Identification of the fenretinide metabolite 4-oxo-fenretinide present in human plasma and formed in human ovarian carcinoma cells through induction of cytochrome P450 26A1

Maria Grazia Villani, Valentina Appierto, Elena Cavadini, Manuela Valsecchi, Sandro Sonnino, Robert W. Curley, Franca Formelli

Research output: Contribution to journalArticle

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Abstract

Purpose: The synthetic retinoid fenretinide (4-HPR) exhibits preventive and therapeutic activity against ovarian tumors. An unidentified polar metabolite was previously found in 4-HPR-treated subjects and in A2780 human ovarian carcinoma cells continuously treated with 4-HPR (A2780/HPR). The metabolite and the enzyme involved in its formation in tumor cells are herein identified. Experimental Design: The metabolite was identified by mass spectrometry in A2780/HPR cell extracts and in plasma from 11 women participating in a phase III trial and treated with 200 mg/d 4-HPR for 5 years. The expression of proteins involved in retinoid metabolism and transport, cytochrome P450 26A1 (CYP26A1), cellular retinol-binding protein I (CRBP-I), and cellular retinoic acid-binding protein I and II (CRABP-I, CRABP-II) were evaluated in tumor cells by reverse transcription-PCR and Western blot analyses. Overexpression of CYP26A1 and retinoic acid receptors (RARs) in A2780 cells were obtained by cDNAs transfection. Results: The polar metabolite was 4-oxo-N-(4-hydroxyphenyl) retinamide (4-oxo-4-HPR) i.e., an oxidized form of 4-HPR with modification in position 4 of the cyclohexene ring. 4-oxo-4-HPR plasma levels were slightly lower (0.52 ± 0.17 μmol/L) than those of the parent drug (0.84 ± 0.53 μmol/L) and of the already identified metabolite N-(4-methoxyphenyl)retinamide (1.13 ± 0.85 μmol/L). In A2780/HPR cells continuously treated with 4-HPR and producing 4-oxo-4-HPR, CYP26A1 and CRBP-I were markedly upregulated compared with A2780 untreated cells. In A2780 cells, not producing 4-oxo-4-HPR, overexpression of CYP26A1 caused formation of 4-oxo-4-HPR, which was associated with no change in 4-HPR sensitivity. Moreover, the addition of 4-oxo-4-HPR to A2780 cells inhibited cell proliferation. Elevated levels of CYP26A1 protein and metabolism of 4-HPR to 4-oxo-4-HPR were found in A2780 cells transfected with RARβ and to a lesser extent in those transfected with RARγ. Conclusions: A new metabolite of 4-HPR, 4-oxo-4-HPR, present in human plasma and in tumor cells, has been identified. The formation of this biologically active metabolite in tumor cells was due to CYP26A1 induction and was influenced by RAR expression. Moreover evidence was provided that 4-HPR up-modulates the expression of CRBP-I transcript, which is lost during ovarian carcinogenesis.

Original languageEnglish
Pages (from-to)6265-6275
Number of pages11
JournalClinical Cancer Research
Volume10
Issue number18 I
DOIs
Publication statusPublished - Sep 15 2004

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Fenretinide
Cytochrome P-450 Enzyme System
Carcinoma
Retinoic Acid Receptors
Cellular Retinol-Binding Proteins
Retinoids
Neoplasms
4-oxofenretinide
Plasmacytoma
Cell Extracts
Reverse Transcription
Transfection
Mass Spectrometry

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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Identification of the fenretinide metabolite 4-oxo-fenretinide present in human plasma and formed in human ovarian carcinoma cells through induction of cytochrome P450 26A1. / Villani, Maria Grazia; Appierto, Valentina; Cavadini, Elena; Valsecchi, Manuela; Sonnino, Sandro; Curley, Robert W.; Formelli, Franca.

In: Clinical Cancer Research, Vol. 10, No. 18 I, 15.09.2004, p. 6265-6275.

Research output: Contribution to journalArticle

Villani, Maria Grazia ; Appierto, Valentina ; Cavadini, Elena ; Valsecchi, Manuela ; Sonnino, Sandro ; Curley, Robert W. ; Formelli, Franca. / Identification of the fenretinide metabolite 4-oxo-fenretinide present in human plasma and formed in human ovarian carcinoma cells through induction of cytochrome P450 26A1. In: Clinical Cancer Research. 2004 ; Vol. 10, No. 18 I. pp. 6265-6275.
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title = "Identification of the fenretinide metabolite 4-oxo-fenretinide present in human plasma and formed in human ovarian carcinoma cells through induction of cytochrome P450 26A1",
abstract = "Purpose: The synthetic retinoid fenretinide (4-HPR) exhibits preventive and therapeutic activity against ovarian tumors. An unidentified polar metabolite was previously found in 4-HPR-treated subjects and in A2780 human ovarian carcinoma cells continuously treated with 4-HPR (A2780/HPR). The metabolite and the enzyme involved in its formation in tumor cells are herein identified. Experimental Design: The metabolite was identified by mass spectrometry in A2780/HPR cell extracts and in plasma from 11 women participating in a phase III trial and treated with 200 mg/d 4-HPR for 5 years. The expression of proteins involved in retinoid metabolism and transport, cytochrome P450 26A1 (CYP26A1), cellular retinol-binding protein I (CRBP-I), and cellular retinoic acid-binding protein I and II (CRABP-I, CRABP-II) were evaluated in tumor cells by reverse transcription-PCR and Western blot analyses. Overexpression of CYP26A1 and retinoic acid receptors (RARs) in A2780 cells were obtained by cDNAs transfection. Results: The polar metabolite was 4-oxo-N-(4-hydroxyphenyl) retinamide (4-oxo-4-HPR) i.e., an oxidized form of 4-HPR with modification in position 4 of the cyclohexene ring. 4-oxo-4-HPR plasma levels were slightly lower (0.52 ± 0.17 μmol/L) than those of the parent drug (0.84 ± 0.53 μmol/L) and of the already identified metabolite N-(4-methoxyphenyl)retinamide (1.13 ± 0.85 μmol/L). In A2780/HPR cells continuously treated with 4-HPR and producing 4-oxo-4-HPR, CYP26A1 and CRBP-I were markedly upregulated compared with A2780 untreated cells. In A2780 cells, not producing 4-oxo-4-HPR, overexpression of CYP26A1 caused formation of 4-oxo-4-HPR, which was associated with no change in 4-HPR sensitivity. Moreover, the addition of 4-oxo-4-HPR to A2780 cells inhibited cell proliferation. Elevated levels of CYP26A1 protein and metabolism of 4-HPR to 4-oxo-4-HPR were found in A2780 cells transfected with RARβ and to a lesser extent in those transfected with RARγ. Conclusions: A new metabolite of 4-HPR, 4-oxo-4-HPR, present in human plasma and in tumor cells, has been identified. The formation of this biologically active metabolite in tumor cells was due to CYP26A1 induction and was influenced by RAR expression. Moreover evidence was provided that 4-HPR up-modulates the expression of CRBP-I transcript, which is lost during ovarian carcinogenesis.",
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T1 - Identification of the fenretinide metabolite 4-oxo-fenretinide present in human plasma and formed in human ovarian carcinoma cells through induction of cytochrome P450 26A1

AU - Villani, Maria Grazia

AU - Appierto, Valentina

AU - Cavadini, Elena

AU - Valsecchi, Manuela

AU - Sonnino, Sandro

AU - Curley, Robert W.

AU - Formelli, Franca

PY - 2004/9/15

Y1 - 2004/9/15

N2 - Purpose: The synthetic retinoid fenretinide (4-HPR) exhibits preventive and therapeutic activity against ovarian tumors. An unidentified polar metabolite was previously found in 4-HPR-treated subjects and in A2780 human ovarian carcinoma cells continuously treated with 4-HPR (A2780/HPR). The metabolite and the enzyme involved in its formation in tumor cells are herein identified. Experimental Design: The metabolite was identified by mass spectrometry in A2780/HPR cell extracts and in plasma from 11 women participating in a phase III trial and treated with 200 mg/d 4-HPR for 5 years. The expression of proteins involved in retinoid metabolism and transport, cytochrome P450 26A1 (CYP26A1), cellular retinol-binding protein I (CRBP-I), and cellular retinoic acid-binding protein I and II (CRABP-I, CRABP-II) were evaluated in tumor cells by reverse transcription-PCR and Western blot analyses. Overexpression of CYP26A1 and retinoic acid receptors (RARs) in A2780 cells were obtained by cDNAs transfection. Results: The polar metabolite was 4-oxo-N-(4-hydroxyphenyl) retinamide (4-oxo-4-HPR) i.e., an oxidized form of 4-HPR with modification in position 4 of the cyclohexene ring. 4-oxo-4-HPR plasma levels were slightly lower (0.52 ± 0.17 μmol/L) than those of the parent drug (0.84 ± 0.53 μmol/L) and of the already identified metabolite N-(4-methoxyphenyl)retinamide (1.13 ± 0.85 μmol/L). In A2780/HPR cells continuously treated with 4-HPR and producing 4-oxo-4-HPR, CYP26A1 and CRBP-I were markedly upregulated compared with A2780 untreated cells. In A2780 cells, not producing 4-oxo-4-HPR, overexpression of CYP26A1 caused formation of 4-oxo-4-HPR, which was associated with no change in 4-HPR sensitivity. Moreover, the addition of 4-oxo-4-HPR to A2780 cells inhibited cell proliferation. Elevated levels of CYP26A1 protein and metabolism of 4-HPR to 4-oxo-4-HPR were found in A2780 cells transfected with RARβ and to a lesser extent in those transfected with RARγ. Conclusions: A new metabolite of 4-HPR, 4-oxo-4-HPR, present in human plasma and in tumor cells, has been identified. The formation of this biologically active metabolite in tumor cells was due to CYP26A1 induction and was influenced by RAR expression. Moreover evidence was provided that 4-HPR up-modulates the expression of CRBP-I transcript, which is lost during ovarian carcinogenesis.

AB - Purpose: The synthetic retinoid fenretinide (4-HPR) exhibits preventive and therapeutic activity against ovarian tumors. An unidentified polar metabolite was previously found in 4-HPR-treated subjects and in A2780 human ovarian carcinoma cells continuously treated with 4-HPR (A2780/HPR). The metabolite and the enzyme involved in its formation in tumor cells are herein identified. Experimental Design: The metabolite was identified by mass spectrometry in A2780/HPR cell extracts and in plasma from 11 women participating in a phase III trial and treated with 200 mg/d 4-HPR for 5 years. The expression of proteins involved in retinoid metabolism and transport, cytochrome P450 26A1 (CYP26A1), cellular retinol-binding protein I (CRBP-I), and cellular retinoic acid-binding protein I and II (CRABP-I, CRABP-II) were evaluated in tumor cells by reverse transcription-PCR and Western blot analyses. Overexpression of CYP26A1 and retinoic acid receptors (RARs) in A2780 cells were obtained by cDNAs transfection. Results: The polar metabolite was 4-oxo-N-(4-hydroxyphenyl) retinamide (4-oxo-4-HPR) i.e., an oxidized form of 4-HPR with modification in position 4 of the cyclohexene ring. 4-oxo-4-HPR plasma levels were slightly lower (0.52 ± 0.17 μmol/L) than those of the parent drug (0.84 ± 0.53 μmol/L) and of the already identified metabolite N-(4-methoxyphenyl)retinamide (1.13 ± 0.85 μmol/L). In A2780/HPR cells continuously treated with 4-HPR and producing 4-oxo-4-HPR, CYP26A1 and CRBP-I were markedly upregulated compared with A2780 untreated cells. In A2780 cells, not producing 4-oxo-4-HPR, overexpression of CYP26A1 caused formation of 4-oxo-4-HPR, which was associated with no change in 4-HPR sensitivity. Moreover, the addition of 4-oxo-4-HPR to A2780 cells inhibited cell proliferation. Elevated levels of CYP26A1 protein and metabolism of 4-HPR to 4-oxo-4-HPR were found in A2780 cells transfected with RARβ and to a lesser extent in those transfected with RARγ. Conclusions: A new metabolite of 4-HPR, 4-oxo-4-HPR, present in human plasma and in tumor cells, has been identified. The formation of this biologically active metabolite in tumor cells was due to CYP26A1 induction and was influenced by RAR expression. Moreover evidence was provided that 4-HPR up-modulates the expression of CRBP-I transcript, which is lost during ovarian carcinogenesis.

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