rescue has the potential to enhance chemotherapeutic efficacy while decreasing adverse effects. This would be a promising approach particularly for hepatoblastomas regardless of cellular CYP2E1 protein level but could also be beneficial in other malignancies.

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Using acetaminophen's toxicity mechanism to enhance cisplatin efficacy in hepatocarcinoma and hepatoblastoma cell lines

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