Cyclooxygenase-2 is essential for HER2/neu to suppress N- (4-hydroxyphenyl)retinamide apoptotic effects in breast cancer cells

We reported that HER2/neu reduces the sensitivity of breast cancer cells to N-(4-hydroxyphenyl)retinamide (4-HPR) by suppressing nitric oxide production. We show that HER2/neu uses Akt to induce cyclooxygenase-2 (COX-2) expression and that inhibition of Akt or COX-2 increases 4-HPR-induced apoptosis and nitric oxide production. Apoptosis induced by the 4-HPR and COX-2 inhibitor combination, although unaffected by an anti-HER2/neu antibody, was reversed by the COX-2 product prostaglandin E(2), indicating that COX-2 is a major mechanism by which HER2/neu suppresses 4-HPR apoptosis in breast cancer cells. Combining 4-HPR with COX-2 inhibitors may be a novel chemopreventive strategy against HER2/neu-overexpressing breast tumors.     

HER2/neu reduces the apoptotic effects of N-(4-hydroxyphenyl)retinamide (4-HPR) in breast cancer cells by decreasing nitric oxide production

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR also known as fenretinide) is a potent inducer of apoptosis in breast cancer cells. We observed a 4.5-fold reduction in 4-HPR-mediated apoptosis in MCF-7 breast cancer cells transfected with HER2/neu (MCF-7/HER2) as compared with the parental MCF-7 (MCF-7/WT) cells. Blocking HER2/neu with trastuzumab (Herceptin) led to a six-fold increase in 4-HPR-induced apoptosis in HER2/neu-overexpressing cells. These data indicate that HER2/neu reduces the sensitivity of breast cancer cells to 4-HPR. We showed previously that nitric oxide (NO) is essential for 4-HPR to induce apoptosis in breast cancer cells. The inhibitory effects of the 4-HPR and trastuzumab combination correlated with the amount of NO produced in HER2/neu-overexpressing cells. When a NO synthase (NOS) inhibitor was used to block NO production, decreased apoptosis by the 4-HPR and trastuzumab combination was observed. Furthermore, 4-HPR-mediated NOSII expression was lower in MCF-7/HER2 than MCF-7/WT cells, but was increased by trastuzumab in HER2/neu-overexpressing cells. Here we report the novel findings that HER2/neu reduces the ability of 4-HPR to induce apoptosis in breast cancer cells, and that one mechanism by which HER2/neu increases the resistance of breast cancer cells to 4-HPR is by decreasing NOSII-mediated NO production.     

Modulation of the insulin-like growth factor-I system by N-(4-hydroxyphenyl)-retinamide in human breast cancer cell lines.

The potent mitogenic activity of insulin-like growth factor I (IGF-I) on breast epithelium is inhibited by retinoic acid in oestrogen receptor-positive (ER+) breast cancer cell lines. We studied and compared the effects of N-(4-hydroxyphenyl)-retinamide (4-HPR) in terms of growth inhibition and modulation of the IGF-I system in ER+ (MCF-7) and oestrogen receptor-negative (ER-) (MDA-MB231) breast cancer cell lines. Treatment with 1-10 microM 4-HPR for up to 96 h induced a dose- and time-dependent inhibition of proliferation in both breast cancer cell lines. Induction of apoptosis was much more evident in MCF-7 than in MDA-MB231 cells (30-40% compared with 0-5% respectively at 5 microM for 48 h). Exogenous human recombinant IGF-I (hr-IGF-I)-stimulated cell proliferation was abolished by 1 microM 4-HPR in MCF-7 cells. Immunoreactive IGF-I-like protein concentration in conditioned medium was reduced by 38% in MCF-7 and by 90% in MDA-MB231 cell lines following treatment for 48 h with 5 microM 4-HPR. Western ligand blot analysis showed a reduction of IGF-binding protein 4 (BP4) and BP5 by 67% and 87%, respectively, in MCF-7, whereas IGF-BP4 and -BP1 were reduced by approximately 20% in MDA-MB231 cells. Exposure to 5 microM 4-HPR for 48 h inhibited [125I]IGF-I binding and Scatchard analysis revealed a decrease of more than 50% in maximum binding capacity (Bmax) and a reduced receptor number/cell in both cancer cell lines. Steady-state type I IGF-receptor mRNA levels were reduced by approximately 30% in both tumour cell lines. We conclude that 4-HPR induces a significant down-regulation of the IGF-I system in both ER+ (MCF-7) and ER- (MDA-MB231) breast cancer cell lines. These findings suggest that, in our model, interference with the ER signalling pathway is not the only mechanism of breast cancer growth inhibition by 4-HPR.     

Cyclosporin A enhances the apoptotic effects of N-(4-hydroxyphenyl)retinamide in breast cancer cells

4HPR, an analogue of ATRA, effectively induces growth inhibition and apoptosis in breast cancer cell lines and animal models but is ineffective against advanced human breast tumors. Different compounds, including tamoxifen, are currently being tested to increase 4HPR efficacy in the clinic. Here, we report that cyclosporin A selectively increases the ability of 4HPR, but not ATRA, to induce growth inhibition and apoptosis in ER(+) and ER(-) breast cancer cell lines. Increased apoptosis by the 4HPR and cyclosporin A combination was correlated with increased production of the free radical nitric oxide. Thus, the 4HPR and cyclosporin A combination may potentially be a novel therapeutic modality against breast tumors.     

Inhibition of N-(4-hydroxyphenyl)retinamide-induced apoptosis in breast cancer cells by galectin-3

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) induces apoptosis in a variety of human cancer cells including breast carcinoma and this property may be important for its chemopreventive and therapeutic effects. Resistance to 4HPR has been described, however, the molecular mechanisms underlying sensitivity or resistance to this retinoid are not clear. Recently, it has been shown that the carbohydrate-binding protein galectin-3, which has been implicated in tumor progression, contains the anti-death motif NWGR present in the anti-apoptotic protein Bcl-2. To determine whether galectin-3 expression can abrogate the effect of 4HPR, we tested the effects of 4HPR on apoptosis of cell clones derived from the galectin-3 deficient human BT549 breast carcinoma cells after transfection with either wild type galectin-3 (BT549Gal-3Wt), galectin-3 inactivated by a point mutation in the NWGR motif (BT549Gal-3Mu), or empty vector control (BT549Vec). Both BT549Vec and BT549Gal-3Mu cells showed a marked decrease in survival after treatment with 4HPR principally due to induction of apoptosis. 4HPR-induced apoptosis in these cells was associated with stimulation of reactive oxygen species generation, decreased levels of Bcl-2 protein, release of cytochrome c into the cytosol, increased caspase-3 activity, and poly(ADP-ribose) polymerase cleavage. In contrast, 4HPR failed to exert any of these effects in the BT549Gal-3Wt cells. The demonstration that galectin-3 suppresses 4HPR-induced apoptosis in human breast carcinoma cells suggests that the increased expression of galectin-3 during cancer progression may be associated with 4HPR resistance.     

Loss of growth inhibitory effects of retinoic acid in human breast cancer cells following long-term exposure to retinoic acid

Although retinoids are known to be inhibitory to breast cancer cell growth, a key remaining question is whether they would remain effective if administered long-term. We describe here the long-term effects of all- trans retinoic acid on two oestrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1. Although both cell lines were growth inhibited by retinoic acid in the short-term in either the absence or the presence of oestradiol, prolonged culture with 1 microM all- trans retinoic acid resulted in the cells acquiring resistance to the growth inhibitory effects of retinoic acid. Time courses showed that oestrogen deprivation of the cell lines resulted in upregulation of the basal non-oestrogen stimulated growth rate such that cells learned to grow at the same rate without as with oestradiol, but the cells remained growth inhibited by retinoic acid throughout. Addition of 1 microM all- trans retinoic acid to steroid deprivation conditions resulted in reproducible loss of growth response to both retinoic acid and oestradiol, although the time courses were separable in that loss of growth response to retinoic acid preceded that of oestradiol. Loss of growth response to retinoic acid did not involve loss of receptors, ER as measured by steroid binding assay or RARalpha as measured by Northern blotting. Function of the receptors was retained in terms of the ability of both oestradiol and retinoic acid to upregulate pS2 gene expression, but there was reduced ability to upregulate transiently transfected ERE- and RRE-linked reporter genes. Despite the accepted role of IGFBP3 in retinoic acid-mediated growth inhibition, progression to retinoic acid resistance occurred irrespective of level of IGFBP3, which remained high in the resistant MCF7 cells. Measurement of AP1 activity showed that the two cell lines had markedly different basal AP1 activities, but that progression to resistance was accompanied in both cases by a lost ability of retinoic acid to reduce AP1 activity. These results warn of potential resistance which could arise on long-term treatment with retinoic acid in a clinical situation and echo the problems of progression to endocrine resistance. It seems that whatever the constraints imposed on growth, these cells have a remarkable ability to escape from growth inhibition. However, the ability of retinoic acid to delay progression to oestrogen resistance is encouraging for endocrine therapy, and the concentration-dependence of retinoic acid resistance suggests that progression is not absolute but could be manipulated by dose.     

Her2/neu induces all-trans retinoic acid (ATRA) resistance in breast cancer cells

We observed that all-trans retinoic acid (ATRA) inhibited the growth of MCF-7 breast cancer cells, but not those transfected with HER2/NEU or its transactivating ligand HEREGULIN. This suggests that Her2/neu causes breast cancer cells to be resistant to the growth inhibitory effects of ATRA. To confirm this observation, MDA-MB-453 and BT-474 cells, which have high levels of Her2/neu and are resistant to ATRA, were incubated with the trastuzumab (Herceptin) antibody so that we could determine whether inhibition of the expression and function of Her2/neu would resensitize these cells to ATRA. Indeed, we found that MDA-MB-453 and BT-474 cells treated with trastuzumab were growth inhibitory by ATRA. We then determined whether Her2/neu uses Grb2 and Akt proteins to induce ATRA resistance. Liposome-incorporated Grb2 antisense oligonucleotides (L-Grb2) and a dominant negative (DN) AKT mutant were used to down-regulate Grb2 expression and inhibit Akt activity, respectively. When incubated with L-Grb2 or transfected with the DN AKT mutant, ATRA-resistant, Her2/neu-overexpressing cells became sensitive to ATRA. Our results indicate that Her2/neu utilizes Grb2 and Akt proteins to induce ATRA resistance in breast cancer cells. ATRA sensitivity was also correlated with RARalpha protein levels since higher RARalpha protein levels were observed in cells in which the Her2/neu pathway was inhibited.     

Cytotoxic responses to N-(4-hydroxyphenyl)retinamide in human pancreatic cancer cells

Purpose  

Although fenretinide (4-HPR) has been studied in breast cancer and in neuroblastoma, little is known regarding its activity in pancreatic cancer, a neoplasm for which there are few therapeutic options. Since pancreatic cancer cells are susceptible to reactive oxygen species (ROS) and ceramide, two hallmarks of 4-HPR cytotoxicity, we investigated the effect of 4-HPR on human pancreatic cancer cells.     

Induction of apoptosis by N-(4-hydroxyphenyl)retinamide in glioma cells.

N-(4-hydroxyphenyl)retinamide (fenretinide) is a synthetic retinoid with anticancer properties. We investigated the effects of fenretinide on the growth of glioma cells. Four glioma cell lines (C6, 9L, Med3 and U87) were treated with fenretinide. Cell viability and independent growth was determined by MTS assay and soft agar assay, respectively. The induction of apoptosis was evaluated by microscopic examination, flow cytometric DNA content analysis, and in situ TdT methods. Fenretinide markedly reduced cell viability of all the glioma cell lines examined at a range of concentrations from 1 to 10 microM. In all cell lines examined, fenretinide also induced morphological changes consistent with apoptosis, including cellular shrinkage, chromatin condensation, and nuclear fragmentation. Flow cytometric analysis also revealed an apoptotic pattern of the DNA content, and in situ detection of apoptosis showed increased incorporation of digoxigenin-nucleotide triphosphate in fenretinide-treated glioma cells. These findings indicate that fenretinide inhibits the growth of glioma cells via the induction of apoptosis, suggesting potential clinical use of fenretinide for treatment of glioma patients.     

N-(4-hydroxyphenyl)retinamide induces growth arrest and apoptosis in HTLV-I-transformed cells.

N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid that inhibits growth and induces apoptosis in many human cell lines. We explored the effects of HPR on human T-cell lymphotropic virus type I (HTLV-I)-positive and HTLV-I-negative malignant T-cell lines, most of which are resistant to all-trans retinoic acid. Clinically achievable concentrations of HPR caused a dramatic inhibition of cell proliferation, G(0)/G(1) arrest, and massive apoptosis in all tested malignant T cells, while no effect was observed on resting or activated normal lymphocytes. Interestingly, HTLV-I-negative cell lines were significantly more sensitive to HPR compared to HTLV-I-positive and Tax-transfected cells. In HTLV-I-negative cells only, HPR-induced apoptosis was associated with ceramide accumulation, sharp decrease in mitochondrial membrane potential, and activation of caspases 8, 9 and 3, and could be partially reverted by the caspase inhibitor z-VAD suggesting that Tax protects infected cells from ceramide accumulation and caspase-mediated apoptosis. In HTLV-I-positive cells, HPR treatment rapidly induced proteasomal-mediated degradation of p21, downregulated cyclin D(1), and upregulated bax protein levels. These findings support a potential therapeutic role for HPR in both HTLV-I-associated adult T-cell leukemia/lymphoma (ATL) and HTLV-I-negative peripheral T-cell lymphomas.     

Inhibition of mammary tumorigenesis in nulliparous C3H mice by chronic feeding of the synthetic retinoid, N-(4-hydroxyphenyl)-retinamide

Feeding of N-(hydroxyphenyl)retinamide (4-HPR) (1.0 mM/kg diet)for 39 weeks to 2 month old nulliparous C3H/Crgl mice significantly(p<0.01) reduced the incidence of mammary tumors; 98 controlmice developed a total of 235 mammary tumors, 100 4-HPR treatedmice developed 171 mammary tumors. Feeding the same dietarylevel of 4-HPR to 4–6 month old multiparous OH mice for14 weeks did not significantly influence the development ofmammary tumors; 78 control mice developed a total of 115 mammarytumors; 78 4-HPR treated mice developed 130 mammary tumors.Body weight gains were comparable among 4-HPR treated mice andcontrol mice. Significant suppression of mouse mammary glandtumorigenesis in vivo by dietary retinoids (nulliparous C3Hmice) has heretofore not been reported.     

N-(4-hydroxyphenyl)retinamide: interactions with retinoid-binding proteins/receptors

The cellular transport, metabolism and biological activity ofretinoids are mediated by their specific binding proteins andnuclear receptors. For an understanding of the mode of actionof retinoids with potential cancer chemopreventive or otherbiological activity, it is important to study their interactionswith these binding proteins and receptors. In our attempts tounderstand the action of N-(4-hydroxyphenyl)retinamide (4HPR)and other retinamides in the prevention of cancer, we observedthat 4HPR binds to a serum protein with a molecular size of     

Prevention of primary prostate cancer in Lobund-Wistar rats by N-(4-hydroxyphenyl)retinamide

We report for the first time that a synthetic retinoid, N-(4-hydroxyphenyl)retinamide, can prevent the development of both primary and metastatic tumors in an animal model of metastasizing primary prostate cancer. Prostatic adenocarcinomas were induced in high incidence in Lobund-Wistar rats by initiation with methylnitrosourea i.v. and promotion with testosterone. Feeding of N-(4-hydroxyphenyl)retinamide to these rats during the latency period markedly diminished the final incidence of both primary and metastatic prostate carcinomas.     

Therapeutic effect of N-(4-hydroxyphenyl)retinamide on N-methyl-N-nitrosourea-induced rat mammary cancer

Virgin Sprague--Dawley rats received a single i.v. injection of 40 mg N-methyl-N-nitrosourea (MNU)/kg body wt. at 50 days of age. After the first palpable mammary tumor reached 10 mm in size, the animals were sequentially allocated to one of 4 groups: (I) placebo diet, (II) 10 micrograms tamoxifen s.c. 3 times per week, (III) 3 mmol N-(4-hydroxyphenyl)retinamide (4-HPR)/kg diet, or (IV) both (II) and (III). Weekly measurements of initial tumors and subsequent tumors were made throughout the study. 4-HPR administration resulted in a complete regression (non-palpable state) of the first mammary tumor in 6 animals (22%) and partial regression or nonprogression in 5 others (19%). Tamoxifen alone induced only partial response in 9 animals (33%). 4-HPR and tamoxifen resulted in 19% total and 26% partial response. The data suggests therapeutic value of 4-HPR in MNU-induced rat mammary carcinoma.     

Efficacy of 9-cis-retinoic acid and N-(4-hydroxyphenyl) retinamide alone and in combination in mammary cancer prevention

As demonstrated in several in vitro and in vivo cancer models, retinoids have chemopreventive activity. The present studies were performed to evaluate the efficacy of 9-cis-retinoic acid (9-cis-RA) and N-(4-hydroxyphenyl) retinamide (4-HPR), alone and combined, in preventing mammary cancers. Female Sprague-Dawley rats received N-methyl-N-nitrosourea (MNU), 50 mg/kg BW, either at 50 days of age (experiment I, young rats) or at 100 days of age (experiment II, older rats). In experiment I, 9-cis-RA (60 mg/kg of diet), 4-HPR (586 mg/kg of diet), or the combination were evaluated; in experiment II, 9-cis-RA (30 mg/kg of diet), 4-HPR (196 mg/kg of diet), or the combination were tested. There were no signs of toxicity in either study. In the young rats, there were only slight reductions (15-20%) in the number of mammary cancers when the agents were given alone. In the older rats, lower doses of 9-cis-RA or 4-HPR alone were highly effective; with 61% and 46% reductions in the number of mammary cancers, respectively. The combination of retinoids in the young rats caused a 49% reduction in mammary cancers, while in the older rats the combination resulted in a 96% reduction. Thus, lower doses of the retinoids caused more striking inhibition of mammary cancers in older rats than the higher doses given to younger animals. In both experiments, the two retinoids in combination produced an additive effect, suggesting that they may inhibit mammary cancers by different mechanisms.