The antiestrogen tamoxifen is thought to antagonize the effects of estrogens by competing with them for estrogen receptor (ER) binding. However, tarnoxifen can also reverse multidrug resistance, synergize with cisplatin cytotoxicity, and inhibit growth in ER-negative lung cancer cells. In addition to ERs, rat and human target tissues contain a second binding macromolecule termed the type II estrogen binding site (type II EBS). It has been shown that tamoxifen and flavonoids, a widely distributed class of natural substances with a variety of biologic actions, bind to type II EBS and inhibit the growth of several tumor cell types. At present, conflicting data about ERs and an absence of data about type II EBSs exist for lung tumors. We have tested non-small-cell lung carcinoma cell lines and primary tumor cells for the presence of ERs and type II EBSs and have evaluated the effects of tamoxifen and quercetin (pentahydroxyflavone) on the growth of these cells. Using a whole-cell assay and nuclear and cytosolic radiobinding experiments with [3H]estradiol as tracer, we have found that SK-LU1, SW900, ChaGo-K-1, H441, H661, and A549 cells, as well as primary tumors, bind estrogen specifically. This binding results mainly from the presence of a large number of type II EBSs, whereas ERs are absent or present at low concentrations. Type II EBSs bound tamoxifen and quercetin with similar affinity. Cell counts and a thymidine incorporation assay showed that both compounds inhibit cell growth in a concentration-dependent manner at concentrations ranging from 10 nM to 1 microM. Neither ipriflavone, an isoflavone, nor rutin, the 3-rhamnosylglucoside of quercetin, bound type II EBSs or inhibited cell growth. These findings suggest that tamoxifen and quercetin could regulate lung cancer cell growth through a binding interaction with type II EBSs. This mechanism could also be active in vivo, in that we have observed that nuclear and cytosolic type II EBSs were present in all primary lung cancers tested (n = 12), and that tamoxifen and quercetin were effective in inhibiting in vitro bromodeoxyuridine (BrdU) incorporation and proliferation-cell nuclear antigen expression by neoplastic cells in these cancers.

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H. pylori infection by a CagA+ strain is associated with the highest production of ROS to which a severe oxidative DNA damage corresponds. This sequence of events could support the hypothesis that the oxygen-free radicals-mediated damage due to H. pylori cytotoxic strains could be a driving force that leads from chronic gastritis to gastric carcinoma.

MethodsSpecimens for hormone receptor analysis were collected at the time of surgery from the tumor mass. Samples were frozen in liquid nitrogen and stored at -80°C until use. From the samples to be assayed for receptor analysis, frozen sections for histological assessment were routinely taken to ensure that there was only a minimum of connective tissue and no normal mucosa in these. Furthermore, some of these sections were used for the immunohistochemical demonstration of nuclear oestrogen receptors with the ERICA kit (Estrogen Receptor Immunochemical Assay, Abbot Laboratories, North Chicago, Illinois).Oestrogen receptors and progesterone receptors were assayed with the use of the dextran coated charcoal (DCC) method, as described previously.'7 Briefly, aliquots of cytosol from the homogenised, ultracentrifuged specimens were incubated with increasing concentrations of [2, 4, 6, Briefly, aliquots of cytosol prepared in 10 mM TRIS-HCI, 1 5 mM EDTA, pH 7-8, at 0°C

Two cases of papillary cystic tumor (PCT) of the pancreas were investigated for the presence of estrogen receptors (ERs) and progesterone receptors (PgRs). Both PCT and normal pancreas are able to specifically bind 3H-estradiol. This binding almost exclusively results from the presence of high levels of type II ER, whereas type I ERs were absent or present at very low levels. Both normal and neoplastic pancreas studied immunohistochemically for the presence of nuclear ER had negative results. This could be explained assuming that anti-ER antibodies are specific for type I binding sites. In conclusion, the presence of specific estrogen as well as progesterone binding may explain the sex and age predilection of PCT and suggest a possible hormone sensitivity for this tumor.

Type II estrogen-binding sites (type II EBS) have been demonstrated in human peripheral blood mononuclear cells (PBMC) using a whole cell assay with [6,7-3H]estradiol [( 3H]E2) as tracer. During whole cell incubations for 60 min at 37 C for type II EBS quantification, we found that PBMC contain 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity, which led to errors in estimating type II EBS concentrations by diminishing, by about 70%, the amount of available labeled E2. On the other hand, after 150 min at 4 C only 16% of the tracer was converted to estrone. Thus, we measured the maximal steady state binding in PBMC by incubating the cells with [3H]E2 at 4 C for 150 min. Equilibrium binding analysis of PBMC yielded sigmoid saturation curves with a saturation point at a ligand concentration of about 40 nmol/L. Scatchard analysis of binding data yielded a concave plot, which together with a Hill coefficient of 2.13, suggests that the type II EBS may have multiple binding sites which display positive cooperativity. The apparent equilibrium dissociation constant (Kd), determined from the [3H]E2 concentration required for half-saturation, was about 22 nmol/L. The type II EBS were estrogen specific, as demonstrated by competition experiments. Only those steroids with estrogenic activity inhibited binding of [3H]E2; nonestrogenic steroids did not. The type II EBS were found to be 3S macromolecules based on analysis of postlabeled fractions prepared by sucrose density gradient centrifugation. The number of type II EBS in PBMC from normal women was highest during the late follicular-early luteal phase of the menstrual cycle. We conclude that human PBMC specifically take up, retain, and metabolize E2.

We examined the levels of activity of methyl-p-hydroxyphenyllactate esterase (MeHPLA-ase) and cytosolic Type-II-estrogen-binding sites (Type-II EBS) in 61 and 71 cases, respectively, of primary ovarian cancer. MeHPLA-ase activity and Type-II EBS were seen to by asymmetrically distributed, in that levels were skewed towards the lower values. A statistically significant direct correlation was found between MeHPLA-ase activity and Type-II EBS. MeHPLA-ase activity and Type-II EBS were inversely correlated with ER and PR levels and showed a trend towards inverse correlation with the percentage of cells in S-phase of the cell cycle. MeHPLA-ase activity and Type-II EBS did not correlate with clinico-pathological parameters. The median MeHPLA-ase activity tended to be higher in responders than in unresponsive patients, but statistical significance was not reached. Higher Type-II-EBS levels were found in cases showing complete and partial response to chemotherapy than in cases which did not respond. A statistically significant relationship was found between high MeHPLA-ase activity and longer overall survival.