Horizontal Transfer of Tamoxifen Resistance in MCF-7 Cell Derivates: Proteome Study.

Using estrogen-dependent MCF-7 breast cancer cells and tamoxifen-resistant MCF-7/T subline we have shown that their co-cultivation lead to increase in tamoxifen resistance in the parent MCF-7 cells. The proteome analysis of MCF-7/T cells and new-generated resistant cells revealed 21 common proteins differently expressed in both the resistant cell lines, among them - 6 proteins were associated with the drug or hormonal resistance. Both resistant lines were characterized with suppression of estrogen receptor and activation of SNAIL1-signaling - mesenchymal pathway playing an important role in the down-regulation of estrogen receptor and maintaining of the estrogen-independent phenotype.

The Mechanism of Adaptation of Breast Cancer Cells to Hypoxia: Role of AMPK/mTOR Signaling Pathway.

We studied the mechanisms of adaptation of human breast cancer cells MCF-7 to hypoxia and analyzed the role of AMPK/mTOR signaling pathway in the maintenance of cell proliferation under hypoxic conditions. It was found that long-term culturing (30 days or more) of MCF-7 cells under hypoxic conditions induced their partial adaptation to hypoxia. Cell adaptation to hypoxia was associated with attenuation of hypoxia-dependent AMPK induction with simultaneous constitutive activation of mTOR and Akt. These findings suggest that these proteins can be promising targets for targeted therapy of tumors developing under hypoxic conditions.

The mechanism of cytostatic effect of a new somatostatin analog cifetrelin on in vitro cultured MCF-7 breast carcinoma cells.

The mechanisms of antitumor effect of cifetrelin have been studied in vitro on cultured MCF-7 breast carcinoma cells. Cifetrelin exhibits higher cytotoxic activity than native somatostatin. In subthreshold concentrations, cifetrelin boosts the apoptotic effects of drugs, e.g. adriamycin. Cifetrelin-induced apoptosis develops by the p53-independent mechanism and is associated with early suppression of NF-kappaB activity. These data attest to high antitumor activity of cifetrelin.

Molecular mechanisms of hormone resistance of breast cancer.

More than 70% malignant mammary tumors contain steroid hormone receptors; this suggests the possibility of hormone therapy in the majority of patients with breast cancer (BC). The main cause of inefficiency of hormone therapy in BC is hormone resistance (tumor resistance to hormonal cytostatics). Here we discuss the main mechanisms of hormone resistance of BC and the mechanisms underlying the formation of hormone resistance of the tumors are analyzed at the molecular level. The data on the signal pathways of estrogen receptors (ER), the key regulators of BC cell proliferation, are presented. The most important factors of BC hormone resistance are: high activity/expression of receptor tyrosine kinases; high activity of proteins regulating cell defense mechanisms (Akt PI3K, mTOR); changes in the activities of cell cycle regulator proteins (Myc, c-Fos, Cyclin D1). Our experiments have demonstrated that estrogen-independent BC cell growth is supported by VEGF/VEGFR2 and EGF/EGFR mitogenic signal pathways. Our data indicate that NF-kappaB transcription factor is directly involved in the regulation of hormone-resistant BC cell growth and survival, while NF-kappaB suppression determines cell sensitivity to apoptotic activity of antitumor compounds. On the whole, the results indicate good prospects of using EGFR, HER-2/neu, mTOR, VEGFR, PI3K/Akt molecular pathways as targets for BC therapy, including therapy for BC resistant forms.

[The role of transcription factor Snail1 in the regulation of hormonal sensitivity of in vitro cultured breast cancer cells].

The loss of hormonal dependency in breast tumor cells is often accompanied by epithelial-mesenchymal transition (EMT) features and an increase in cell metastasizing and invasiveness. Here we studied the role of transcription factors Snail1--the central mediator of EMT, in the progression of hormonal resistance of breast cancer cells. The experiments were performed on the estrogen receptor(ER)-positive estrogen-dependent MCF-7 breast cancer cells, ER-positive estrogen-resistant MCF-7/LS subline generated through long-term cultivation of the parental cells in steroid-free medium, and ER-negative estrogen-resistant HBL-100 breast cancer cells. We found that decrease in the estrogen dependency of breast cancer cells is accompanied by an increase in Snail1 expression and activity, and demonstrated the Snail1 involvement in the negative regulation of ER. NF-kappaB was found to serve as a positive regulator of Snail1 in breast cancer cells, and simultaneous inhibition of NF-kappaB and Snail1 by RNA interference resulted in marked increase of cell response to antiestrogen tamoxifen. In general, the results obtained demonstrate that direct inhibition of NF-kappaB and Snail1 partially restores the estrogen receptor machinery, and show that Snail1 and NF-kappaB may serve as an important targets in the treatment of breast cancer.

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.

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.

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.

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.

[Regulation of the transcription of the tyrosine aminotransferase gene by glucocorticoids in Morris hepatoma 7777 and 8994 cells]. / Reguliatsiia transkriptsii gena tirozinaminotransferazy gliukokortikoidami v kletkakh gepatomy Morrisa 7777 i 8994.

Kinetics of tyrosine aminotransferase (TAT) mRNA synthesis in Morris rat hepatoma cell lines 7777 and 8994 after dexamethasone treatment (10(-7) M) was studied by molecular hybridization of the RNA with cloned fragments of TAT gene from rat liver cells. It was demonstrated that initiation of TAT gene transcription increased 20 minutes after glucocorticoid treatment. The level of TAT mRNA was not induced by dexamethasone in rat hepatoma cell line 8994. Actinomycin D prevented the deinduction of TAT by stabilization of TAT mRNA.

[Signaling pathways that control the growth and survival of prostate tumor cells in the absence of androgens]. / Signal'nye puti, kontroliruiushchie rost i vyzhivaemost' kletok raka predstatel'noi zhelezy v otsutstvie androgenov.

Androgen-dependent human prostate adenocarcinoma cell line LNCaP was used to study the effect of androgen deprivation on the cell response to TNF-related cytokines. Several signaling pathways were implicated in cell survival in the absence of androgens. In androgen-deprived LNCaP cells, TNF-alpha and TRAIL stimulated the cell growth and activated the mitogenic and antiapoptotic signaling pathways involving NF-kappa B, STAT3, PI3K, and beta-catenin. The results suggested a role of cytokines in the survival of prostate adenocarcinoma cells deprived of androgens in vitro.

[Evaluation of the effectiveness of tamoxifen in malignant breast tumors: new approaches]. / Opredelenie éffektivnosti deistviia tamoksifena na zlokachestvennye opukholi molochnoi zhelezy: novye podkhody.

It has been shown that 17 beta-estradiol stimulates the cycle of phospholipid conversion in the breast tumor cells. The action of tamoxifen on antiestrogen cells is not limited by weakening the stimulating effect of 17 beta-estradiol on the exchange of intracellular phospholipids, but gives rise to a more complicated pattern of changes: inhibited incorporation of 32-P-phosphatidylcholine (PC) and activated exchange of phosphoinositides (PI). The experimental findings of 53 breast tumors have indicated that in 47.2% of cases Tamoxifen alters the PC/PI ratio and causes its 2-fold increase. Such alterations have been found to be induced by the ability of Tamoxifen to suppress the activity of protein kinase C that regulates the synthesis of PC and PI. It is suggested that the revealed capacity of Tamoxifen to change the rate of intracellular phospholipid conversion might be used for evaluating the efficiency of this agent on malignant tumors.

[The molecular mechanism responsible for the adaptation of malignant tumors to hormonal drugs: a role of phophatidylinositol-3-kinase and phosphoinositide-dependent proteins]. / Molekuliarnyi mekhanizm adaptatsii zlokachestvennykh opukholei k gormonal'nym preparatam: rol' fosfatidilinozit-3-kinazy i fosfoinozitidzavisimykh belkov.

Phophatidylinositol-3-kinase (PI3K) is a major intracellular protein that is responsible for the transmission of an antiapoptotic signal and controls the survival of tumor cells upon exposure to damaging agents. Experiments using different tumor cell cultures have shown that the resistance of cells to the antiproliferative action of dexamethasone, caused by their long cultivation with the hormone, is associated with the activation of PI3K and the transcription factor STATS. The activation of PI3K and STAT3 in the dexamethasone-resistant cells correlates with the increase in the total thyrosine kinase activity and with the decrease in the sensitivity of cells to exogenous proliferative agents, such as 17beta-estradiol. The long exposure of hormone-sensitive cells to nonhormonal factors that activate the PI3K/STAT3 signaling pathway, hypoxia in particular, has been shown to suffice to reduce the degree of hormonal tumor cell dependence. VEGF-A, an angiogenic peptide whose action was partially realizes through the PI3K-signalling pathway, has been demonstrated to be involved in the maintenance of cell growth, including the growth of hormone-independent cells. The findings suggest that complex changes in the antiapoptotic and mitogenic signaling pathways associated with PI3K, which ensures the autonomic, hormone-independent growth of tumor cells, may underlie the decreased hormonal dependence of tumor cells. Whether PI3K may be used to suppress the growth of hormone-independent tumors is discussed.

[The effect of steroid hormones and tamoxifen on the rate of phospholipid turnover in the cells of uterine and breast tumors]. / Vliianie steroidnykh gormonov i tamoksifena na skorost' obrashcheniia fosfolipidov v kletkakh opukholei matki i molochnoi zhelezy.

The cycle of phospholipid turnover has been found to be under the negative control of hormonal cytostatics (progesterone) and under the positive control of proliferation stimulants (17 beta-estradiol, epidermal growth factor). Specific changes in the synthesis of phospholipids are shown when tamoxiphen, an antiestrogen and an inhibitor of protein kinase C, was used. The findings suggest that changes in the turnover rate of phospholipids are one of the key stages of steroid action on target cells and may be regarded as an additional criterion of tumor genetic sensibility.

[Role of phosphatidylinositol signalling path in developing hormonal resistance in tumor cells]. / Rol' fosfatidilinozitol'nogo signal'nogo puti v razvitii gormonal'noi razistentnosti opukholevykh kletok.

Phosphatidylinositol 3-kinase (PI3K) is a key regulatory protein which is responsible for anti-apoptotic signal transduction regulating cell survival during exposure to damaging factors. The report deals with the role of the PI3K signaling pathway in regulating cellular response to hormones and, particularly, in development of resistance as a result of long-term exposure of cells to steroid cytostatic hormones. In our study, even a short-term exposure of transformed fibroblasts of hamster (line 2PK) resulted in an activation of main PI3K effectors (MAP-kinase and protein kinase B (PKB)) which appeared against the background of hormone-induced inhibition of cellular growth. A long-term (3 months) cell culturing with dexamethasone was followed by formation of subpopulations of cells which were refractory to the growth inhibition by hormone and were characterized by high levels of activity of PI3K, MAP-kinases and PKB. Activation of PI3K and PI3K-dependent enzymes correlated with enhancement of synthesis of c-jun, a component of the AP-1 transcriptional factor, was observed both in short- and long-term application of dexamethasone. We believe that, during long-term exposure of cells to cytostatic hormones, continuous activation of PI3K and PI3K-dependent transcriptional factors may result in a significant restructuring of intracellular signal pathway, and, finally, constitutive PI3K-signal pathway and partial overcoming the proliferative block by cells.

[The role of protein kinase PAK1 in the regulation of estrogen-independent growth of breast cancer].

The main goal of this work was to study the intracellular signaling pathways responsible for the development of hormone resistance and maintaining the autonomous growth of breast cancer cells. In particular, the role of PAK1 (p21-activated kinase 1), the key mitogenic signaling protein, in the development of cell resistance to estrogens was analyzed. In vitro studies were performed on cultured breast cancer cell lines: estrogen-dependent estrogen receptor (ER)-positive MCF-7 cells and estrogen-resistant ER-negative HBL-100 cells. We found that the resistant HBL-100 cells were characterized by a higher level of PAK1 and demonstrated PAK1 involvement in the maintaining of estrogen-independent cell growth. We have also shown PAK1 ability to up-regulate Snail1, one of the epithelial-mesenchymal transition proteins, and obtained experimental evidence for Snail1 importance in the regulation of cell proliferation. In general, the results obtained in this study demonstrate involvement of PAK1 and Snail1 in the formation of estrogen-independent phenotype of breast cancer cells showing the potential role of both proteins as markers of hormone resistance of breast tumors.

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.

Development of differential sensitivity of CaOv ovarian adenocarcinoma cells to antitumor agents under conditions of hypoxia.

We studied the role of VEGF signal pathway in autocrine regulation of tumor cell growth and survival under conditions of hypoxia. Hypoxia-resistant CaOv/H substrain with high level of VEGF-A secretion was obtained by long-term culturing of CaOv ovarian adenocarcinoma cells with CoCl2 (hypoxia inductor). VEGF-A directly participates in autocrine regulation of CaOv cell growth, including the maintenance of cell growth under conditions of hypoxia or cytostatic treatment. On the other hand, CaOv/H cells retain high apoptotic potential and are characterized by high expression of p27Kip1 (cyclin-dependent kinase inhibitor), which attests to possible involvement of this inhibitor into the regulation of apoptotic response of cells under conditions of hypoxia.