Synthesis, structure-activity relationships, and RARgamma-ligand interactions of nitrogen heteroarotinoids.

Three heteroarotinoids containing a nitrogen atom in the first ring and a C-O linking group between the two aryl rings were synthesized and evaluated for RAR and RXR retinoid receptor transactivation, tumor cell growth inhibition, and transglutaminase (TGase) induction. Ethyl 4-(N,4,4-trimethyl-1,2,3,4-tetrahydroquinolinyl)benzoate (1) contained an N-CH(3) group and activated all retinoid receptors except for RARgamma. Inceasing the hydrophobicity around the rings with analogues ethyl 4-(N,4,4,7-tetramethyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (2) [7-methyl group added] and ethyl 4-(4,4-dimethyl-N-isopropyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (3) [NCH(CH(3))(2) group at C-4] increased the potency and specificity for RARalpha, RARbeta, and RXRalpha, compared to 1, but had little effect on RXRbeta and RXRgamma activation. Although 1 and 3 were unable to activate RARgamma, 2 did activate this receptor with efficacy and high potency equal to that of 9-cis-retinoic acid (9-c-RA). All three heteroarotinoids exhibited 5-8-fold greater specificities for RARbeta over RARalpha. In addition, esters 1-3 inhibited the growth of two cell lines each derived from cervix, vulvar, ovarian, and head/neck tumors with similar efficiencies to that of 9-c-RA through a mechanism independent of apoptosis. The vulvar cell lines were the most sensitive, and the ovarian lines were the least sensitive. Ester 2 was similar to 1 and 3 except that 2 was a much more potent growth inhibitor of the two vulvar cell lines, which is consistent with strong RARgamma activation by 2 (but not by 1 and 3) and the high levels of RARgamma expression in skin. All three heteroarotinoids induced production of TGase, a marker of retinoid activity in human erythroleukemic cells. Esters 2 and 3 were the more potent TGase activators than 1, in agreement with the stronger activation of the RAR receptors by 2 and 3. The biological activities of these agents, and the RARgamma potency of 2 in particular, demonstrate the promise of these compounds as pharmaceutics for cancer and skin disorders.

Heteroarotinoids inhibit head and neck cancer cell lines in vitro and in vivo through both RAR and RXR retinoic acid receptors.

A class of less toxic retinoids, called heteroarotinoids, was evaluated for their molecular mechanism of growth inhibition of two head and neck squamous cell carcinoma (HNSCC) cell lines SCC-2 and SCC-38. A series of 14 heteroarotinoids were screened for growth inhibition activity in vitro. The two most active compounds, one that contained an oxygen heteroatom (6) and the other a sulfur heteroatom (16), were evaluated in a xenograph model of tumor establishment in nude mice. Five days after subcutaneous injection of 10(7) SCC-38 cells, groups of 5 nu/nu mice were gavaged daily (5 days/week for 4 weeks) with 20 mg/kg/day of all-trans-retinoic acid (t-RA, 1), 10 mg/kg/day of 6, 10 mg/kg/day of 16, or sesame oil. After a few days, the dose of t-RA (1) was decreased to 10 mg/kg/day to alleviate the side effects of eczema and bone fracture. No significant toxic effects were observed in the heteroarotinoid groups. All three retinoids caused a statistically significant reduction in tumor size as determined by the Student t-test (P < 0. 05). Complete tumor regression was noted in 3 of 5 mice treated with t-RA (1), 4 of 5 mice treated with 16, 1 of 5 mice treated with 6, and 1 of 5 mice treated with sesame oil. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine that the expression levels of RARalpha, RXRalpha, and RXRbeta were similar in the two cell lines, while RARbeta expression was higher in SCC-2 over SCC-38, and RARgamma expression was higher in SCC-38 over SCC-2. Receptor cotransfection assays in CV-1 cells demonstrated that 16 was a potent activator of both RAR and RXR receptors, while 6 was selective for the RXR receptors. Transient cotransfection assays in CV-1 cells using an AP-1 responsive reporter plasmid demonstrated that t-RA (1), 6, and 16 each inhibited AP-1-driven transcription in this cell line. In conclusion, the growth inhibition activity of the RXR-selective 6 and the more potent growth inhibition activity of the RAR/RXR pan-agonist 16 implicate both RARs and RXRs in the molecular mechanism of retinoid growth inhibition. Moreover, the chemoprevention activity and the lack of toxicity of heteroarotinoids demonstrate their clinical potential in head and neck cancer chemoprevention.

Systemic administration of interferon-gamma impairs wound healing.

Interferon-gamma (IFN-gamma), a cytokine that has been shown to upregulate macrophage function, has recently been demonstrated to improve outcome when exogenously administered in several animal models of injury. Because the macrophage is also important in the events that govern wound healing, we evaluated the effects of IFN-gamma upon wound healing in a murine model. IFN-gamma was administered in doses of 937.5-22,500 u synchronous with the creation of a left paraspinous wound and then daily. At Day 10, wounds were harvested, evaluated for wound disruption strength (WDS), and subjected to morphometric analysis. Wounds were also subjected to 36-hr formalin fixation to maximally cross-link collagen fibrils and retested for WDS. We found that IFN-gamma impaired wound healing at all doses relative to control, and WDS was impaired in a dose-dependent fashion. Our highest dose of IFN-gamma (22,500 u) produced a WDS only 65% of the control. Morphometric studies demonstrated less collagen deposition and a lower degree of neovascularity in IFN-gamma-treated animals. In addition, formalin fixation studies suggested that IFN-gamma may impair collagen cross-linking. The potential benefits of IFN-gamma in the multiply injured patient must be weighed against the possibility that IFN-gamma might deleteriously effect events fundamental to wound healing.