Apoptosis induction in cancer cells by a novel analogue of 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid lacking retinoid receptor transcriptional activation activity.

The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) is reported to have anticancer activity in vivo. Induction of cell cycle arrest and apoptosis in cancer cell lines refractory to standard retinoids suggests a retinoid-independent mechanism of action for AHPN. Conformational studies suggested that binding of AHPN does not induce an unusual conformation in retinoic acid receptor (RAR) gamma. The 3-chloro AHPN analogue MM11453 inhibited the growth of both retinoid-resistant (HL-60R leukemia, MDA-MB-231 breast, and H292 lung) and retinoid-sensitive (MCF-7 breast, LNCaP prostate, and H460 lung) cancer cell lines by inducing apoptosis at similar concentrations. Before apoptosis, MM11453 induced transcription factor TR3 expression and loss of mitochondrial membrane potential characteristic of apoptosis. MM11453 lacked the ability to significantly activate RARs and retinoid X receptor alpha to initiate (TREpal)(2)-tk-CAT reporter transcription. These results, differential proteolysis-sensitivity assays, and glutathione S-transferase-pulldown experiments demonstrate that, unlike AHPN or the natural or standard synthetic retinoids, MM11453 does not behave as a RAR or retinoid X receptor alpha transcriptional agonist. These studies strongly suggest that AHPN exerts its cell cycle arrest and apoptotic activity by a signaling pathway independent of retinoid receptor activation.

Retinoic acid (RA) receptor transcriptional activation correlates with inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase (ODC) activity by retinoids: a potential role for trans-RA-induced ZBP-89 in ODC inhibition.

Evaluation of retinoic acid receptor (RAR) subtype-selective alpha and gamma agonists and antagonists and a retinoid X receptor (RXR) class-selective agonist for efficacy at inhibiting both induction of ornithine decarboxylase (ODC) by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis and rat tracheal epithelial cells and the appearance of papillomas in mouse epidermis treated in the 2-stage tumor initiation-promotion model indicated that (i) RXR class-selective transcriptional agonists, such as MM11246, were not involved in ODC inhibition; (ii) RAR-selective agonists that induce gene transcription from RA-responsive elements (RAREs) were active at low concentrations; (iii) RAR-selective antagonists that bind RARs and inhibit AP-1 activation on the collagenase promoter but do not activate RAREs to induce gene transcription were less effective inhibitors; and (iv) RARgamma-selective retinoid agonists were more effective inhibitors of TPA-induced ODC activity than RARalpha-selective agonists. These results suggest that RARE activation has a more important role in inhibition of ODC activity than RXR activation or AP-1 inhibition and that RARgamma-selective agonists would be the most useful inhibitors of epithelial cell proliferation induced by tumor promoters. The natural retinoid all-trans-RA induced expression of transcription factor ZBP-89, which represses activation of the GC box in the ODC promoter by the transcription factor Sp1.

Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3.

TR3, an immediate-early response gene and an orphan member of the steroid-thyroid hormone-retinoid receptor superfamily of transcription factors, regulates apoptosis through an unknown mechanism. In response to apoptotic stimuli, TR3 translocates from the nucleus to mitochondria to induce cytochrome c release and apoptosis. Mitochondrial targeting of TR3, but not its DNA binding and transactivation, is essential for its proapoptotic effect. Our results reveal a mechanism by which a nuclear transcription factor translocates to mitochondria to initiate apoptosis.

sp2-bridged diaryl retinoids: effects of bridge-region substitution on retinoid X receptor (RXR) selectivity.

RXR class selectivity and RXR transcriptional activation activity compared to those for the retinoic acid receptor subtypes were enhanced on the 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenylethenyl)be nzoic acid scaffold and its 3-methyl analogue by replacing their 1,1-ethenyl bridge by a 1,1-(2-methylpropenyl) or cyclopropylidenylmethylene group.

4-[3-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)phenyl]benzoic acid and heterocyclic-bridged analogues are novel retinoic acid receptor subtype and retinoid X receptor alpha agonists.

Aromatic retinoids having a meta-substituted aromatic ring bridge, such as 4-[3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)phenyl]benzo ic acid and its 3,5-diaryl-substituted 4,5-dihydroisoxazole analogue, function as retinoid receptor panagonists by activating both retinoic acid and retinoid X receptors to induce gene transcription, and thereby provide novel scaffolds for retinoid drug development. Both classes of these ligand-inducible transcription factors are involved in mediating the inhibitory effects of retinoids on cancer cell growth.

Antioxidant activity of michellamine alkaloids.

The alkaloids michellamines A, B, and C are natural products isolated from a Central African tropical plant Ancistrocladus korupensis. We have investigated the radical scavenging ability of these compounds. The alkaloids inhibited the azo-induced oxidation of beta-phycoerythrin with IC50 values in the 0.5- to 0.8-microM range. Michellamine B also protected rat liver mitochondria against lipid peroxidation induced by adenosine diphosphate and Fe2+. The alkaloids were more potent antioxidants in these assays than several compounds being considered clinically as chemoprevention agents.

Michellamine alkaloids inhibit protein kinase C.

Michellamines A, B, and C have shown antiviral activity against HIV-1 and HIV-2 in cell culture. They act in a complex manner by at least two reported antiviral mechanisms, inhibition of HIV reverse transcriptase and inhibition of HIV-induced cellular fusion. On the basis of their structural similarity to other protein kinase C (PKC) inhibitors, we have investigated another possible mechanism-inhibition of PKC. The michellamines were found to inhibit rat brain PKC with IC50 values in the 15-35 microM range. Michellamine B was a noncompetitive PKC inhibitor with respect to ATP with a Ki value of 4-6 microM, whereas mixed-type inhibition was observed when the peptide concentration was varied. Michellamine B inhibited the kinase domain of PKC similarly. These results indicate that the michellamines bind to the PKC kinase domain and not its regulatory domain. Molecular modeling showed that all three michellamines can bind in the active site cleft of the PKC kinase domain, to block both the ATP and the peptide substrate subsites.

Effects of novel RAR- and RXR-selective retinoids on myeloid leukemic proliferation and differentiation in vitro.

Retinoids such as all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA) have an important role in many aspects of proliferation and differentiation of hematopoietic cells. They exert their effects by binding to retinoic acid receptors (RARs) and/or retinoid X receptors (RXRs). We studied the effects of novel retinoids on proliferation and differentiation of HL-60 and NB4 myeloid leukemic cells, as well as acute promyelocytic leukemia (APL) cells from patients. RXR-selective SR11345 (Retinoid C) had little ability to inhibit the clonal growth and to induce the differentiation of either HL-60 or NB4 cells. However, SR11276 (Retinoid E), which activated both the RAR and RXR classes, and SR11278 (Retinoid D), which activated the RAR subtypes alpha, beta, and gamma, could inhibit clonal growth of both cell types, as well as leukemic cells from APL patients. The combination of ATRA and either SR11276 or SR11278 additively inhibited APL cell proliferation. SR11302 (Retinoid A), with reported anti-AP-1 activity and no activation of RARs and RXR and SR11363 (Retinoid B), which selectively activated RARbeta and gamma, were inactive. The clonal proliferation of both HL-60 and NB4 cells that were pulse-exposed to 10(-9) mol/L ATRA, SR11276, SR11278, or SR11345 for 3 days, washed, and plated in methylcellulose culture were inhibited by 0%, 51%, 21%, and 1% for HL-60 cells and 43%, 41%, 35%, and 1% for NB4, respectively, compared with nontreated control cells. When the HL-60 cells were pulse-exposed to 10(-9) mol/L of either SR11278 or SR11276, plus 10(-9) mol/L ATRA for 3 days, colony numbers were reduced by 46% and 64%, respectively. Induction of leukemic cell differentiation as determined by the nitroblue tetrazolium (NBT) assay showed that the combination of 10(-7) mol/L of either SR11278 or SR11276 with 10(-7) mol/L ATRA had additive effects on HL-60 cells, NB4 cells, and fresh APL cells. Induction of CD11b expression on both HL-60 and NB4 cells occurs during their differentiation. Expression of this antigen was synergistically augmented by the combination of either 10(-7) to 10(-8) mol/L SR11278 or 10(-7) to 10(-9) mol/L SR11276 with 10(-9) mol/L ATRA compared with either analog alone in HL-60 cells. Expression of the novel myeloid specific transcription factor C/EBPepsilon was increased by SR11278 and SR11276 in both the HL-60 and NB4 cell lines. We conclude that retinoids or combination of retinoids with specificities for both RAR and RXR may markedly enhance the ability of ATRA to inhibit clonal growth and induce differentiation of HL-60 and NB4 leukemic cells. This occurs in the absence of continuous contact with retinoids.

Molecular determinants of AHPN (CD437)-induced growth arrest and apoptosis in human lung cancer cell lines.

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437), originally identified as a retinoic acid receptor gamma-selective retinoid, was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study, we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis, a process that is accompanied by rapid induction of c-Jun, nur77, and p21(WAF1/CIP1). In addition, we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein, a dominant-negative inhibitor of c-Jun, in A549 cells, nur77 expression and apoptosis induction by AHPN/CD437 were impaired, whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore, overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus, expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together, our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer.

Regulation of RAR beta expression by RAR- and RXR-selective retinoids in human lung cancer cell lines: effect on growth inhibition and apoptosis induction.

Retinoids regulate the growth and differentiation of human tracheobronchial epithelial cells. In this study, we investigated the effects of all-trans-retinoic acid (trans-RA) and receptor class-selective retinoids on the growth and apoptosis of human lung cancer cell lines. Trans-RA significantly inhibited the growth of Calu-6 and H460 cells, accompanied by induction of RA receptor (RAR) beta expression. In contrast, it had little effect on the growth of H292, SK-MES-1 and H661 lung cancer cell lines, in which RAR beta expression was not induced. Stable expression of RAR beta in RAR beta-negative, trans-RA-resistant SK-MES-1 and H661 lung cancer cells led to recovery of trans-RA-induced growth inhibition, which occurred, however, only at low serum concentration. Using fluorescent microscopy and the terminal deoxyribonucleotidyl transferase (TdT) assay, we demonstrated that induction of apoptosis by trans-RA contributed to its growth-inhibitory effect in trans-RA-sensitive lung cancer cell lines. Analysis of RAR-selective and retinoid X receptor (RXR)-selective retionoids showed that activation of both RARs and RXRs could induce growth inhibition in trans-RA-sensitive lung cancer cells. Also, an additive synergistic effect on growth inhibition and RAR beta induction was observed when cells were treated with combinations of RAR-selective and RXR-selective retinoids. Together, our results show that expression of RAR beta plays a role in mediating retinoid response in lung cancer cells and that activation of RARs or RXRs contributes to induction of RAR beta, growth inhibition and apoptosis by retinoids.

Effects of trans-retinoic acid, 9-cis-retinoic acid, 1alpha,25-(dihydroxy)vitamin D3 and a novel apoptosis-inducing retinoid on breast cancer and endothelial cell growth.

Breast cancer cell growth inhibition was not synergistically enhanced by trans-retinoic acid (RA) or 9-cis-RA plus 1alpha,25-(dihydroxy)vitamin D3 (DHVD). The retinoid/DHVD combinations did lower their 50% effective concentrations for inhibiting retinoid-sensitive MCF-7, but not retinoid-refractory BT-20, breast cancer cell growth. In contrast, the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) and its analog SR11389 inhibited the growth of both cell lines. Unlike RA, 9-cis-RA and DHVD, AHPN and SR11389 also potently inhibited human umbilical vascular endothelial cell growth. These results on AHPN and SR11389 suggest that angiogenesis of tumor microvasculature should also be an effective therapeutic target for this new compound class.

Inhibition of trans-retinoic acid-resistant human breast cancer cell growth by retinoid X receptor-selective retinoids.

All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs). Previous studies demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen-independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, we evaluated several synthetic retinoids for their ability to induce growth inhibition and apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines. Our results demonstrate that RXR-selective retinoids, particularly in combination with RAR-selective retinoids, could significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but had low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. Using gel retardation and transient transfection assays, we found that the effects of RXR-selective retinoids on MDA-MB-231 cells were most likely mediated by RXR-nur77 heterodimers that bound to the RA response element in the RARbeta promoter and activated the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR-selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurred through RXR-RAR alpha heterodimers that also bound to and activated the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids were suppressed, while the effects of RAR-selective retinoids were enhanced. Together, our results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR-mediated growth inhibition in breast cancer cells.

Effects of receptor class- and subtype-selective retinoids and an apoptosis-inducing retinoid on the adherent growth of the NIH:OVCAR-3 ovarian cancer cell line in culture.

Comparison of the adherent growth inhibition of NIH:OVCAR-3 ovarian cancer cells by retinoid receptor class-selective and subtype-selective compounds with their receptor binding affinities and transcriptional activation activities indicated no correlation for RAR alpha and RAR gamma although both receptors are present. Retinoids that activated RXR alpha inhibited cell growth in the range as all-trans-retinoic acid and 9-cis-retinoic acid. The most potent inhibitor was 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN), which has been found to inhibit breast and lung cancer and leukemia cell growth and induce cancer cell apoptosis through a pathway independent of the retinoid receptors.

Correlation of retinoid binding affinity to retinoic acid receptor alpha with retinoid inhibition of growth of estrogen receptor-positive MCF-7 mammary carcinoma cells.

Both anchorage-dependent growth and anchorage-independent growth of the estrogen receptor-positive mammary carcinoma cell line MCF-7 are inhibited by all-trans-retinoic acid. This cell line has nuclear retinoic acid receptors (RARs) alpha and gamma. The natural retinoids all-trans-retinoic acid and 9-cis-retinoic acid and a series of 12 conformationally restricted retinoids, which showed a range of binding selectivities for these receptors and had either agonist or antagonist activity for gene transcriptional activation by the RARs, were evaluated for their abilities to inhibit anchorage-dependent (adherent) and anchorage-independent (clonal) growth of MCF-7 cells. Correlation analyses were performed to relate growth inhibition by these retinoids with their binding affinity to RAR alpha or RAR gamma. Inhibition of anchorage-dependent growth in culture after 7 days of retinoid treatment correlated with binding to RAR alpha (n = 14; P < or = 0.001) and not to RAR gamma (n = 14; P > 0.1). Both the RAR alpha-selective retinoid agonists and the two RAR antagonists that were evaluated inhibited adherent cell growth. The RAR gamma-selective agonists had very low growth inhibitory activity (< 10%) at concentrations as high as 12.5 microM. These results suggest that RAR alpha is the retinoid receptor involved in the inhibition of adherent cell growth by retinoids and that transcriptional activation by this receptor on a RAR response element does not appear to be required for this process to occur. For this series of retinoids, inhibition of anchorage-independent growth after 21 days of retinoid treatment only correlated (n = 12; P < or = 0.005) with binding affinity to RAR alpha for the retinoid agonists, although the RAR gamma-selective retinoids displayed weak activity. The RAR antagonists were very poor inhibitors of growth. These results suggest that activation of gene transcription by RAR alpha appears to be required for inhibition of anchorage-independent growth by retinoids in this estrogen receptor-positive mammary carcinoma cell line.

A new class of retinoids with selective inhibition of AP-1 inhibits proliferation.

Retinoids regulate many biological processes, including differentiation, morphogenesis and cell proliferation. They are also important therapeutic agents, but their clinical usefulness is limited because of side effects. Retinoid activities are mediated by specific nuclear receptors, the RARs and RXRs, which can induce transcriptional activation through specific DNA sites or by inhibiting the transcription factor AP-1 (refs 12-15), which usually mediates cell proliferation signals. Because the two types of receptor actions are mechanistically distinct, we investigated whether conformationally restricted retinoids, selective for each type of receptor action, could be identified. Here we describe a new class of retinoids that selectively inhibits AP-1 activity but does not activate transcription. These retinoids do not induce differentiation in F9 cells but inhibit effectively the proliferation of several tumour cell lines, and could thus serve as candidates for new retinoid therapeutic agents with reduced side effects.

Identification of retinoids with nuclear receptor subtype-selective activities.

Retinoic acid (RA) and its synthetic analogues, retinoids, have shown promising results in the prevention of epithelial carcinogenesis and in the treatment of acute promyelocytic leukemia and various proliferative skin disorders. Retinoid action on gene regulation is mediated by three distinct nuclear retinoic acid receptor subtypes, RA receptors alpha, beta, and gamma. The existence of multiple RA receptors has raised the possibility that receptor subtype-specific retinoids with reduced side effects can be developed. To analyze the activity of retinoids at the molecular level, we used a receptor activation assay. RA and 22 retinoids were compared on the three receptor subtypes. We found the alpha receptor to be least sensitive to activation by RA and the gamma receptor to be most sensitive. Compared with RA, one of the retinoids showed increased activity for the alpha and beta receptors. Three retinoids revealed no gene activation activity and showed no antagonistic effects when assayed in the presence of RA. Surprisingly, several of the retinoids were efficient activators of the beta and gamma receptors but poor activators or nonactivators of the alpha receptor. Our data demonstrate that the three RA receptor subtypes have differential ligand activation specificities and that the design of receptor subtype-selective retinoids is possible.

Inhibition of mammary carcinoma growth by retinoidal benzoic acid derivatives.

The growth of many breast carcinoma cell lines is inhibited by vitamin A, and derivatives as well as synthetic retinoids. New retinoidal derivatives have recently been synthesized. These retinoidal benzoic acid derivatives displayed enhanced potency in their ability to reverse hamster tracheal keratinization and inhibit ornithine decarboxylase induction in mouse epidermis. We therefore screened a series of analogues of these compounds for their ability to inhibit human breast carcinoma cell proliferation utilizing three estrogen receptor-positive and two estrogen receptor-negative cell lines. The compound (E)-4-2-(5,6,7,8)tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)prop enyl benzoic acid (Ro 1374-10) was approximately 2-3 orders of magnitude more potent than all-trans-retinoic acid in inhibiting breast carcinoma cell proliferation while the compound SRI-6409-40, which differs from Ro 1374-10 only by the position of a methyl group, was 50-fold more potent than Ro 1374-10. All of the compounds tested displayed were inactive against the estrogen receptor-negative breast carcinoma lines.

Synthesis of the 7-hydroxy metabolites of methotrexate and 10-ethyl-10-deazaaminopterin.

The potent folate antagonists methotrexate and 10-ethyl-10-deazaaminopterin are oxidized in vivo to their 7-hydroxy derivatives. A specific cyanation at the C-7 position of the pteridine ring system using diethyl phosphorocyanidate converted the dimethyl esters of methotrexate and 10-ethyl-10-deazaaminopterin to the corresponding 7-cyano dimethyl ester derivatives, which were hydrolyzed to the 7-hydroxy metabolites.

Quantification of retinoic acid by gas-liquid chromatography-mass spectrometry: total versus all-trans-retinoic acid in human plasma.

An assay based on negative ion chemical ionization mass spectrometry has been developed to quantify retinoic acid in plasma or serum. The lower limit of detection is 75 pg (240 fmol); normal values of retinoic acid can be determined on as little as 40 microliters of human plasma. The plasma concentrations of total retinoic acid in 12 healthy male volunteers taking no medication or vitamin supplementation ranged from 2.8 to 6.6 ng/ml; the mean was 4.9 ng/ml. The assay can be manipulated to measure all-trans-retinoic acid alone; about 75% of retinoic acid in human plasma or rat serum is all-trans-retinoic acid. Both retinol and retinoic acid can be quantified on the same 0.1-ml sample; the concentration of retinoic acid in human plasma or rat serum is at least 150-fold less than that of retinol.

Conformational restrictions of the retinoid skeleton.

A series of conformationally restricted retinoids has been synthesized and assayed for biological activity. These compounds have aromatic rings in place of selected double bonds of the tetraene side-chain of retinoic acid and could be considered as analogues of retinoic acid in which some of the double bonds possess s-cis topology. Thus far, analogues in which the bonds corresponding to the (5,7E)-, (7,9E)-, (9,11,13E)- and (11,13E)-double bond systems of retinoic acid are restricted to a cisoid conformation have been studied. Analogues were screened for their ability to reverse keratinization in hamster tracheal organ culture and to inhibit the induction of ornithine decarboxylase in mouse epidermis. Selected compounds were also screened in the antipapilloma assay in mice. The toxicity of some analogues on intraperitoneal injection in mice was determined.