Wan-Ru Chao

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.

Aromatic retinoic acid analogs. Synthesis and pharmacological activity

Aromatic analogues of (all-E)- and 13(Z)-retinoic acids have been synthesized as potential chemopreventive agents for the treatment of epithelial cancer. In the E series, (1E,3E)-1-(4-carboxyphenyl)-2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1,3- butadiene (7a), its ethyl ester 5a, and the epoxy ethyl ester 14 displayed excellent activity in the assay for the inhibition of tumor promotor-induced mouse epidermal ornithine decarboxylase, while (1E,3E)-1-(4-carboethoxy-3-methylphenyl)-2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1,3-butadiene (5b) was inactive. The 13(Z) analogues, (E)-1-(2-carboxyphenyl)-4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1,3,5-he xatriene (19) and (E)-1-(2-hydroxyphenyl)-4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1,3,5-he xatriene (27), had minimal activity.

A novel steroidal inhibitor of estrogen-related receptor α (ERRα)

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRalpha, bound to a co-activator protein (e.g., peroxisome proliferator receptor gamma co-activator-1alpha, PGC-1alpha), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRalpha is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRalpha, but not to ERRbeta or ERRgamma, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRalphas transcriptional activity in reporter gene assays, and prevents endogenous PGC-1alpha and ERRalpha from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30mg/kg given once daily and 100mg/kg given once weekly. In a combination study, SR16388 (10mg/kg, once daily) and paclitaxel (7.5mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRalpha biology.

5′-AMP-activated Protein Kinase (AMPK) Supports the Growth of Aggressive Experimental Human Breast Cancer Tumors

Background: 5′-AMP-activated protein kinase (AMPK) is a ubiquitous regulator of cellular energy homeostasis. Results: AMPK contributes to the growth and core glucose metabolism of aggressive (rapidly growing) experimental human tumors. Conclusion: AMPK can be activated by metabolic stress to support the growth of aggressive tumors. Significance: AMPKs role in tumor biology could be strongly influenced by microenvironmental stress. Rapid tumor growth can establish metabolically stressed microenvironments that activate 5′-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [13C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.

Effects of trans-retinoic acid, 9-cis-retinoic acid, 1α,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.

Identification of a unique binding protein specific for a novel retinoid inducing cellular apoptosis.

The retinoid 6‐[3‐(1‐adamantyl)‐4‐hydroxyphenyl]‐2‐naphthalenecarboxylic acid (AHPN, CD437) induces apoptosis in a variety of cell types, many of which are cancer cells that resist the antiproliferative and/or differentiating effects of retinoids. While the retinoids exert their effects by binding to the retinoic acid nuclear receptors (RARs) or retinoid X receptors (RXRs), AHPN (CD437) binds to another protein with different ligand specificity. In nuclear extracts from HL‐60R cells the binding of AHPN (CD437) was only minimally competed by either retinoic acid (tRA)or 9‐cis‐retinoic acid (9‐cis‐RA), the natural ligands for the RARs and RXRs, respectively. Moreover, AHPN (CD437) was unable to compete with either tRA or 9‐cis‐RA for binding to endogenous retinoid receptors in nuclear extracts from the MDA‐MB‐468 breast carcinoma cell line. Size exclusion chromatography revealed AHPN binding to a 95 kDa protein(s) which is neither an RAR or RXR. Our results suggest that apoptosis induction by AHPN (CD437) may occur through interaction with another protein and is independent of the RAR/RXR‐signaling pathways. Int. J. Cancer 86:474–479, 2000.

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 α 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.

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.

Biologically active aromatic retinoids bearing azido photoaffinity-labeling groups and their binding to cellular retinoic acid-binding protein

Retinoids bearing azido photoaffinity-labeling groups (azidoretinoids) have potential as probes for investigating the molecular mechanisms of action of all-trans-retinoic acid (RA) as mediated by its cellular retinoic acid-binding protein (CRABP) and nuclear receptor proteins. Two new azidoretinoids, 3-azido-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1E- propen-1-yl]-benzonic acid and 4-(4-azido-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)be nzoic acid were synthesized, and evaluated for their in vitro biological potency, and binding affinity for CRABP. Like RA, these aromatic azides had significant activity in modulating cell differentiation in retinoid-deficient hamster tracheal organ culture (ED500.02 nM and 0.03 nM, respectively) and in the inhibition of the induction of ornithine decarboxylase in mouse epidermis (ED50 7.0 nmol and 0.5 nmol, respectively). They also possessed high binding affinity for CRABP (ID50 0.9 microM and 0.85 microM, respectively). The tritiated aromatic azides were further evaluated for their ability to bind covalently to CRABP after photolysis. On photolysis at -78 degrees C, the two radiolabeled azidoretinoids formed stable adducts with CRABP. Treatment of the adducts with either RA or p-chloromercuriphenylsulfonic acid (CMPS) and subsequent dialysis did not cause any dissociation, indicating the formation of a covalent bond. In contrast, treatment of the unirradiated complexes with RA or CMPS led to dissociation of the complex. Synthesis of affinity labels and characterization of CRABP-retinoid complexes should provide useful information on the ligand-binding regions and insights into the mechanism of action of RA.

Comparison of the inhibitory effects of retinoids on 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activities in CD-1 and Sencar mice

Linear regression analysis of the dose levels of a series of retinoids required ornithine decarboxylase (ODC) induction by 50% indicated that there was no significant difference in inhibitory activity between CD-1 and Sencar mice (r = 0.9699; P less than 0.001). The ID50 values (nmol) found were as follows: retinoic acid (0.20, CD-1; 0.29, Sencar); 13-cis-retinoic acid (1.4, CD-1; 1.9, Sencar); 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1E-propenyl]benzoic acid (0.04, CD-1; 0.03; Sencar); 2-[1-(4-carboxyphenyl)-1E-propeny-2-yl]-4,5,6,7-tetrahydro-4, 4-dimethylbenzothiophene (0.08, CD-1; 0.14, Sencar); 2-[1-(4-carboxyphenyl)-1E-propen-2-yl]3, 4-dihydro-4,4-dimethyl-2H-1-benzothiopyran (0.56, CD-1; 0.70, Sencar); 6-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-2-naphthalenecarboxylic acid (1.0, CD-1; 0.90, Sencar).