NF-kappaB suppression provokes the sensitization of hormone-resistant breast cancer cells to estrogen apoptosis.

The progression of breast cancer cells to estrogen-independent growth may be accompanied with the paradoxical cell sensitization to estrogen apoptotic action; however, the mechanism of this phenomenon is still unclear. In the present study, we have shown that the sensitization of hormone-resistant breast cancer cells to estrogen apoptotic action is accompanied with the gradual NF-kappaB suppression. Using the chemical inhibitors of NF-kappaB as well as the dominant-negative NF-kappaB constructs, we have proved the sufficiency of NF-kappaB inhibition for the sensitization of the resistant cells to estrogen apoptosis. Estradiol treatment results in the additional suppression of NF-kappaB, demonstrating the possible NF-kappaB involvement in the regulation of cell response to estrogens. Totally, the results presented suggest that the constitutive NF-kappaB suppression in the estrogen-independent cells may be considered as one of the factors resulting in a imbalance between pro- and anti-apoptotic pathways and enhancement in estrogen apoptotic action in the cells.

Role of phosphatidylinositol signaling pathway in the development of cross-resistance of tumor cells to hormonal cytostatics and hypoxia.

Long-term in vitro culturing of CaOv ovarian adenocarcinoma cells in the presence of a synthetic analogue of glucocorticoid hormone dexamethasone allowed us to obtain a subline of CaOv/D cells resistant to the antiproliferative effect of dexamethasone and characterized by high resistance to hypoxia. It was found that CaOv/D cells are characterized by constitutive increase in phosphatidylinositol 3-kinase expression and hypersecretion of vascular endothelial growth factor VEGF-A. Culturing of cells under hypoxic conditions was accompanied by a significant increase in phosphatidylinositol 3-kinase expression and VEGF-A synthesis. Experiments with cell transfection with phosphatidylinositol 3-kinase catalytic subunit proved its participation in the regulation of VEGF-A synthesis and maintenance of cell growth under condition of hypoxia. Our results indicate that coordinated activation of phosphatidylinositol 3-kinase and VEGF-A can be a factor determining the development of cross-resistance of tumor cells to hormonal cytostatics and hypoxia.

Mechanism of estrogen-induced apoptosis in breast cancer cells: role of the NF-kappaB signaling pathway.

The ability of sex steroid hormones to up-regulate the apoptotic signaling proteins is well documented; however, the apoptotic potential of sex hormones is not remarkable and fully compensated by their growth stimulatory action to target cells. In the present study using the long-term cultivation of estrogen-dependent MCF-7 breast cancer cells in steroid-free medium, we have established a cell subline, designed as MCF-7/LS, which was characterized by the resistance to growth stimulatory estradiol action and hypersensitivity to estrogen-induced apoptosis. We have demonstrated that estrogen treatment of the cells does not influence on the level of TNF-R1 or Fas, but dramatically decreases the transcriptional activity of NF-kappaB. Importantly, the MCF-7/LS cells, which are insensitive to growth stimulatory estrogen action, retain the ability to decrease in the NF-kappaB activity in response to estrogen stimulus. Furthermore, the significant increase in the basal (in the absence of ligand) estrogen receptor (ER)-dependent transcriptional activity in the MCF-7/LS cells was revealed and reciprocal transcriptional antagonism between ER and NF-kappaB was demonstrated. Finally, we proved the possible involvement of phosphatidylinositol-3 kinase (PI3K) in the ligand-independent ER activation. In general, the results presented suggest that long-term growth of MCF-7 breast cancer cells in steroid-free medium is accompanied with the increase in the basal ER-dependent transcriptional activity as well as the maintenance of the negative regulatory loop ER-NF-kappaB. The latter may be considered as one of the factors resulting in a disbalance between pro- and anti-apoptotic pathways and enhancement in estrogen apoptotic action in the cells.

Sensitization of MCF-7 breast cancer cells to the apoptotic effect of estradiol.

A new substrain of hormone-resistant MCF-7/T breast cancer cells was selected after long-term culturing of estrogen-dependent MCF-7 cells in the presence of tamoxifen. These cells were resistant to the growth-stimulating and cytostatic effects of estradiol and tamoxifen, respectively. MCF-7/T cells gained paradoxical sensitivity to the apoptotic effect of estradiol. Estradiol stimulated p53 expression and decreased DNA-binding activity of NF-kappaB. Our findings provide indirect evidence that these proteins are involved in the regulation of estrogen-induced apoptosis. These results indicate that tamoxifen-resistant breast cancer cells can be sensitized to the apoptotic effect of estradiol. The data form a basis for the development of new methods of endocrine therapy for breast cancer patients.

Role of phosphatidylinositol-3 kinase in regulation of differential sensitivity of melanoma cells to antitumor agents. A model for hormone resistance development in tumor cells.

Phosphatidylinositol-3 kinase (PI3K) belongs to one of the most important cellular proteins involved in the transmission of anti-apoptotic signal and regulation of survival pathways in tumor cells. Earlier we have found that prolonged treatment of melanoma cells with dexamethasone results in formation of a cell subline which was resistant to growth inhibitory dexamethasone action. We showed that constitutive activation of PI3K can be considered as one of the factors that regulate cell resistance to dexamethasone. Here we demonstrate that increased level of PI3K protein in dexamethasone-resistant cells correlates with partial decrease in expression of down-stream target of PI3K--protein kinase B (PKB). Study of the cell's sensitivity to various damaging agents showed that the cells after prolonged dexamethasone treatment are characterized by increased level of the resistance to both hormonal drugs and hypoxia, and at the same time with high sensitivity to ultraviolet (UV) radiation or anti-tumor agents such as adriamycin. As revealed, hypoxic conditions or short-term dexamethasone treatment of the resistant cells lead to a substantial increase in the PKB level, whereas neither UV radiation nor adriamycin affects the PKB level in these cells. We demonstrate that long-term dexamethasone treatment of melanoma cells results in the accumulation of the active form of mitogen-transducing signaling protein STAT3 (Signal Transducer and Activator of Transcription-3), which also contributes to inducing the melanoma cell's resistance to antiproliferative action of dexamethasone. We suggest that decreased level of PKB in combination with an activation of PI3K/STAT3 signaling in the melanoma cells after prolonged dexamethasone treatment may be one of the mechanisms of different sensitivity of these cells to hormonal drugs and damaging agents. The model of the progression of hormonal resistance of in vitro cultured tumor cells is presented.

Expression of phosphatidylinositol-3 kinase in lung cancer.

The expression of phosphatidylinositol-3 kinase in tumors and homologous tissues from 29 patients with lung cancer, 5 patients with lung metastases of various tumors, and some non-tumorous pulmonary diseases was studied by Western blot analysis. The expression of phosphatidylinositol-3 kinase was increased in these tumors in comparison with histologically intact lung tissue in 5 patients with non-small-cell cancer. In 20 patients expression of phosphatidylinositol-3 kinase was the same as in homologous tissue and in 4 patients it was decreased. No relationship between phosphatidylinositol-3 kinase expression and clinical and morphological characteristics of lung cancer was revealed.

Phospatidylinositol 3-kinase expression in human breast cancer.

Phospatidylinositol 3-kinase (PI 3-kinase) expression was analysed by Western blotting with monoclonal antibodies to the p85 subunit in a series of tumour and adjacent mammary gland samples collected at surgery from 33 breast cancer patients. Seventy-nine percent of the investigated pairs of the samples were characterised by an increased level of PI 3-kinase in the tumour in comparison with the adjacent mammary gland. PI 3-kinase activation was not associated with tumour steroid receptor status, histologic grade and other clinico-morphological characteristics. Furthermore, immunoblotting of epidermal growth factor receptor (EGFR) in the tumours with increased PI 3-kinase and corresponding adjacent tissues revealed no association between EGFR and PI 3-kinase activation. Thus, increased PI 3-kinase expression appears to be a widespread feature of breast cancer not associated with the main biological markers of its prognosis and hormone sensitivity.

The role of phosphatidylinositol 3-kinase in the regulation of cell response to steroid hormones.

Phosphatidylinositol 3-kinase (PI-3 kinase) has been implicated in the regulation of many cellular processes, including growth and transformation. We describe the effect of glucocorticoids on cell growth, phosphoinositide formation and PI-3 kinase activity in Rous sarcoma virus-transformed hamster fibroblasts (HET-SR). Using a prolonged dexamethasone treatment of HET-SR cells we have selected a new glucocorticoid receptor-positive cell subline, HET-SR(h), that was resistant to growth inhibitory action of dexamethasone and/or non-hormonal drugs (vinblastine, adriamycin) and was characterized by higher levels of phosphoinositide formation and increased PI-3 kinase activity. Study of the short-term hormone action has shown that both dexamethasone-sensitive and -resistant sublines responded to hormone by a decrease in phospholipid turnover rate. At the same time, in both cell lines activation of PI-3 kinase after dexamethasone addition was revealed. Dexamethasone-dependent activation of PI-3 kinase was more significant and maintained for a longer period in HET-SR(h) cells than in parent HET-SR cells. Finally, by transfecting p110*, a constitutively active catalytic subunit of PI-3 kinase, into hormone-sensitive HET-SR cells, we have found a marked increase in cell resistance to growth inhibitory dexamethasone action. These results suggest that PI-3 kinase may serve as one of the factors providing cell resistance to cytostatic drugs.

Comparative analysis of the sensitivity of endometrial cancer cells to epidermal growth factor and steroid hormones.

BACKGROUND: Epidermal growth factor (EGF) and EGF-regulated processes play an important role in steroid signal transduction. Comparative analysis of EGF and steroid receptor expression and the sensitivity of early stages of proliferation induction, such as activation of phospholipid turnover to EGF and steroids, may provide a useful new approach to characterizing the sensitivity of endometrial cancer to hormone therapy. METHODS: Progesterone (PR), estradiol (ER), and EGF receptor (EGFR) content was measured by radioligand competitive methods in surgically excised tumors from 26 patients with primary endometrial cancer. In short term cell cultures isolated from 11 of these tumors, the influence of a 10-minute treatment with 10(-8)M EGF either alone or combined with 10(-8)M progesterone on 32P-incorporation into phospholipids was studied. Phospholipids were fractionated by thin-layer chromatography and were located by autoradiography, and quantification of the labeled compounds was made by densitometric scanning of the autoradiograms. RESULTS: Epidermal growth factor receptor was found in 15 of 26 (58%) endometrial cancer samples. Eighty-two percent of the tumors studied contained PR, and 81% contained ER. No significant correlations were revealed between EGFR and ER/PR status or concentration. Epidermal growth factor stimulated 32P-incorporation by more than 120% of the control level in five of seven EGFR-positive and in one of four EGFR-negative endometrial cancer samples. An inverse relationship was revealed between EGFR content and the percentage of EGF-induced stimulation of phospholipid turnover in endometrial cancer cells (r = -0.6; P = 0.15) and between EGFR content in EGFR-positive samples and the extent of progesterone suppression of EGF-induced turnover (r = -0.77; P = 0.04). CONCLUSIONS: Determination of EGF sensitivity on a receptor and a functional level may provide important additional information about the hormonal sensitivity of endometrial cancer.

Regulation of phospholipid turnover by steroid hormones in endometrial carcinoma and breast cancer cells.

To study the early effects of steroid hormones on cells we investigated the influence of the sex steroids and tamoxifen on phospholipid turnover in endometrial carcinoma and breast cancer cells. Studies were performed on 19 human uterine adenocarcinomas and 29 breast cancer tumors. Progesterone in a final concentration of 10(-7) mol/l caused a twofold decrease of 32P incorporation into phospholipids (phosphatidylcholine and phosphoinositides) in 85% of the uterine adenocarcinomas where the progesterone receptor (PR) content was more than 100 nmol/kg and only in 30% of the tumors where the PR content was less than 100 nmol/kg. Treatment of the cells with 10(-8) mol/l 17 beta-estradiol or 10(-8) mol/l epidermal growth factor led to an increase in 32P incorporation into phospholipids. Analysis of the hormonal responsiveness of 29 human breast cancers showed that 17 beta-estradiol increased 32P incorporation into phospholipids in 47% of the tumors where the estradiol receptor (ER) content was more than 10 nmol/kg and in 21% of the receptor-negative tumors (ER < 10 nmol/kg) The results show that phospholipid turnover in uterine and breast cells can be regulated by sex steroids. Treatment of the breast cancer cells with the antiestrogen tamoxifen (10(-6) mol/l) led to an increase of 32P incorporation into phosphoinositides and a decrease of 32P incorporation into phosphatidylcholine. Addition of an activator of protein kinase C, i.e. 2 x 10(-7) mol/l 12-0-tetradecanoylphorbol-13-acetate, weakened the inhibitory effect of tamoxifen on phosphatidylcholine turnover. These findings suggest that tamoxifen action can be mediated via an alteration of the growth signal transducing system.

Glucocorticoid regulation of phospholipid turnover and protein kinase C activity in mouse hepatoma 22 cells.

Glucocorticoids induce growth inhibition in certain sensitive hepatoma cells. To investigate how glucocorticoids interact with growth-factor-dependent pathways, we studied the effects of dexamethasone (Dex) on the DNA synthesis, protein kinase C (PKC) activity and phospholipid turnover in mouse hepatoma 22 cells. Dex was found to reduce DNA synthesis in slowly growing hepatoma cells, whereas exponentially growing cells were Dex-insensitive. Direct measurements of PKC activity in the hormone-sensitive hepatoma 22 cells showed a rapid inhibition (within 30 min) when treated with Dex. Dex addition to hormone-sensitive but not to hormone-insensitive hepatoma 22 cells for 30 min caused a significant decrease of 32P-incorporation into the major cellular phospholipids: phosphatidylcholine, phosphatidylglycerol and phosphoinositides. At the same time, the analysis of the correlation between changes in PKC activity and phospholipid turnover showed that synthesis of phosphatidylcholine and phosphatidylglycerol was under positive control of PKC activity. The data suggest that suppression of phospholipid turnover in hormone-sensitive hepatoma 22 cells is one of the early events caused by glucocorticoids, whereas the decrease of PKC activity induced by the hormone is mediated, probably, via changes in phospholipid metabolism.