Hydrolysis of 4-HPR to atRA occurs in vivo but is not required for retinamide-induced apoptosis

doi:10.1016/j.abb.2003.09.001

Archives of Biochemistry and Biophysics

Volume 419, Issue 2, 15 November 2003, Pages 234-243

https://doi.org/10.1016/j.abb.2003.09.001 Get rights and content

Abstract

The retinamide, N-(4-hydroxyphenyl)retinamide (4-HPR), has shown promising anti-tumor activity, but it is unclear whether this compound is hydrolyzed to all-trans retinoic acid (atRA) and if so, whether this plays any role in its chemotherapeutic activity. To address this issue, the ability of 4-hydroxybenzylretinone (4-HBR), a carbon-linked analog of 4-HPR, to support growth in vitamin A-deficient (VAD) animals and to activate an atRA-responsive gene in vivo was compared to 4-HPR and atRA. Further, the non-hydrolyzable 4-HBR analog was used to determine whether the presence of the labile amide linkage in 4-HPR is essential for the induction of apoptosis in cultured MCF-7 breast cancer cells. Studies in VAD rats showed that 4-HPR, like atRA, supports animal growth and induces CYP26B1 mRNA expression in lung whereas 4-HBR does not. Analysis of plasma from 4-HPR- and atRA-treated VAD animals revealed the presence of atRA whereas it was not detected in plasma from animals given 4-HBR. To determine whether hydrolysis to atRA is necessary for apoptosis induced by 4-HPR in MCF-7 breast cancer cells, morphological and biochemical assays for apoptosis were performed. 4-HBR, like 4-HPR, induced apoptosis in MCF-7 cells. Apoptosis was not induced even at high concentrations of atRA, showing that 4-HPR and 4-HBR act in cells via a distinct signaling pathway. These results show that although limited hydrolysis of 4-HPR occurs in vivo, the ability to liberate atRA is not required for these 4-hydroxyphenyl retinoids to induce apoptosis in MCF-7 breast cancer cells. Thus the non-hydrolyzable analog, 4-HBR, may have significant therapeutic advantage over 4-HPR because it does not liberate atRA that can contribute to the adverse side effects of drug administration in vivo.

Section snippets

Chemicals

[20-methyl ³H]-all-trans-RA (72 Ci/mmol) was obtained from New England Nuclear (Dupont, Boston, MA). atRA was purchased from Sigma Chemical (St. Louis, MO). 4-HPR was a gift from McNeil Pharmaceuticals (Springhouse, PA). Retinoids were dissolved in ethanol and the purity was checked by HPLC (>99%) followed by storage at −70 °C until use.

4-HBR synthesis

4-HBR was prepared as briefly outlined previously [35] with some modifications. Care was taken to avoid exposure of 4-HBR to trace acid because of its much

4-HPR, but not 4-HBR, supports the growth of VAD rats

The structures of the retinoids used in these studies are shown in Fig. 1. In a preliminary growth study, we observed that VAD rats given vehicle alone exhibited steady weight loss over a 5-day test period, whereas maximal growth was fully supported in animals fed atRA in amounts greater than or equal to 0.08 μmol/day (data not shown). Consequently, in the experiment shown in Fig. 2, a dose of atRA at a level just below that required to support maximal growth (0.04 μmol/day) and two higher doses

Discussion

4-HPR has positive chemotherapeutic effects in mammary tumor models as evidenced by a reduction in both the number and size of existing DMBA-induced mammary tumors [27] and it also inhibits the growth of mammary tumor cells in culture [56], [57]. The possibility that atRA is liberated by hydrolysis from 4-HPR and contributes to its activity continues to be raised in the literature [21], [29], [30]. To evaluate the vitamin A activity of 4-HPR in vivo, a highly sensitive VAD rat bioassay was

Acknowledgements

We thank Ron Merrill for generating the CYP26B1 plasmid and Adam Steinberg in the Biochemistry Media Lab for the artwork. The F9 reporter cell line was a kind gift from Dr. M. Wagner and Dr. T. Jessell. This work was supported by a Grant (CA49837) from the National Cancer Institute.

References (66)

P McCaffery et al.
Cytokine Growth Factor Rev.
(2000)
B Amos et al.
Methods Enzymol.
(1990)
J.J DiGiovanna
J. Am. Acad. Dermatol.
(2001)
C.N Ellis et al.
J. Am. Acad. Dermatol.
(2001)
H Nau
J. Am. Acad. Dermatol.
(2001)
R.G Mehta et al.
Eur. J. Cancer
(1991)
A.N Fanjul et al.
J. Biol. Chem.
(1996)
R.R Mehta et al.
Eur. J. Cancer
(1998)
K.L Weiss et al.
Bioorg. Med. Chem. Lett.
(2001)
T Suda et al.
J. Nutr.
(1970)

P Chomczynski et al.

Anal. Biochem.

(1987)

M Clagett-Dame et al.

Arch. Biochem. Biophys.

(1993)

M.M Bradford

Anal. Biochem.

(1976)

J.A White et al.

J. Biol. Chem.

(1996)

W.J Ray et al.

J. Biol. Chem.

(1997)

G MacLean et al.

Mech. Dev.

(2001)

A.H Wyllie et al.

Int. Rev. Cytol.

(1980)

R Bhatnagar et al.

Biochem. Pharmacol.

(1991)

M Zile et al.

J. Nutr.

(1968)

M Poot et al.

Exp. Cell Res.

(2002)

W.Y Zhu et al.

Exp. Cell Res.

(1997)

M Clagett-Dame et al.

Annu. Rev. Nutr.

(2002)

S.-Y Sun et al.

Drugs Future

(1998)

J.A Fontana et al.

Leukemia

(2002)

P Prakash et al.

Nutr. Rev.

(2000)

W.K Hong et al.

L.A Hansen et al.

Carcinogenesis

(2000)

R.B Armstrong et al.

M.D Collins et al.

Annu. Rev. Pharmacol. Toxicol.

(1999)

F Formelli

D Delia et al.

Cancer Res.

(1993)

A Di Vinci et al.

Int. J Cancer

(1994)

A Mariotti et al.

J. Natl. Cancer Inst.

(1994)

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Hydrolysis of 4-HPR to atRA occurs in vivo but is not required for retinamide-induced apoptosis

Abstract

Section snippets

Chemicals

4-HBR synthesis

4-HPR, but not 4-HBR, supports the growth of VAD rats

Discussion

Acknowledgements

Cytokine Growth Factor Rev.

Methods Enzymol.

J. Am. Acad. Dermatol.

J. Am. Acad. Dermatol.

J. Am. Acad. Dermatol.

Eur. J. Cancer

J. Biol. Chem.

Eur. J. Cancer

Bioorg. Med. Chem. Lett.

J. Nutr.

Anal. Biochem.

Arch. Biochem. Biophys.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Mech. Dev.

Int. Rev. Cytol.

Biochem. Pharmacol.

J. Nutr.

Exp. Cell Res.

Exp. Cell Res.

Annu. Rev. Nutr.

Drugs Future

Leukemia

Nutr. Rev.

Carcinogenesis

Annu. Rev. Pharmacol. Toxicol.

Cancer Res.

Int. J Cancer

J. Natl. Cancer Inst.