Effect of 4-hydroxyphenylretinamide and retinoic acid on proliferation and cell cycle of cultured human breast cancer cells

The synthetic retinoid 4-hydroxyphenylretinamide (HPR) showed antiproliferative effect on cultured human breast cancer cells, which were sensitive to retinoic acid (RA) too. Investigation of the cell cycle by flow cytophotometry showed a significant increase of cells in the S-phase of the cell cycle after 24 hours of RA treatment, whereas HPR did not show this effect. After 4-7 days of retinoid treatment, the percentage of resting cells increased. The influence on the cell cycle and the antiproliferative effect were more pronounced for RA than for HPR treatment in both cell lines investigated (ZR-75.1 and 734 B).     

Effects of retinoic acid and fenretinide on the c-erbB-2 expression, growth and cisplatin sensitivity of breast cancer cells.

We investigated the effects of all-trans retinoic acid (ATRA) and fenretinide (4-HPR) on c-erbB-2 expression in SK-BR-3, BT-474 and MCF-7 breast cancer cells and on the growth, differentiation, apoptosis and cisplatin (CDDP) sensitivity of SK-BR-3 cells. It has been reported that oestrogen inhibits c-erbB-2 in oestrogen receptor-positive breast cancer cells. Using ELISA, Western and Northern analysis we have demonstrated that ATRA and 4-HPR exert similar effects down-regulating c-erbB-2 protein and mRNA in c-erbB-2-overexpressing SK-BR-3 and BT-474 and in normally expressing MCF-7 cells. Both retinoids inhibit SK-BR-3 cell growth. ATRA induces cellular enlargement and flattening, suggesting epithelial differentiation. 4-HPR causes nuclear and cytoplasmic condensation, DNA fragmentation and externalization of phosphatidylserine, indicating apoptosis. c-erbB-2 expression/activity has been linked to sensitivity against CDDP. Therefore, combinations of ATRA or 4-HPR with CDDP were tested for their anti-proliferative activity. Retinoid-conditioned cells were either exposed to retinoid and CDDP (schedule I, ''continuous retinoid treatment'') or to CDDP alone (schedule II, ''retinoid pretreatment''). This retinoid-conditioning followed by CDDP +/- retinoid yields stronger growth inhibition compared with unconditioned cells, which were exposed to CDDP +/- retinoid (schedule III, ''no retinoid pretreatment''). The inefficacy of schedule III indicates that retinoid-conditioning is essential for the improvement of the antiproliferative effect. The interactions in schedules I and II are synergistic for ATRA and CDDP, but slightly antagonistic for 4-HPR and CDDR However, 4-HPR + CDDP is more effective in growth inhibition than each drug alone.     

Unique anti-activator protein-1 activity of retinoic acid receptor beta

The anticancer effects of retinoids are mainly mediated by two classes of nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are encoded by three distinct genes (alpha, beta, and gamma). Recent studies have demonstrated that RARbeta plays a critical role in mediating anticancer effects of retinoids. However, how RARbeta exerts its potent anticancer effects remains largely unknown. In this study, we investigated anti-Activator Protein-1 (AP-1) activity of RARbeta. In a transient transfection assay, all three RAR subtypes, RARalpha, RARbeta, and RARgamma, could effectively inhibit phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity and the activity of oncogenes c-Jun and c-Fos on AP-1 containing reporter genes in the presence of retinoic acid (RA). However, RARbeta showed a strong RA-independent inhibition of AP-1 activity, whereas inhibition of AP-1 activity by RARalpha and RARgamma was RA dependent. By using several hybrid receptors that contain either the COOH-terminal portion or the NH2-terminal portion of RARbeta, we demonstrated that the NH2-terminal portion of RARbeta, the A/B domain, was mainly responsible for the RA-independent inhibition of AP-1 activity. This activity was not attributable to constitutive AF-1 activity of RARbeta, because it did not activate several RA response element-containing reporter genes. In addition, inhibition of histone deacetylase activity by trichostatin A did not overcome the inhibitory effect of RARbeta. In cancer cells, stable transfection of RARbeta exhibited strong inhibition of AP-1 activity, even in the absence of RA. Moreover, expression of endogenous AP-1-responsive gene collagenase I was strongly repressed in cancer cells stably transfected with RARbeta. In studying the antitransforming activity of RARbeta, we observed that the growth of breast cancer MDA-MB231 cells in soft agar was significantly repressed in a RA-independent manner when cells were stably transfected with RARbeta but not RARalpha. Together, our results demonstrate that RARbeta may exert its potent anticancer effect in part through its unique anti-AP-1 activity.     

Nuclear retinoid receptors are involved in N-(4-hydroxyphenyl) retinamide (Fenretinide)-induced gene expression and growth inhibition in HL-60 acute myeloid leukemia cells

N-(4-hydroxyphenyl) retinamide (Fenretinide, 4-HPR) inhibits cell growth by inducing apoptosis in numerous tumor cell types including all-trans-retinoic acid (ATRA)-resistant tumor cells. However, the mechanism(s) by which 4-HPR mediates its anti-proliferative effects remains unclear. Here, we determined whether 4-HPR induced growth inhibition and gene expression involve retinoid receptors in human acute myeloid leukemia (AML) cells (HL-60). We treated HL-60 and ATRA-resistant HL-60 (HL-60R) cells that express mutated RARalpha and very low levels of RARbeta, RARgamma and RXRalpha with 4-HPR (2 microM) for 3 days. 4-HPR showed significant anti-proliferative effects against both cell types and induced growth inhibition (92.7%) in HL-60 cells. However, at the same dose, 4-HPR induced only 53.4% growth inhibition in HL-60R cells. Growth inhibition by 4-HPR was significantly enhanced in HL-60R cells that were retroviraly transduced to express human RARalpha, RARbeta or RXRalpha (95.6%, 97.1%, and 75.6%, respectively), in comparison to HL-60R cells (P < 0.05), but not in HL-60R cells expressing RARgamma. Although ATRA and 4-HPR induced expression of CYP26, an ATRA-inducible gene encoding a cytochrome P450 enzyme, in HL-60 cells, both retinoids failed to induce CYP26 in HL-60R cells. However, induction of CYP26 mRNA by 4-HPR was restored in HL-60R cells expressing RARalpha and RARgamma, but not RARbeta and RXRalpha. In conclusion, our data suggest that nuclear retinoid receptors are involved in 4-HPR-induced growth inhibition and gene expression, and that 4-HPR can mediate its anti-proliferative effects through retinoid receptor-dependent mechanisms in HL-60 cells.     

Stage-specific effect of N-(4-hydroxyphenyl)retinamide on cell growth in squamous cell carcinogenesis

Squamous cell carcinoma (SCC) is the most prevalent form of epithelial cancer. SCC results when normal epithelial cells undergo multiple neoplastic changes that culminate in the evolution of an invasive cancer. Retinoids are commonly used as chemopreventive and treatment agents in skin cancer; however, SCC progression is accompanied by a gradual loss of retinoid responsiveness. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has shown promising anti-neoplastic activity in a variety of tumor cells, including those that are resistant to all-trans retinoic acid (t-RA). We investigated the effect of HPR on growth and apoptosis of squamous cells at different stages of carcinogenesis. We then determined if retinoic acid receptor (RAR) overexpression affected the outcome of HPR treatment. To model SCC malignant progression, we used a panel of murine keratinocytes representing different stages of squamous cell carcinogenesis. This panel consisted of primary keratinocytes, SP1 and 308 papilloma cell lines, the PAM-212 squamous carcinoma cell line, and the spindle I7 cell line. With the exception of the primary keratinocytes, all cells were unresponsive to t-RA treatment. Pharmacological concentrations of HPR were non-cytotoxic to all keratinocytes tested and HPR sensitivity was stage-dependent, with the papilloma cell lines being the most sensitive, and the spindle cells being the most resistant. Overexpression of RARgamma in SP1 papilloma cells enhanced growth suppression and apoptosis induction by HPR. HPR-induced growth suppression was accompanied by a simultaneous block in the G(1) phase of the cell cycle in RAR-transduced and control SP1 cells and differential regulation of cell cycle and apoptotic mediators.     

Tyrphostins and retinoids cooperate during inhibition of in vitro growth of ovarian cancer cells

Chemoresistance of ovarian cancer can be overcome by co-administration of retinoids, albeit clinical proof of this hypothesis is pending. Moreover, growth factor/c-erbB signaling is crucial for ovarian tumor growth/chemosensitivity. Retinoids and c-erbB modulators therefore represent promising drugs for ovarian cancer. We demonstrate that c-erbB-1 (RG-14620, AG1517) and c-erbB-2 selective tyrphostins (AG825, AG879), and all-trans and 9-cis retinoic acid inhibit ovarian cancer cell proliferation (HOC-7, OVCAR-3). Unlike retinoids, AG1517 and AG879 induce apoptosis. The antiproliferative activity of AG1517 is enhanced by all-trans retinoic acid suggesting that c-erbB and retinoid pathways interact. Thus, these agents cooperate during ovarian cancer cell growth inhibition.     

The antiproliferative activity of all-trans-retinoic acid catabolites and isomers is differentially modulated by liarozole-fumarate in MCF-7 human breast cancer cells.

The clinical use of all-trans-retinoic acid (ATRA) in the treatment of cancer is significantly hampered by the prompt emergence of resistance, believed to be caused by increased ATRA catabolism. Inhibitors of ATRA catabolism may therefore prove valuable for cancer therapy. Liarozole-fumarate is an anti-tumour drug that inhibits the cytochrome P450-dependent catabolism of ATRA. ATRA, but also its naturally occurring catabolites, 4-oxo-ATRA and 5,6-epoxy-ATRA, as well as its stereoisomers, 9-cis-RA and 13-cis-RA, show significant antiproliferative activity in MCF-7 human breast cancer cells. To further elucidate its mechanism of action, we investigated whether liarozole-fumarate was able to enhance the antiproliferative activity of ATRA catabolites and isomers. Liarozole-fumarate alone up to a concentration of 10(-6) M had no effect on MCF-7 cell proliferation. However, in combination with ATRA or the ATRA catabolites, liarozole-fumarate (10(-6) M) significantly enhanced their antiproliferative activity. On the contrary, liarozole-fumarate (10(-6) M) was not able to potentiate the antiproliferative activity of the ATRA stereoisomers, most probably because of the absence of cytochrome P450-dependent catabolism. Together, these findings show that liarozole-fumarate acts as a versatile inhibitor of retinoid catabolism in that it not only blocks the breakdown of ATRA, but also inhibits the catabolic pathway of 4-oxo-ATRA and 5,6-epoxy-ATRA, thereby enhancing their antiproliferative activity.     

Increasing the intracellular availability of all-trans retinoic acid in neuroblastoma cells

Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 microM) or R116010 (1 or 10 microM) in combination with either 10 microM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma.     

Inhibition of cancer cell growth by all- trans retinoic acid and its analog N-(4-hydroxyphenyl) retinamide: a possible mechanism of action via regulation of retinoid receptors expression

In order to better understand the mechanisms that underlie the antiproliferative effect of retinoids, we have examined the response of human carcinoma cell lines to all-trans retinoic acid (RA) and N-(4-hydroxyphenyl) retinamide (4HPR) in terms of cell growth, apoptosis and regulation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mRNA. GLC82 (lung adenocarcinoma), BGC823 (stomach adenocarcinoma) and EC109 (esophageal squamous carcinoma) cells were treated with 10 μM of RA or 4HPR for various length of time and analyzed. The results show that growth inhibition by RA and 4HPR in GLC82 and BGC823 cells correlates with the induction of RARβ2 gene, whereas RA resistance in EC109 cells parallels loss of RARβ2 induction. Exogenous RARβ2 expression did not restore RA responsiveness in EC109 cells, but potentiated 4HPR-induced growth inhibition, suggesting that 4HPR acts at least in part via the RARβ receptor. We speculate that the loss of RARβ2 inducibility in EC109 cells may be due to an unknown repressor. Int. J. Cancer 78:248–254, 1998.© 1998 Wiley-Liss, Inc.     

Induction of apoptosis by retinoids in human cervical-carcinoma cell-lines

Retinoids can inhibit the growth and modulate the differentiation of a variety of tumor cell types in vitro and in vivo. All-trans retinoic acid (ATRA) and N-(4-hydroxyphenyl)retinamide (4HPR) are currently being evaluated in clinical trials for their potential use in cancer chemoprevention and therapy. We compared the effects of these retinoids on 10 human cervical carcinoma cell lines. Four of the 10 cell lines showed dramatic morphological changes and the other 5 exhibited decreased cell density after treatment with 10 mu M 4HPR, whereas few changes were induced by 10 mu M ATRA. Cell rounding and detachment were also observed in four of the cell lines. An analysis of DNA from both detached and attached cells after retinoid treatment has demonstrated the formation of a DNA ladder after electrophoresis in agarose gels, which indicated that some of the cell lines had undergone apoptosis. Induction of DNA fragmentation by 4HPR but not by other retinoids (ATRA, 13-cis-RA, and 9-cis-RA) was further evidenced as early as 24 h after treatment by a quantitative assay based on the degradation of [H-3]-thymidine-labeled DNA. Ln addition, morphological changes of nuclei associated with apoptosis such as chromatin condensation were observed by propidium iodide staining of the nuclei after 4HPR treatment. These results demonstrate that 4HPR causes apoptosis in several cervical carcinoma cell lines and that it is more potent in this effect than ATRA or other RA isomers.     

N-(4-hydroxyphenyl)-retinamide selectively increases All-TRANS retinoic acid inhibitory effects in HER2/NEU-overexpressing breast cancer cells.

We previously reported that overexpression of the HER2/NEU oncogene induces all-TRANS retinoic acid (ATRA) resistance in breast cancer cells. N-(4-hydroxyphenyl)-retinamide (4HPR), a synthetic analogue of ATRA, has been shown to repress the expression of HER2/neu and its family member, epidermal growth factor receptor (EGFR). We investigated whether 4HPR, by suppressing HER2/neu or EGFR expression, could sensitize breast cancer cells to ATRA. At 1.3 micro M concentration (a clinically pharmacologically achievable dose), 4HPR increased ATRA sensitivity synergistically in HER2/NEU-overexpressing BT-474, MDA-MB-453, and MCF-7/Her2 breast cancer cells. However, 4HPR did not sensitize EGFR-overexpressing MDA-MB-468, Hs578T, and MCF-7/EGFR breast cancer cells to ATRA. The increased inhibitory effects in HER2/NEU-overexpressing cells were not correlated with increases in expression levels of p21(WAF1/CIP1) or retinoblastoma protein. Combining 4HPR with ATRA may lead to a novel, selective therapeutic or chemopreventive strategy against HER2/NEU-overexpressing breast tumors.