In vitro and in vivo effects of easily administered, low-toxic retinoid and phenylacetate compounds on human neuroblastoma cells.

We have investigated the effects of the low-toxic retinoid, all-trans retinoyl beta-glucuronide (RAG) alone and in combination with the phenylacetate (PA) derivative 4-chloro-phenylacetate (4-CPA) on the human neuroblastoma cell line, LA-N-5. In vitro studies demonstrated that RAG and 4-CPA treatments alone showed differentiation-inducing activity on LA-N-5 cells, with 4-CPA found to be about three-fold more potent than the PA parent compound in inducing morphologic differentiation and growth inhibition. As previously reported for retinoic acid (RA) and PA, RAG and 4-CPA were significantly more effective in their antiproliferative effects on the cells than either agent alone. Pharmacologic studies of 4-CPA in mice demonstrated that blood plasma levels reached peak concentrations 4 h after bolus administration of the compound and showed slow clearance characteristics with an apparent half-life of 4-8 h. As opposed to PA, 4-CPA was found to be essentially odourless and readily consumed in drinking water, giving rise to steady-state blood plasma levels of 4-CPA in the near mM range. Continuous consumption of 4-CPA in this manner for up to 5 months demonstrated no apparent adverse effects on the mice. Long-term RAG- and/or 4-CPA-treatment of nude mice injected with LA-N-5 cells demonstrated that both compounds alone exhibit potent antitumour activity. Together, RAG plus 4-CPA was the most effective treatment for inhibiting established tumour growth. In contrast, 4-CPA alone was equally as effective as the combination for preventing tumour development. The potent in vivo antitumour effects of 4-CPA could not be accounted for by the known ability of PA compounds to induce expression of the RA nuclear receptor beta (RARbeta) suppressor gene. Taken together, these findings demonstrate the possibility that RAG and/or 4-CPA may serve as effective, less-toxic alternatives to 13-cis RA, which is presently being utilised for nb therapy.

Inhibition of estrogen-dependent breast cell responses with phenylacetate.

The aromatic fatty acid phenylacetate (PA) and its analogs have come under intense investigation due to their ability to cause the growth arrest of a variety of neoplasia, including human breast cancer. We have determined that PA and its halide derivative 4-chlorophenylacetate (4-CPA) showed marked antiproliferative activity on 3 of 6 human breast cancer cell lines tested. Interestingly, the 3 cell lines that were growth inhibited by PA and 4-CPA were estrogen receptor (ER) positive (T47-D, MCF-7 and ZR-75-1) whereas those that were little affected by these compounds were ER-negative (MDA-MB-157, MDA-MB-231 and SK-Br-3). Dose response studies indicated that 4-CPA inhibited the growth of the sensitive (ER+) cell lines with a potency 3-4 times that of PA. These findings suggest that there is "cross-talk" between the PA and estrogen signaling pathways such that PA can directly inhibit estrogen-dependent events. This hypothesis was directly tested in vitro using ER+ MCF-7 cells that were stably transfected with a luciferase reporter construct driven by the full length (1745 bp) cyclin D1 promoter (MCF-7-D1). Our experiments with MCF-7-D1 cells indicated that PA and 4-CPA inhibited basal and estrogen-induced reporter gene activity by up to 90%, resulting in almost complete elimination of estrogen-dependent cyclin D1 gene activation. Using a reporter gene construct (ERE(V)-tk-Luc) containing a canonical estrogen response element that was transiently transfected into MCF-7 and MDA-MB-231 cells, we have also demonstrated inhibition of promoter activity by PA and 4-CPA that was directly mediated by blockage of activity through the ERE. Taken together, these findings indicate that PA analogs possess potent antiestrogen properties that may, at least partly, account for their antiproliferative effects on ER+ breast cancer cells. The data suggests a novel mechanism of action that might bypass some of the limitations of conventional antiestrogen therapy.

RU486-induced growth inhibition of human endometrial cells.

OBJECTIVE: To determine the direct action of RU486 on endometrial cell proliferation and to differentiate whether the antioxidant or the antiprogesterone property of RU486 is predominately responsible for its effect on cell growth. DESIGN: In vitro study comparing the effects of RU486 (antiprogesterone and antioxidant), reduced RU486 (antioxidant), ZK112,993 (antiprogesterone), and lazaroid U74,500A (antioxidant) on endometrial cell growth. The human endometrial cell line EM42 was used in transient transfection assays to confirm the relative antiprogesterone potency of the various compounds. SETTING: Academic medical center PATIENT(S): Women presenting with pelvic pain or infertility and diagnosed with endometriosis at time of surgery or women desiring tubal ligation with a normal pelvis (controls). INTERVENTION(S): Endometrial cell cultures were treated with RU486, reduced RU486, lazaroid U74,500A, and ZK112,993. MAIN OUTCOME MEASURE(S): Tritiated thymidine incorporation was used to assess cell growth. Inhibition of progesterone induction of transiently transfected reporter plasmids was used to measure antiprogesterone activity of compounds studied. RESULT(S): RU486 reduced cell growth in a dose-dependent fashion of the endometrial cell lines EM42 and RL95-2 and of endometrial and endometriosis cells from primary culture. After being reduced, RU486 lost most of its antiprogesterone activity but retained its antiproliferative properties. ZK112,993 was similar in potency to RU486 as a progesterone antagonist but did not significantly modify endometrial cell growth. Lazaroid U74,500A was devoid of antiprogesterone activity but was shown to be a potent antiproliferative agent. CONCLUSION(S): RU486 has a direct inhibitory effect on human endometrial cell growth. This activity appears to be at least partly mediated through its antioxidant property.

Copper/zinc-Superoxide dismutase is required for oxytetracycline resistance of Saccharomyces cerevisiae.

Saccharomyces cerevisiae, along with other eukaryotes, is resistant to tetracyclines. We found that deletion of SOD1 (encoding Cu/Zn superoxide dismutase) rendered S. cerevisiae hypersensitive to oxytetracycline (OTC): a sod1Delta mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 microg ml(-1), whereas concentrations of up to 1,000 microg ml(-1) had no effect on the growth of the wild type. OTC resistance was restored in the sod1Delta mutant by complementation with wild-type SOD1. The effect of OTC appeared to be cytotoxic and was not evident in a ctt1Delta (cytosolic catalase) mutant or in the presence of tetracycline. SOD1 transcription was not induced by OTC, suggesting that constitutive SOD1 expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and sod1Delta strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the sod1Delta mutant, but not the wild type, to OTC. We propose that Sod1p protects S. cerevisiae against a mode of OTC action that is dependent on oxidative damage.