Primary hamster tracheal epithelial cells growing on a collagen gel matrix produce hig molecular weight mucins indistinguishable from mucins produced in vivo. Using a modified version of these confluent cultures, we have demonstrated here that (i) release of mucins can be stimulated by human neutrophil elastase (HNE; EC 3.4.21.37); (ii) HNE can degrade mucins, and both mucin release and degradation by HNE require an active catalytic site; and (iii) there are at least two pools of mucins in these cells: one is a rapidly turning-over spontaneously releasable constitutive pool, the other is a slowly turning-over HNE-releasable pool. We provide evidence that the HNE-releasable mucins are membrane bound and associated with the secretory cell apical surface. (4,5). Our knowledge of the regulation of surface epithelial mucin secretion in airways has been hindered by lack of suitable in vitro systems. Most secretion studies have been performed in organ cultures (4-6), where it is difficult to determine whether a particular agent has acted directly on the secretory cell or indirectly via an effect on neighboring cells such as underlying smooth muscle cells. In addition, the stability of these in vitro systems and the biochemical nature of their secretions have not been well characterized.Primary cell cultures from hamster tracheal epithelium growing on a collagen gel produce high molecular weight mucins at confluence that are indistinguishable in size, charge heterogeneity, carbohydrate composition (7), and density from mucins produced in vivo. In this communication we show that mucin release by this confluent cell culture can be enhanced by human neutrophil elastase (HNE; EC 3.4.21.37).We have found that the HNE-induced mucin release derives from a pool of mucins differing from that involved in spontaneous mucin secretion, and we provide evidence that the HNE-susceptible pool of mucins is likely bound to the apical cell surface membrane. Possible mechanisms ofairway goblet cell mucin secretion as well as the HNE-induced secretory cell metaplasia are discussed. MATERIALS AND METHODSMaterials. All the materials were purchased commercially except the following: HNE was purified as previously described from purulent sputum and was 98% active by active site titration (8,9). Porcine pancreatic elastase (PPE) was purified by the method of Shotton (10) and characterized (95% active) as previously described (11). Inactive HNE (HNE-CMK) was prepared by incubating HNE with a molar excess of the elastase inhibitor succinyl-Ala-Ala-Pro-ValCH2Cl [a chloromethyl ketone (CMK)] followed by dialysis to remove unreacted CMK (12). The HNE-CMK preparation exhibited 0.025% residual elastase activity.Cell Culture and Metabolic Labeling of Mucins. Cultures were prepared as described previously (13). Cultures were metabolically labeled by incubating with 0.1 ml/cm2 of the complete growth medium containing [6-3H]glucosamine (39.2 Ci/mmol, New England Nuclear; 1 Ci = 37 GBq) at 10 pCi/ml for 24 hr. The spent medium was collected, centr...

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Resveratrol has been shown to have anticarcinogenic activity. We previously found that resveratrol inhibited growth and induced apoptosis in 2 human melanoma cell lines. In this study we determined whether resveratrol would inhibit human melanoma xenograft growth. Athymic mice received control diets or diets containing 110 micromol/L or 263 micromol/L resveratrol, 2 wk prior to subcutaneous injection of the tumor cells. Tumor growth was measured during a 3-wk period. Metabolism of resveratrol was assayed by bolus gavage of 75 mg/kg resveratrol in tumor-bearing and nontumor-bearing mice. Pellets containing 10-100 mg resveratrol were implanted into the mice, next to newly palpated tumors, and tumor growth determined. We also determined the effect of a major resveratrol metabolite, piceatannol, on experimental lung metastasis. Resveratrol, at any concentration tested, did not have a statistically significant effect on tumor growth. The higher levels of resveratrol tested (0.006% in food or 100 mg in slow-release pellets) tended to stimulate tumor growth (P = 0.08-0.09). Resveratrol and its major metabolites, resveratrol glucuronide and piceatannol, were found in serum, liver, skin, and tumor tissue. Piceatannol did not affect the in vitro growth of a murine melanoma cell line, but significantly stimulated the number of lung metastases when these melanoma cells were directly injected into the tail vein of the mouse. These results suggest that resveratrol is not likely to be useful in the treatment of melanoma and that the effects of phytochemicals on cell cultures may not translate to the whole animal system.

Retinoid response pathways involve retinoic acid receptors (RARs) and retinoid X receptors. N-(4-hydroxyphenyl) retinamide (4-HPR), a derivative of all-trans-retinoic acid (RA) is currently in clinical trials as a chemopreventive agent for breast cancer. The issue whether 4-HPR mediates its biological actions via classical retinoid receptor pathways remains to be investigated. In this study, we provide several lines of evidence that 4-HPR mediates its biological actions via a novel pathway(s) that does not involve the classical retinoid receptor pathways. For example, 4-HPR was more potent than RA as an antiproliferative agent and inhibited growth of otherwise RA-resistant human breast carcinoma cells. Exposure to 4-HPR resulted in the generation of DNA fragmentation with subsequent cell death in both RA-positive estrogen receptor (ER)-positive as well as RA-refractory ER-negative breast carcinoma cell lines. N-(4-Methoxyphenyl)retinamide (4-MPR), which is the major 4-HPR metabolite in circulation, was biologically inert in this system. 4-HPR and 4-MPR bound poorly to the RAR alpha, beta and gamma in vitro and only minimally activated the retinoic acid receptor element (RARE) and retinoid X receptor response elements (RXREs) in human breast carcinoma cells. Neither 4-HPR nor 4-MPR are metabolized to any of the known conventional retinoids. In addition, 4-HPR or 4-MPR transactivation of RAREs or RXREs transfected into MCF-7 and MDA-MB-231 cells was not noted at 48 h. Nevertheless 4-HPR-mediated cell death was observed at 48 h, further suggesting that neither 4-HPR nor 4-MPR are metabolized to retinoids which activate the RAREs or RXREs in breast carcinoma cells. Furthermore, unlike RA, which exhibited anti-AP1 activity, 4-HPR inhibition of growth did not involve anti-AP1 activity. These results suggest that 4-HPR acts by a unique pathway that is not mediated by retinoid receptors.