The antiproliferative effect of opioid receptor agonists on the T47D human breast cancer cell line, is partially mediated through opioid receptors

The aim of this paper is twofold: first, to present empirical findings, using data drawn from a comprehensive two year study of biomedical practice and patients perceptions of treatment and recovery done in a Mexico City public hospital, identifying specific aspects of the doctor-patient relationship that impact on patient perceived recovery. Second, and flowing from the first, the objective is to consider broader theoretical issues relevant to the doctor-patient relationship, the role it plays in the healing process and in biomedical practice, especially in a developing nation such as Mexico. The paper is divided into two sections: in Section I, the problem is defined and the methodology and findings are described, employing statistical and qualitative analyses. While enormous importance has been given to the doctor-patient relationship in biomedicine, the results of the study using statistical analysis, reveal that only particular components of the physician-patient encounter significantly influence differential treatment outcomes. In Section II the significance of the findings are discussed and theoretical issues are addressed bearing on the physician-patient relationship, and patients' perceptions of treatment. To illuminate the differential perceptions of treatment outcomes by patients with non-life-threatening disorders, the concept of life's lesions is proposed.     

Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists and antagonists in T47D human breast cancer cells

Indole-3-carbinol (I3C) is a major component of Brassica vegetables, and diindolylmethane (DIM) is the major acid-catalyzed condensation product derived from I3C. Both compounds competitively bind to the aryl hydrocarbon (Ah) receptor with relatively low affinity. In Ah-responsive T47D human breast cancer cells, I3C and DIM did not induce significantly CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity or CYP1A1 mRNA levels at concentrations as high as 125 or 31 microM, respectively. A 1 nM concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced EROD activity in these cells, and cotreatment with TCDD plus different concentrations of I3C (1-125 microM) or DIM (1-31 microM) resulted in a > 90% decrease in the induced response at the highest concentration of I3C or DIM. I3C or DIM also partially inhibited (< 50%) induction of CYP1A1 mRNA levels by TCDD and reporter gene activity, using an Ah-responsive plasmid construct in transient transfection assays. In T47D cells cotreated with 5 nM [3H]TCDD alone or in combination with 250 microM I3C or 31 microM DIM, there was a 37 and 73% decrease, respectively, in formation of the nuclear Ah receptor. The more effective inhibition of induced EROD activity by I3C and DIM was due to in vitro inhibition of enzyme activity. Thus, both I3C and DIM are partial Ah receptor antagonists in the T47D human breast cancer cell line.     

Modulation of cell cycle control by vitamin D3 and its analogue, EB1089, in human breast cancer cells

OBJECTIVES: Receptive anal intercourse but not orogenital sex has been identified as a major risk factor for transmission of HIV-1. Recent studies using simian immunodeficiency virus (SIV) in rhesus macaques have demonstrated relatively efficient infection following oral administration, indicating that modes of transmission may vary between HIV-1 and SIV. Here, we investigate whether HIV-1 infection of macaques via the oral route is more efficient than via the rectal route. DESIGN: Eleven Macaca nemestrina neonates were exposed to HIV-1 via different routes (four oral, two intravenous, and five rectal). One animal was orally inoculated with a sham inoculum and two control animals were not exposed. METHODS: All animals were followed for virological signs of infection, and for pathogenesis associated with HIV-1 infection by general physical examinations, complete blood cell counts and lymphocyte subset analysis, and full necropsies. RESULTS: Three out of five rectally exposed macaques and both of the intravenously inoculated animals became infected with HIV-1, whereas none of the orally exposed animals showed evidence of HIV-1 infection. Clinical observations following exposure included failure to thrive in the orally inoculated animals and low CD4/CD8 ratios in the rectally exposed macaques. CONCLUSIONS: The finding that, contrary to what has been reported for SIV, transmission of HIV-1 via the oral route is not more efficient than via the rectal route, indicates important biological differences between HIV-1 and SIV, with direct implications for the spread of HIV and associated AIDS, and for development of anti-HIV-1 vaccines.     

Insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 share a common nuclear transport pathway in T47D human breast carcinoma cells

Insulin-like growth factor-binding proteins (IGFBPs) play an integral role in modifying insulin-like growth factor actions in a wide variety of cell types. Recent evidence suggests that IGFBP-3 and IGFBP-5 also have effects on cell growth that are insulin-like growth factor-independent. In investigating possible mechanisms for this effect, the intracellular trafficking of IGFBP-3 and IGFBP-5, both of which contain sequences with the potential for nuclear localization, was studied in T47D cells. Nuclear uptake of fluorescently labeled IGFBP-3 and IGFBP-5 was observed in a proportion of T47D cells that appeared to be rapidly dividing. IGFBP-1 and IGFBP-2, which do not possess the putative domain for nuclear translocation, were not transported to the nuclei of T47D cells. When T47D cells were preincubated with excess unlabeled IGFBP-3, nuclear localization of labeled IGFBP-3 or IGFBP-5 was not detected, indicating that their nuclear translocation involves a common pathway. Inhibition of receptor-mediated endocytosis did not affect nuclear uptake of IGFBP-3, suggesting that it uses an alternative non-classical import pathway for transport across the plasma membrane. In addition, a variant form of IGFBP-3 with a mutation in the putative nuclear localization sequence was unable to translocate to the nuclei of T47D cells, suggesting that nuclear translocation of IGFBP-3 was dependent on these carboxyl-terminal basic residues.     

Inverse effects of 20K and 22K human growth hormones on the growth of T-47D human breast cancer cells in culture and in nude mice

The major form of human growth hormone (22K hGH) stimulates the growth of T-47D human breast cancer cells in culture and in nude mice by binding to their receptors for growth hormone and prolactin. Another isoform of hGH having a smaller molecular mass (20K hGH) is known to show different binding affinities to these receptors. In this study, we have analyzed the effects of 20K hGH on the growth of T-47D cells in vitro and in vivo. 20K hGH (50 ng/ml) inhibited the proliferation and DNA synthesis of T-47D cells cultured in the presence and absence of 17 beta-estradiol (100 ng/ml), while 22K hGH (50 ng/ml) promoted the cellular growth. In estradiol-treated nude mice, 22K hGH (100 micrograms) remarkably promoted the growth of T-47D tumor, but 20K hGH again suppressed the tumor growth significantly. The results suggest the presence of different signal pathways for these two hGH isoforms and imply a possible clinical application for 20K hGH.     

Effects of estrogen and antiestrogen on DNA polymerase in human breast cancer

We have investigated the effects of estrogens and antiestrogens on cellular DNA-dependent DNA polymerase activity in human breast cancer, using as a model the MCF-7 human breast cancer cell line which contains estrogen receptor. 17 beta-Estradiol had little if any effect on cytosol DNA polymerase activity or growth (total DNA per flask) of MCF-7 cells. Incubation of the cells for 4 to 6 days with the antiestrogen nafoxidine, however, resulted in a dose-dependent reduction in cytosol DNA polymerase activity to one-half that observed in untreated cells. Enzyme activity in antiestrogen-treated cells was restored to levels contained in untreated cells by removing antiestrogen from the growth medium and incubating the cells for an additional 4 days with 17 beta-estradiol. The restoration required estrogenic steroids specifically, and the time course, magnitude, and dose dependence of the response were similar to estrogen-stimulated increases in DNA polymerase activity described in other estrogen target tissues. Estrogen-mediated reversal of antiestrogen suppression of DNA polymerase activity was paralleled by increases in total DNA synthesis.     

Involvement of apoptosis and cyclin D1 gene repression in growth inhibition of T-47D human breast cancer cells by methylglyoxal bis(cyclopentylamidinohydrazone)

In 2 forested areas near Belém (Para State, Brazil), 2 Haemagogus and 6 Sabethes species were marked released and recaptured in May 1989 and in April 1993. The recapture rates were high, 4.9 and 13.1% for Haemagogus and Sabethes spp., respectively. For Haemagogus janthinomys Dyar, females were recaptured until 27 d after release. The duration of the gonotrophic cycle was between 5.0 and 9.5 d and the survival rate was 0.90-0.92. Haemagogus leucocelaenus (Dyar & Shannon) was recaptured once, 21 d after release. Twelve Sabethes chloropterus (Von Humboldt) were recaptured, with a peak at 15-18 d; 1 female was recaptured at 44 d, indicating extended survival. Seven Sabethes amazonicus Gordon & Evans and 7 Sabethes cyaneus (F.) were recaptured, mostly at 14-39 d. These results indicate that Haemagogus and Sabethes spp. have a gonotrophic cycle in nature longer than inferred from laboratory studies, and that cycle length varies seasonally. The capacity of these species to sustain epizootics or epidemics of arboviruses may depend on local weather, with risk greatest at the end of the rainy season.     

Classification of breast cancer cells on the basis of a functional assay for estrogen receptor

BACKGROUND: The receptor (ER) for estrogen (E2) is routinely assayed as a marker to determine the feasibility of anti-hormone therapy against breast cancer because ER-positive (ER+) tumors are much more likely to respond to anti-hormone therapy than are ER-negative (ER-). However 40% of ER+ breast cancer patients do not respond to anti-hormone therapy. We suggest that this unpredictability of therapeutic responses lies in the current ER assays, which measure only an initial component of the E2-responsive pathway, and that the difference depends upon altered downstream processes. We propose a functional criterion that subclassifies breast cancers on the basis of specific binding of ER to its cognate DNA sequence, the estrogen response element (ERE). MATERIALS AND METHODS: ER was identified in breast cancer cell lines by immunofluorescence assay, Western blot analysis, identification of ER-specific mRNA, and by interaction of the ER-ERE complex with three different ER-specific antibodies. ER-ERE complex formation was measured by electrophoretic mobility shift assay (EMSA). Transactivation of the E2-responsive gene was studied by transfection of cells with fusion gene construct with the promoter-containing ERE sequence and assay of reporter gene activity in the cell extracts. RESULTS: The growth of ER+ T47D cells was sensitive to tamoxifen, ICI-182,780, and ethynyl estradiol (EE2), whereas another ER+ breast cancer cell line, 21 PT, was resistant to these compounds. The estrogen receptor (ER) in the nuclear extracts of MCF-7 and T47D demonstrated hormone-dependent interaction with the response element (ERE) and also downstream transactivation of the E2-responsive PS2 promoter. But in the 21 PT cell line that was designated as ER- on the basis of ligand-binding assay and was found to be ER+ by all the other ER assays, ER-ERE interaction and PS2 promoter transactivation were independent of hormone. CONCLUSIONS: On the basis of the downstream functional assay of ER interaction with ERE, ER+ breast tumor cells can be subclassified into two categories. The first is E2-dependent (ERd+) and these cells should respond to anti-hormone therapy. The second type of ER interacts with ERE independent of E2 (ERi+) and constitutively transactivates responsive genes. It is predicted that the latter type of breast cancers will not respond to antihormone therapy.     

Tenascin expression in cancer cells and stroma of human breast cancer and its prognostic significance

Sections of formalin-fixed, paraffin-embedded tissues from 210 human breast cancers were immunohistochemically examined using the mAb against human tenascin (TN) RCB1. Immunoreactive TN was detected in the breast cancer stroma in 77 (36.7%) cases, whereas the remaining 133 (63.3%) were negative. Of the 77, 12 (5.7%) cases also showed positive staining in the carcinoma cell cytoplasm. The positive cells were often observed in the margin of the cancer nests at the site adjacent to the stroma. According to the staining pattern of TN, the breast cancer cases were classified into the three groups of cancer cell TN(+)/stromal TN(+), cancer cell(-)/stromal TN(+), and cancer cell(-)/stromal TN(-). Analysis of the relationship of these TN patterns with various clinicopathological characteristics of the tumors and the patient outcome revealed that, in comparison to the cancer cell(-)/stromal TN(-) group, the cancer cell TN(+)/stromal TN(+) group exhibited increased frequency of lymph node metastasis and exceptionally poor outcome, and the cancer cell(-)/stromal TN(+) group also showed more frequent metastasis and poorer outcome. Most of the cancer cell TN(+)/stromal TN(+) cases were c-erbB-2 positive and estrogen receptor negative. Furthermore, in situ hybridization of freshly obtained breast cancer tissues demonstrated that both cancer cells and stromal cells express TN mRNA. These results indicate that the TN in breast cancer is produced by cancer epithelial cells as well as by stromal mesenchymal cells, and that cancer cell TN might be involved in cancer spreading, resulting in unfavorable patient prognosis.     

Assay of DNA content and estrogen receptor status in human breast cancer

IVOX was named as an acronym for intravascular oxygenator. The device does not need a blood pomp like an extracorporeal membrane oxygenator (ECMO), and performs intracorporeal gas exchange to be a small elongated, hollow fiber membrane oxygenator designed to lie within the subject's venae cavae so that circulating venous blood can flow freely over and around the external surfaces of the hollow fibers. The amount of gas exchange in IVOX is less than ECMO, however, the equipment is simple and there is no effect to hemodynamics and body temperature. IVOX has been utilized in the management of 165 clinical trials patients in 31 international critical care centers. Currently the gas transfer rate by means of the IVOX device constitutes 1/4 to 1/3 the total metabolic requirement of adult acute respiratory failure patients. Therefore, intentional hypoventilation to limit airway pressures (mild permissive hypercapnia) is recommended to improve CO2 removal with increasing mixed venous CO2 concentrations. In the future, improvements of design, function, and methods of utilization of IVOX device are expected to increase the amount of gas exchange and to enlarge the indications for its use.     

C-myc gene chromatin of estrogen receptor positive and negative breast cancer cells

Expression of the c-myc protooncogene is estrogen regulated in estrogen receptor (ER) positive, hormone-dependent human breast cancer cells, but it is constitutively active in ER negative, hormone-independent breast cancer cells. To determine whether these differences are reflected in c-myc chromatin, DNase I hypersensitive sites (DHS) were mapped. Six DHS were detected in all cell lines studied, with DHS 3(2) being more prominent than DHS 3(1). The accessibility of DHS 2 was markedly greater in ER negative cells than in ER positive cells, and this relative accessibility remained unchanged when cells were grown in estrogen free medium. DHS 2, 3(1) and 3(2) map near the P0, P1 and P2 promoters, respectively. An analysis of promoter usage demonstrated that P2 was the preferred promoter. Thus, the differences in the accessibility of DHS 2 in c-myc chromatin of ER positive and negative cells likely reflects alterations in DNA-protein interactions in this region.     

Comparative effect of embryonic mouse fibroblasts (Balb/c-3T3) on the proliferation of hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) human breast cancer cell lines

An increase in the intensity and distinct spectral changes of ultraweak luminescence from the yeast Saccharomyces cerevisiae were measured when the metabolism of cells was drastically altered. A small emission peak and a red emission band 680-850 nm appeared when air-dried cells were imbibed in water. Lethal concentrations of HCHO (0.01%-10%) elicited a 2500 fold increase of the emission intensity and distinct spectral alterations. A transient 500-580 nm emission appeared in the initial phase of interaction. Then a gradually increasing long-lasting red emission band centered around 620 nm predominated in the total spectral range covering 470-850 nm. These emissions were not correlated with minor changes in fluorescence emission and excitation spectra originating from tryptophan, flavins, and unidentified emitters.     

Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGFbeta1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta1 neutralizing antibody confirms the correlation between induction of active TGFbeta1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGFbeta1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGFbeta1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.     

Vitamin D derivatives inhibit the mitogenic effects of IGF-I on MCF-7 human breast cancer cells

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and four novel synthetic analogues (EB1089, KH1060, KH1230 and CB1093) on IGF-I-stimulated growth of MCF-7 human breast cancer cells have been determined. A significant time- and dose-dependent inhibition of IGF-I-stimulated cell growth was seen with EB1089, such that after 7 days of treatment with 10(-8) M EB1089, the mitogenic effect of IGF-I (30 ng/ml) was negated. Comparison with 1,25(OH)2D3 showed the synthetic analogues to be more potent. The anti-oestrogen ICI 182,780 similarly inhibited IGF-I-stimulated growth of these cells and in combination with EB1089 exerted additional inhibitory effects. Retinoids (all-trans-retinoic acid or the isomer 9-cis-retinoic acid) were less effective in limiting MCF-7 cell responsiveness to IGF-I but, in combination with EB1089, a co-operative effect was achieved. Using radioligand-binding techniques, we observed that 1,25(OH)2D3 and EB1089 down-regulated the levels of 125I-IGF-I binding to MCF-7 cell membranes. Scatchard analysis showed that EB1089 decreased maximal binding approximately 2-fold. Vitamin D derivatives were also demonstrated to reduce IGF-I receptor expression in MCF-7 cells by Western analysis. Our findings demonstrate that vitamin D derivatives limit responsiveness of MCF-7 cells to the mitogenic effects of IGF-I, which may be mediated by reduction of IGF-I receptor expression.     

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.     

Integrin-associated protein (CD47) is a comitogenic molecule on CD3-activated human T cells

IAP is a glycoprotein functionally and physically associated with some integrins, i.e., the leukocyte response integrin and the beta3 integrin chain on placenta, platelets, and polymorphonuclear cells. IAP may act as a transducer element in activation mediated via these integrins. Since IAP is present at high density on peripheral T lymphocytes we have investigated its involvement in T cell activation. We tested three mAbs against IAP, namely B6H12, BRIC126, and 2D3, which recognize two distinct epitopes. IAP cross-linking with B6H12 or BRIC126, but not 2D3, transduces costimulatory signals within highly purified CD3-activated T lymphocytes, i.e., enhancement of proliferation, CD25 expression, and IL-2 secretion, while no effect was observed upon CD2 stimulation. However, we could not observe any functional association between IAP and integrins on peripheral T cells. In an attempt to explore further the activation signal delivered by IAP, we show here that IAP cross-linking with the comitogenic B6H12 mAb induces the phosphorylation on tyrosine of several proteins, one of which is identified as p56(lck) protein tyrosine kinase. Moreover, we observed that IAP is associated with p56(lck) on PMA-activated, but not on resting, T cells. These data suggest that on T cells, IAP may be involved directly via a specific ligand in cell-matrix or cell-cell interactions. Such interactions could trigger protein tyrosine phosphorylation pathways, which play an important role in both maturation and activation of T cells.     

Inhibition of breast cancer tissue aromatase activity and estrogen concentrations by the third-generation aromatase inhibitor vorozole

In about one-third of advanced breast cancers, estrogen deprivation causes tumor regression. Estrogen concentrations in tumor tissue seem to depend largely on local production. The aromatase enzyme complex is thought to be the key enzyme in this respect. In the present study, the effect of the new third-generation nonsteroidal aromatase inhibitor vorozole (Rivizor) on tumor tissue aromatase activity and estrogen concentrations was evaluated. During 7 days preceding mastectomy, 11 postmenopausal breast cancer patients were treated with 2.5 mg of vorozole once daily. Eight patients could be evaluated. Intratumoral aromatase activity and estrone and estradiol levels were measured and compared to the values of nine untreated postmenopausal breast cancer patients. In treated patients, median tissue aromatase activity was 89% lower than that in controls (P < 0.001). Similarly, median tissue estrone and estradiol concentrations were 64 and 80% lower, respectively, in treated patients (P = 0.001 and P < 0.05, respectively). These results support the hypothesis that depleting the tumor of estrogens, thus impairing estrogenic stimulation, is an important mechanism in the antitumor activity of aromatase inhibitors.