[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.

[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.

[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.

[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.

[Effect of specific activation of phosphatidylcholine metabolism in hamster fibroblasts transformed by Rous sarcoma virus]. / Effekt spetsificheskoi aktivatsii obmena fosfatidilkholina v fibroblastakh khomiaka, transformirovannykh virusom sarkomy Rausa.

Transformation of embryonic hamster fibroblasts by the Rous sarcoma virus results in sharp increase of the turnover rate of one of cellular phospholipids-phosphatidylcholine. The decrease in the rate of virus-transformed cells (HETSR strain) during the monolayer formation is attended by additional activation of phosphatidylcholine turnover. A similar effect is observed after prolonged culturing of cells with dexamethasone. Addition of the tyrosine kinase inhibitor, genistein, to cells leads to selective inhibition of phosphatidylcholine synthesis without any effect of phosphoinositide synthesis. Immunoblotting analysis of p60-src, the product of the viral oncogen v-src related to the tyrosine kinase family failed to produce any significant changes in protein synthesis and activity during dexamethasone-induced inhibition of HETSR cell growth. The data obtained testify to selective activation of phosphatidylcholine metabolism in src-transformed cells which enhances with a decrease in the rate of cell growth. The presence in HETSR cells of p60-src whose synthesis is not controlled by dexamethasone may be responsible for increased phosphatidylcholine metabolism and sustaining cell growth under conditions of limited activity of growth-promoting compounds.

[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.

[Regulation of the cycle of phospholipid turnover in hamster fibroblasts transformed by v-src and N-ras oncogenes]. / Reguliatsiia tsikla obrashcheniia fosfolipidov v fibroblastakh khomiaka, transformirovannykh onkogenami v-src i N-ras.

The phospholipid turnover has been studied in two lines of golden hamster cells: in cells transformed by the Rous sarcoma virus (line HET-SR) and in cells additionally transfected with the activated oncogene N-ras (line HET-SR-N-ras, clone 6). It has been found that HET-SR cells are distinguished by a high level of phosphatidylcholine turnover and a relatively low level of phosphoinositide turnover. Transfection of cells with the activated N-ras (line HET-SR-N-ras) leads to the inhibition of phosphatidylcholine synthesis and activation of phosphoinositide metabolism. Both cell lines preserve their sensitivity to serum growth factors stimulating the rate of phospholipid turnover. In both cell lines dexamethasone decreases the rate of DNA synthesis and inhibits the phosphatidylcholine and phosphoinositide turnover. At the same time, dexamethasone does not influence the predominant activation of phosphatidylcholine synthesis in HET-SR cells or the activation of phosphoinositide synthesis characteristic of HET-SR-N-ras cells. The data obtained suggest that the transmission of the mitogenic signal from growth factor in HET-SR and HET-SR-N-ras cells occurs via the activation of the phospholipid turnover and is controlled by steroid hormones. The role of v-src and N-ras oncogens in the transmission of the mitogenic signal seems to be insignificant; their activity is not controlled by dexamethasone.

[The effect of progesterone and tamoxifen on EGF-dependent activation of phospholipid turnover in uterine and breast tumor cells]. / Vliianie progesterona i tamoksifena na EFR-zavisimuiu aktivatsiiu obrashcheniia fosfolipidov v kletkakh opukholei matki i molochnoi zhelezy.

The in vitro effects of the epidermal growth factor (EGF) and progesterone on phospholipid turnover in cells of 19 human adenocarcinomas (postsurgical material) have been studied. In 58% of tumours EGF increased the 32P incorporation into two basic cell phospholipids--phosphatidylcholine and phosphoinositides. In EGF-insensitive cells progesterone induced no noticeable changes in the basal level of phospholipid metabolism. However, in 10 out of 11 positively responding to EGF adenocarcinomas progesterone inhibited the EGF-dependent activation of 32P incorporation into the phospholipids already on the 15th min after its addition to the cells. Analysis of effects of EGF and the anti-estrogen drug tamoxifen on phospholipid turnover in 22 human mammary tumours did not reveal any significant differences in tamoxifen effect on tumour cells differing in their sensitivity to EGF. Independently of cell sensitivity to EGF, tamoxifen caused some decrease in the 32P incorporation into phosphatidylcholine but increased the label incorporation into phosphoinositides. Tamoxifen added to tumour cells prestimulated with EGF or 17 beta-estradiol failed to abrogate the effect of these compounds on phospholipid turnover. At the same time, treatment of cells with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate fully inhibited the effect of tamoxifen on phospholipid metabolism. The results obtained suggest that the EGF-dependent activation of intracellular phospholipid turnover is under the negative control of progesterone. As for tamoxifen, its effect on cells is independent of EGF and consists, apparently, in the inhibition of protein kinase C activity.

[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.

[Regulation of the phospholipid turnover rate and protein kinase C activity as a necessary stage in the realization of the growth-inhibiting effect of dexamethasone on hepatoma 22 cells]. / Reguliatsiia skorosti obrashcheniia fosfolipidov i aktivnosti proteinkinazy C kak neobkhodimyi étap v realizatsii rost-ingibiruiushchego deistviia deksametazona.

The role of protein kinase C and phospholipid turnover in the realization of the cytostatic effect of dexamethasone on hormone-sensitive cells of mouse hepatoma 22 has been studied. It was found that dexamethasone added to hepatoma cells induces a rapid (within 30 min) inhibition of the protein kinase C activity with a simultaneous decrease of the 32P incorporation into the major phospholipids (phosphatidylglycerol, phosphatidylcholine, and phosphoinositides). Analysis of correlation between the protein kinase C activity and phospholipid turnover rate revealed that phosphatidylglycerol and phosphatidylcholine synthesis is under the positive control of protein kinase C, whereas that of phosphoinositides is not controlled by the enzyme. A proportional decrease in the rates of metabolism of all the three major phospholipids after addition of the hormone to hepatoma cells suggests that inhibition of phospholipid turnover is one of the primary manifestations of the dexamethasone effect. The hormone-induced decrease in the protein kinase C activity may be regarded as being due to these changes.

[Growth inhibiting effect of glucocorticoids and progestins on tumor cells in vitro: effect on the rate of phospholipid reversal]. / Rost-ingibiruiushchee deistvie gliukokortikoidov i progestinov na opukholevye kletki in vitro: vliianie na skorost' obrashcheniia fosfolipidov.

The growth-inhibiting effect of dexamethasone was estimated by the ability of the hormone to inhibit the proliferative activity of in vitro cultured hepatoma 22 cells. The effect of another steroid proliferation inhibitor, progesterone, was studied in primary cell cultures of human uterine carcinoma. The cytostatic effect of dexamethasone was observed only in slowly proliferating cells and was rapidly reversed by stimulation of cell division with fresh sera. Dexamethasone did not induce any conspicuous changes in the rate of 32P incorporation into hepatoma phospholipids. In 9 out of 14 human uterine carcinomas progesterone inhibited, whereas 17 beta-estradiol stimulated the 32P incorporation into phospholipids (phosphatidylinositol and phosphatidylcholine); this effect was manifested already after 15-min incubation of cells with the hormone. The resistance of uterine carcinoma cells to steroids was paralleled, as a rule, with the increase in the basal level of 32P incorporation into the phospholipids typical of actively proliferating cells. It was assumed that the inhibition of phospholipid exchange rate is related to the earliest manifestations of the growth-inhibiting effect of steroid hormones, at least progestins. In its turn, stimulation of the proliferative activity of cells may relieve this effect of steroids, eventually resulting in a temporary decrease of the cell sensitivity to hormones.

[Polypeptide growth factors and their interrelation with hormones in the blood plasma of patients with primary bone tumors]. / Polipeptidnye faktory rosta i ikh vzaimosviaz' s gormonami v plazme krovi bol'nykh s pervichnymi opukholiami kostei.

A relationship between blood plasma levels of polypeptide growth factors and those of peptide and sex steroid hormones, as assayed radioimmunologically, was studied in 91 patients with bone tumors of various histology and 45 healthy donors. The levels of insulin-like growth factor (IGF-1) and somatotropic hormone were significantly higher in cases of chondrosarcoma and patients suffering osteogenic sarcoma in the late puberal period as compared to controls and cases of fibrous histiocytoma, giant-cell tumor, benign tumors and tumor-like lesions of the bone. The peak levels of IGF-1, somatotropic hormone and insulin were registered in osteogenic sarcoma patients who developed pulmonary metastases either in the course or after the completion of combined treatment. Somatostatin level was significantly lower in patients with osteogenic sarcoma aged 11-20 years as compared to healthy adolescents, the lowest level being observed in adolescents suffering osteogenic sarcoma with metastases to the lungs. No relationship was established between total testosterone level, on the one hand, and those of IGF-1 and epidermal growth factor, on the other. A reverse correlation was established between concentrations of IGF-1 and total estradiol. The role of polypeptide growth factor antagonists in combined treatment of bone sarcomas is discussed.

[Status of steroid receptors in the uterus, placenta and fetal membranes]. / Sostoianie steroidnykh retseptorov v matke, platsente i plodovykh obolochkakh.

Receptors of the myometrium, placenta and fetal membranes have been examined during deliveries at different gestational ages. No direct correlation was seen between plasma levels of steroid hormones and steroid receptor concentrations in target organs.