fenretinide and Carcinoma--Embryonal

Fenretinide (4-HPR) is a retinoid analogue with antitumor and chemopreventive activities. In addition to 4-HPR, there are several other new phenylretinamides bearing hydroxyl, carboxyl, or methoxyl residues on carbons 2, 3, and 4 of the terminal phenylamine ring [N-(2-hydroxyphenyl)retinamide (2-HPR), N-(3-hydroxyphenyl)retinamide, N-(2-carboxyphenyl)retinamide, N-(3-carboxyphenyl)retinamide, N-(4-carboxyphenyl)retinamide, and N-(4-methoxyphenyl)retinamide (4-MPR) ]. It is hypothesized that these agents can act independent of the nuclear retinoid receptor pathway. To test this hypothesis directly, we have analyzed the activity of these phenylretinamides in vitro on a panel of F9 murine embryonal carcinoma cell lines, which includes wild-type (F9-WT) and mutant cells that have disrupted genes for both retinoid X receptor alpha and retinoic acid receptor gamma retinoid receptors (F9-KO). The F9-KO cells lack almost all measurable response to all-trans-retinoic acid, the primary biologically active retinoid. Two distinct effects of retinamides were identified. The first is a rapid, dose-dependent induction of cell growth inhibition (reduced cell viability), and the second is a slower induction of differentiation and accumulation of cells in the G(1) phase of the cell cycle that was observed with a concentration of 1 micro M, for only those phenylretinamides bearing charged (hydroxyl or carboxyl) groups on the terminal phenylamine ring. The induction of differentiation and G(1) accumulation was only observed in the F9-WT cells, indicating that this effect is receptor-dependent. 4-MPR, a major metabolite of 4-HPR, lacks a charged group on the terminal phenylamine ring and did not induce retinoid receptor-dependent effects, but did induce cell growth inhibition. Thus, 4-MPR may play a role in the clinical activity of 4-HPR. This study further reveals the mechanism of action of these novel phenylretinamides and supports continued investigation into their development as chemopreventive drugs.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Carcinoma, Embryonal; Cell Death; Cell Differentiation; Fenretinide; G1 Phase; Immunoenzyme Techniques; Mice; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Retinoid X Receptors; S Phase; Signal Transduction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Fenretinide (N-[4-hydroxyphenyl]retinamide (4HPR)) is a retinoid analogue with antitumor and chemopreventive activities. The mechanism of action of 4HPR is not fully understood, but it is hypothesized that this compound acts independently of the nuclear retinoid receptor pathway. To test this hypothesis directly, we have analyzed the activity of 4HPR on a panel of F9 embryonal carcinoma cell lines, which includes wild-type and mutant lines that lack expression of retinoic acid receptor gamma, retinoid X receptor alpha, or both. 4HPR (10 microM) treatment resulted in a rapid induction of cell death in F9 cells, which was responsible for their near elimination by 48 h. This effect occurred in the receptor-null cell lines as well. Treatment of the wild-type cells for 4 days with 1 microM 4HPR also resulted in a primitive endodermal differentiated phenotype that is normally seen upon all-trans-retinoic acid treatment and is characterized by the up-regulation of laminin B1 and type IV collagen. This differentiation response did not occur in the receptor-null cells. Therefore, two distinct effects of 4HPR were identified in this system: a rapid induction of cell death and a slower induction of differentiation, which are likely to be receptor independent and dependent, respectively.

Topics: Animals; Antineoplastic Agents; Carcinoma, Embryonal; Cell Death; Cell Differentiation; Fenretinide; Mice; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Retinoid X Receptors; Signal Transduction; Transcription Factors; Tumor Cells, Cultured

Retinoids signal biological effects through retinoic acid receptors (RAR) and retinoid X receptors (RXR) and their co-regulators. We previously reported that all-trans retinoic acid (RA) triggers terminal differentiation in the human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1), through an RARgamma dependent pathway. RARgamma repression in NT2/D1-R1 cells accounts for RA resistance in this line. This report finds RARgamma repression is due to selective repression of RARgamma but not RARbeta transcription in NT2/D1-R1 cells. The repression is neither due to mutations in RARgamma nor its promoter containing the RA response element. Prior work was confirmed and extended by demonstrating that an RARgamma selective agonist preferentially signals differentiation of NT2/D1 cells, while RARalpha/beta, RARbeta, RXR agonists and an RAR pan-antagonist do not even when NT2/D1 cells are treated with these retinoids at 10 microM dosages. None of these examined retinoids induced differentiation of the RA resistant NT2/D1-R1 cells. In contrast, N-(4-hydroxyphenyl)retinamide (4HPR), a reported transcriptional activator of RARgamma was shown to potently induce growth inhibition and apoptosis in both NT2/D1 and NT2/D1-R1 cells. 4HPR-induced apoptosis was unaffected by co-treatment of both cell lines with equimolar RAR antagonist. Semi-quantitative reverse transcription-polymerase chain reaction (RT - PCR) assays of total RNA from 4HPR-treated NT2/D1 and NT2/D1-R1 cells did not reveal RARgamma induction. Since 4HPR signals in RA-resistant NT2/D1-R1 cells having an RARgamma transcriptional block, these results indicate that 4HPR triggers apoptosis but not differentiation through an RARgamma independent pathway. Taken together, these findings implicate a therapeutic role for 4HPR mediated apoptosis in germ cell tumors even when a maturation block is present.

Topics: Anticarcinogenic Agents; Apoptosis; Carcinoma, Embryonal; Cell Differentiation; DNA Fragmentation; Fenretinide; Humans; Neurons; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Retinoids; RNA, Messenger; Teratocarcinoma; Time Factors; Tretinoin; Tumor Cells, Cultured