Irreversible inhibition of estrogen receptor α signaling and the emergence of hormonal resistance in MCF7 breast cancer cells induced by DNA damage agents.

Combining chemotherapy and hormone therapy is a prevalent approach in breast cancer treatment. While the cytotoxic impact of numerous chemotherapy drugs stems from DNA damage, the exact role of these DNA alterations in modulating estrogen receptor α (ERα) machinery remains elusive. The present study aimed to analyze the impact of DNA damage agents on ERα signaling in breast cancer cells and assess the signaling pathways mediating the influence of DNA damage drugs on the ERα machinery. Cell viability was assessed using the MTT method, while the expression of signaling proteins was analyzed by immunoblotting. ERα activity in the cells treated with various drugs (17ß-estradiol, tamoxifen, 5-fluorouracil) was assessed through reporter gene assays. In vitro experiments were conducted on MCF7 breast cancer cells subjected to varying durations of 5-fluorouracil (5-FU) treatment. Two distinct cell responses to 5-FU were identified based on the duration of the treatment. A singular dose of 5-FU induces pronounced DNA fragmentation, temporally suppressing ERα signaling while concurrently activating AKT phosphorylation. This suppression reverses upon 5-FU withdrawal, restoring normalcy within ten days. However, chronic 5-FU treatment led to the emergence of 5-FU-resistant cells with irreversible alterations in ERα signaling, resulting in partial hormonal resistance. These changes mirror those observed in cells subjected to UV-induced DNA damage, underscoring the pivotal role of DNA damage in shaping estrogen signaling alterations in breast cancer cells. In summary, the results of the present study suggested that the administration of DNA damage agents to cancer cells can trigger irreversible suppression of estrogen signaling, fostering the development of partial hormonal resistance. This outcome may ultimately impede the efficacy of combined or subsequent chemo- and hormone therapy strategies.

Honokiol inhibits the growth of hormone-resistant breast cancer cells: its promising effect in combination with metformin.

Background and purpose: Primary and metastatic breast cancers still represent an unmet clinical need for improved chemotherapy and hormone therapy. Considerable attention has been paid to natural anticancer compounds, especially lignans. The study aimed to evaluate the activity of several lignans against breast cancer cells and assess the effect of leading lignans on signaling pathways in combination with metformin. Experimental approach: Human breast cancer cell lines MCF7 (hormone-dependent), MDA-MB-231, and SKBR3 (hormone-independent) were used. A hormone-resistant MCF7/hydroxytamoxifen (HT) subline was obtained by long-term cultivation of the MCF7 line with hydroxytamoxifen. Antiproliferative activity was assessed by the MTT test; the expression of signaling pathway proteins was evaluated by immunoblotting analysis. Findings/Results: We evaluated the antiproliferative activity of lignans in breast cancer cells with different levels of hormone dependence and determined the relevant IC50 values. Honokiol was chosen as the leading compound, and its IC50 ranged from 12 to 20 µM, whereas for other tested lignans, the IC50 exceeded 50 µM. The accumulation of cleaved PARP and a decrease in the expression of Bcl-2 and ERα in MCF7/HT were induced following the combination of honokiol with metformin. Conclusions and implications: Honokiol demonstrated significant antiproliferative activity against both hormone-dependent breast cancer cells and lines with primary and acquired hormone resistance. The combination of honokiol with metformin is considered an effective approach to induce death in hormone-resistant cells. Honokiol is of interest as a natural compound with antiproliferative activity against breast cancers, including resistant tumors.

Secosteroid thiosemicarbazides and secosteroid-1,2,4-triazoles as antiproliferative agents targeting breast cancer cells: Synthesis and biological evaluation.

A convenient and selective approach to 13,17-secoestra-1,3,5(10)-trien-17-oic acid [N'-arylcarbothioamido]hydrazides and hybrid molecules containing secosteroid and 1,2,4-triazole fragments was disclosed and these novel types of secosteroids were screened for cytotoxicity against hormone-dependent human breast cancer cell line MCF-7. Most of secosteroid-1,2,4-triazole hybrids showed significant cytotoxic effect comparable or superior to that of the reference drug cisplatin. Hit secosteroid-1,2,4-triazole hybrids 4b and 4h were characterized by high cytotoxicity and good selectivity towards MCF-7 breast cancer cells. PARP cleavage (marker of apoptosis) and ERα and cyclin D1 downregulation were discovered in MCF-7 cells treated with lead secosteroid-1,2,4-triazole hybrid 4b. The synthesized secosteroids may be considered as new promising anticancer agents.

Exosomes are involved in the intercellular transfer of rapamycin resistance in the breast cancer cells.

Introduction: Resistance to chemotherapy and/or irradiation remains one of the key features of malignant tumors, which largely limits the efficiency of antitumor therapy. In this work, we studied the progression mechanism of breast cancer cell resistance to target drugs, including mTOR blockers, and in particular, we studied the exosome function in intercellular resistance transfer. Methods: The cell viability was assessed by the MTT assay, exosomes were purified by successive centrifugations, immunoblotting was used to evaluate protein expression, AP-1 activity was analyzed using reporter assay. Results: In experiments on the MCF-7 cell line (breast cancer) and the MCF-7/Rap subline that is resistant to rapamycin, the capability of resistant cell exosomes to trigger a similar rapamycin resistance in the parent MCF-7 cells was demonstrated. Exosome-induced resistance reproduces the changes revealed in MCF-7/Rap resistant cells, including the activation of ERK/AP-1 signaling, and it remains for a long time, for at least several months, after exosome withdrawal. We have shown that both the MCF-7 subline resistant to rapamycin and cells having exosome-triggered resistance demonstrate a stable decrease in the expression of DNMT3A, the key enzyme responsible for DNA methylation. Knockdown of DNMT3A in MCF-7 cells by siRNA leads to partial cell resistance to rapamycin; thus, the DNMT3A suppression is regarded as one of the necessary elements for the development of acquired rapamycin resistance. Conclusion: We propose that DNA demethylation followed by increased expression of key genes may be one of the factors responsible for the progression and maintenance of the resistant cell phenotype that includes exosome-induced resistance.

Targeting hormone-resistant breast cancer cells with docetaxel: a look inside the resistance.

Aim: The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen (HT) on their sensitivity to tubulin polymerization inhibitor docetaxel. Methods: The analysis of cell viability was performed by the MTT method. The expression of signaling proteins was analyzed by immunoblotting and flow cytometry. ERα activity was evaluated by gene reporter assay. To establish hormone-resistant subline MCF7, breast cancer cells were treated with 4-hydroxytamoxifen for 12 months. Results: The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen, and the resistance index was 2. Increased Akt activity (2.2-fold) and decreased ERα expression (1.5-fold) were revealed in MCF7/HT cells. The activity of the estrogen receptor α was reduced (1.5-fold) in MCF7/HT. Evaluation of class III ß-tubulin expression (TUBB3), a marker associated with metastasis, revealed the following trends: higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells (P < 0.05). The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells (MCF7/HT < MCF7 < MDA-MB-231, approximately 1:2:4). High TUBB3 expression strongly correlated with docetaxel resistance: IC50 value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells, whereas resistant MCF7/HT cells were the most sensitive to the drug. The accumulation of cleaved PARP (a 1.6-fold increase) and Bcl-2 downregulation (1.8-fold) were more pronounced in docetaxel-treated resistant cells (P < 0.05). The expression of cyclin D1 decreased (2.8-fold) only in resistant cells after 4 nM docetaxel treatment, while this marker was unchanged in parental MCF7 breast cancer cells. Conclusion: Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising, especially for cancers with low TUBB3 expression.

Antiproliferative effects of 13α/ß-steroids on triple-negative MDA-MB-231 breast cancer cells: unraveling intracellular signaling without ERα

Abstract This study aimed to investigate the activities of novel 20(R)-3,20-dihydroxy-19-norpregn-1,3,5(10)-trienes (kuz7 and kuz8b) of natural 13ß- and epimeric 13α-series against triple-negative MDA-MB-231 breast cancer cells. High antiproliferative activity of synthesized compounds kuz8b and kuz7 against MDA-MB-231 triple-negative cancer cells was revealed. The steroid kuz7 of natural 13ß-configuration was more active against MDA-MB-231 cells than the 13α-steroid kuz8b. Cell cycle analysis revealed common patterns for the action of both tested compounds. The number of cells in the subG1 phase increased in a dose-dependent manner, indicating induction of apoptosis, which was also verified by PARP cleavage. In contrast, the number of cells in the G0/G1 phase decreases with increasing compound concentration. Steroid kuz7 at micromolar concentrations reduced the expression of GLUT1, a glucose transporter. High efficacy of the combination of kuz7 with biguanide metformin was shown, and synergistic effects on MDA-MB-231 cell growth and expression of the anti-apoptotic protein Bcl-2 were revealed. According to the obtained results, including the high activity of kuz7 against triple-negative cancer cells, the detected induction of apoptosis, and the decrease in GLUT1 expression, 13ß-steroid kuz7 is of interest for further preclinical studies both alone and in combination with the metabolic drug metformin

Indoline-5-Sulfonamides: A Role of the Core in Inhibition of Cancer-Related Carbonic Anhydrases, Antiproliferative Activity and Circumventing of Multidrug Resistance.

The overexpression and activity of carbonic anhydrase (CA, EC 4.2.1.1) isoforms CA IX and CA XII promote the accumulation of exceeding protons and acidosis in the extracellular tumor environment. Sulfonamides are effective inhibitors of most families of CAs. In this study, using scaffold-hopping, indoline-5-sulfonamide analogs 4a-u of the CA IX-selective inhibitor 3 were designed and synthesized to evaluate their biological properties. 1-Acylated indoline-5-sulfonamides demonstrated inhibitory activity against tumor-associated CA IX and XII with KI values up to 132.8 nM and 41.3 nM. Compound 4f, as one of the most potent inhibitors of CA IX and XII, exhibits hypoxic selectivity, suppressing the growth of MCF7 cells at 12.9 µM, and causes partial inhibition of hypoxia-induced CA IX expression in A431 skin cancer cells. 4e and 4f reverse chemoresistance to doxorubicin of K562/4 with overexpression of P-gp.

Design, Synthesis and In Vitro Investigation of Cabozantinib-Based PROTACs to Target c-Met Kinase.

(1) Background: This investigation aimed at developing a series of c-Met-targeting cabozantinib-based PROTACs. (2) Methods: Purification of intermediate and target compounds was performed using column chromatography, in vitro antiproliferation activity was measured using a standard MTT assay and a c-Met degradation assay was performed via the immunoblotting technique. (3) Results: Several compounds exhibited antiproliferative activity towards different cell lines of breast cancer (T47D, MDA-MB-231, SKBR3, HCC1954 and MCF7) at the same level as parent cabozantinib and 7-demethyl cabozantinib. Two target conjugates, bearing a VHL-ligand as an E3-ligase binding moiety and glycol-based linkers, exhibited the effective inhibition of c-Met phosphorylation and an ability to decrease the level of c-Met in HCC1954 cells at micromolar concentrations. (4) Conclusions: Two compounds exhibit c-Met inhibition activity in the nanomolar range and can be considered as PROTAC molecules due to their ability to decrease the total level of c-Met in HCC1954 cells. The structures of the offered compounds can be used as starting points for further evaluation of cabozantinib-based PROTACs.

Synthesis and biological activity of 21,22-cyclosteroids and their derivatives.

Synthesis of 21,22-cyclosteroids has been achieved starting from pregnenolone acetate. The key transformation was the Kulinkovich reaction of 17-vinyl steroids with esters. The resulting cyclopropanols were further subjected to three-membered ring-opening under various conditions including to base-, palladium or visible light-promoted isomerization and cross-coupling reaction. A number of steroidal Δ2-6-ketones and 3ß-hydroxy-Δ5-enes with functional groups at C-21 - C-23 have been synthesized via the 21,22-cyclosteroids. The antiproliferative and antihormonal activity of the obtained compounds on the cell lines of prostate (22Rv1) and breast (MCF-7) cancer was studied. The androgen receptor activity was assessed by reporter assay when the expression of signalling proteins was evaluated by immunoblotting. (20S,22R)-22-Acetoxy-21,22-cyclo-5α-cholest-5-ene with the moderate antiandrogenic potency revealed IC50 values of 18.4 ± 1.2 and 14.6 ± 1.4 µM against MCF-7 and 22Rv1 cells, respectively, and its effects on the expression of AR-V7, cyclin D1 and BCL2 were explored.

Synthesis, Molecular Docking, In Vitro and In Vivo Studies of Novel Dimorpholinoquinazoline-Based Potential Inhibitors of PI3K/Akt/mTOR Pathway.

A (series) range of potential dimorpholinoquinazoline-based inhibitors of the PI3K/Akt/mTOR cascade was synthesized. Several compounds exhibited cytotoxicity towards a panel of cancer cell lines in the low and sub-micromolar range. Compound 7c with the highest activity and moderate selectivity towards MCF7 cells which express the mutant type of PI3K was also tested for the ability to inhibit PI3K-(signaling pathway) downstream effectors and associated proteins. Compound 7c inhibited the phosphorylation of Akt, mTOR, and S6K at 125-250 nM. It also triggered PARP1 cleavage, ROS production, and cell death via several mechanisms. Inhibition of PI3Kα was observed at a concentration of 7b 50 µM and of 7c 500 µM and higher, that can indicate minority PI3Kα as a target among other kinases in the titled cascade for 7c. In vivo studies demonstrated an inhibition of tumor growth in the colorectal tumor model. According to the docking studies, the replacement of the triazine core in gedatolisib (8) by a quinazoline fragment, and incorporation of a (hetero)aromatic unit connected with the carbamide group via a flexible spacer, can result in more selective inhibition of the PI3Kα isoform.

Synthesis, biological evaluation, and in silico studies of potential activators of apoptosis and carbonic anhydrase inhibitors on isatin-5-sulfonamide scaffold.

Carbonic anhydrase IX is a promising target for the search for new antitumor compounds with improved properties. Using the molecular hybridization approach, on the basis of structures of a selective carbonic anhydrase IX inhibitor 3 and an activator of apoptosis 2 (1), a series of 1-substituted isatin-5-sulfonamides 5a-5u were designed and synthesized. The study of the inhibitory activity of isatin-5-sulfonamides showed the ability to inhibit I, II, IX, XII isoforms at nano- and micromolar concentrations. Docking of compounds 5e and 5k into the active site of II and IX carbonic anhydrase isoforms showed the coordination of sulfonamidate anions with zinc cations, as well as a number of additional hydrophobic interactions. The trifluoromethylthio derivative 5r suppressed the growth of tumor cells at low micromolar concentrations, maintaining activity on resistant lines and under hypoxic conditions. Immunoblotting of MCF7 cells treated with the 5r revealed its antiestrogenic activity and ability to activate apoptosis in tumor cells.

Effective synthesis of novel dihydrobenzisoxazoles bearing the 2-aminothiazole moiety and evaluation of the antiproliferative activity of their acylated derivatives.

An effective method for the synthesis of 8-aryl-4,5-dihydrothiazolo[4',5':3,4]benzo[1,2-c]isoxazol-2-amines was developed. This method includes the α-keto bromination of 3-aryl-6,7-dihydrobenzo[c]isoxazol-4(5H)-ones followed by the condensation of the obtained bromo derivatives with thiourea in acetonitrile. Using virtual screening, a series of acylated derivatives of the obtained compounds were selected as potential HSP90 inhibitors. These compounds were prepared and evaluated as antiproliferative agents against three cancer cell lines (A431, 22Rv1, and MCF-7). Compounds 8b, 8c and 8q exhibiting high antiproliferative potency against MCF-7 breast cancer cells with IC50 values ranging from 2.3 to 9.5 µM were chosen for in-depth evaluation. The selected compounds had remarkable effects on HSP90 client proteins, including steroid hormone receptors and the anti-apoptotic factor BCL2. The obtained compounds are of interest for anticancer drug development.

Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance.

Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.

Glucose starvation greatly enhances antiproliferative and antiestrogenic potency of oligomycin A in MCF-7 breast cancer cells.

Energy imbalance is one of the key properties of tumour cells, which in certain cases supports fast cancer progression and resistance to therapy. The simultaneous blocking of glycolytic processes and oxidative phosphorylation pathways seems to be a promising strategy for antitumor therapies. The study aimed to evaluate the effect of glucose starvation on the antiproliferative and antiestrogenic potency of oligomycin A against hormone-dependent breast cancer cells. Cell viability was assessed by the MTT test. Estrogen receptor alpha (ERα) activity was evaluated by reporter assay. mTOR, AMPK, Akt, and S6 kinase expression was assessed by immunoblotting. Glucose starvation caused multiple increases in the antiproliferative potency of oligomycin A in the hormone-dependent breast cancer MCF-7 cells, while its effect on the sensitivity of the second hormone-dependent cancer cell line, named T47D, was weak and limited. Glycolytic inhibitors, 3-bromopyruvate and 2-deoxyglucose, greatly enhanced the antiproliferative potency of oligomycin A in MCF-7 cells. Glucose starvation leads to remarkable activation of Akt in MCF-7 cells, whereas oligomycin A enhances its effect. The mTOR, S6 kinase, and AMPK signalling pathways are significantly modulated by oligomycin A under glucose starvation. Oligomycin A demonstrates more pronounced antiestrogenic effects under glucose starvation. Thus, glucose starvation and pharmacological inhibition of glycolysis are of interest for revealing the antitumor potential of macrolide oligomycin A against hormone-dependent breast cancers.

Secretion of Mutant DNA and mRNA by the Exosomes of Breast Cancer Cells.

Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes' ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the PIK3CA gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes' ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of PIK3CA) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes' ability to transfer their cargo into the heterologous cells.

Discovery of derivatives of 6(7)-amino-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides: novel, hypoxia-selective HIF-1α inhibitors with strong antiestrogenic potency.

In this article, we describe the synthesis of 3-phenylquinoxaline-2-carbonitrile 1,4-dioxides bearing cyclic diamine residues at positions 6 or 7; the synthesis is based on the nucleophilic substitution of halogens. All synthesized 6(7)-aminoquinoxaline-2-carbonitrile 1,4-dioxides 3-6 demonstrated higher cytotoxicity and hypoxia selectivity compared to the reference agent tirapazamine against breast adenocarcinoma cell lines (MCF7, MDA-MB-231). The structure and position of the diamine residue considerably affects the antiproliferative properties of the quinoxaline-2-carbonitrile 1,4-dioxides. The introduction of a halogen atom at position 7 in the quinoxaline ring of 4a considerably increases the cytotoxicity of compounds 5a and 6a under both normoxic and hypoxic conditions. However, the most hypoxia-selective derivatives were non-halogenated 7-aminosubstituted 3-phenylquinoxaline-2-carbonitrile 1,4-dioxides 3a-j. Of the 32 novel synthesized derivatives, approximately 20 of the 6(7)-amino-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides demonstrated high antiproliferative potency against wild type leukemia cells K562 and drug-resistant subline K562/4 with the expression of p-glycoprotein (p-gp) compared to the reference agent doxorubicin, which exhibited one order of magnitude lower activity towards K562/4 cells than towards K562 cells. Lead compounds 5a and 3f inhibited HIF-1α expression and activity and induced apoptosis in hypoxic tumor cells, which was confirmed by poly(ADP-ribose)polymerase (PARP) cleavage. Moreover, 5a and 3f showed strong antiestrogenic potencies in MCF7 breast cancer cells. Thus, the described series of quinoxaline 1,4-dioxides has high anticancer potential and good aqueous solubility. Therefore, these compounds are promising for further drug development of hypoxia-targeted anticancer agents.

Novel steroidal 1,3,4-thiadiazines: Synthesis and biological evaluation in androgen receptor-positive prostate cancer 22Rv1 cells.

A flexible approach to previously unknown spirofused and linked 1,3,4-thiadiazine derivatives of steroids with selective control of heterocyclization patterns is disclosed. (N-Arylcarbamoyl)spiroandrostene-17,6' [1,3,4]thiadiazines and (N-arylcarbamoyl)17-[1',3',4']thiadiazine-substituted androstenes, novel types of heterosteroids, were prepared from 16ß,17ß-epoxypregnenolone and 21-bromopregna-5,16-dien-20-one in good to high yields by the treatment with oxamic acid thiohydrazides. The synthesized compounds were screened for antiproliferative activity against the human androgen receptor-positive prostate cancer cell line 22Rv1. Most of (N-arylcarbamoyl)17-[1',3',4']thiadiazine-substituted androstenes exhibit better antiproliferative potency (IC50 = 2.1-6.6 µM) than the antiandrogen bicalutamide. Compounds 7d with IC50 = 3.0 µM and 7j with IC50 = 2.1 µM proved to be the most active in the series under study. Lead synthesized compound 7j downregulates AR expression and activity in 22Rv1 cells. NF-κB activity is also blocked in 7j-treated 22Rv1 cells. Apoptosis is considered as a possible mechanism of 7j-induced cell death.

The phenomenon of acquired resistance to metformin in breast cancer cells: The interaction of growth pathways and estrogen receptor signaling.

Metformin, a biguanide antidiabetic drug, is used to decrease hyperglycemia in patients with type 2 diabetes. Recently, the epidemiological studies revealed the potential of metformin as an anti-tumor drug for several types of cancer, including breast cancer. Anti-tumor metformin action was found to be mediated, at least in part, via activation of adenosine monophosphate-activated protein kinase (AMPK)-intracellular energy sensor, which inhibits the mammalian target of rapamycin (mTOR) and some other signaling pathways. Nevertheless, some patients can be non-sensitive or resistant to metformin action. Here we analyzed the mechanism of the formation of metformin-resistant phenotype in breast cancer cells and its role in estrogen receptor (ER) regulation. The experiments were performed on the ER-positive MCF-7 breast cancer cells and metformin-resistant MCF-7 subline (MCF-7/M) developed due to long-term metformin treatment. The transcriptional activity of NF-κB and ER was measured by the luciferase reporter gene analysis. The protein expression was determined by immunoblotting (Snail1, (phospho)AMPK, (phospho)IκBα, (phospho)mTOR, cyclin D1, (phospho)Akt and ERα) and immunohistochemical analysis (E-cadherin). We have found that: 1) metformin treatment of MCF-7 cells is accompanied with the stimulation of AMPK and inhibition of growth-related proteins including IκBα, NF-κB, cyclin D1 and ERα; 2) long-term metformin treatment lead to the appearance and progression of cross-resistance to metformin and tamoxifen; the resistant cells are characterized with the unaffected AMPK activity, but the irreversible ER suppression and constitutive activation of Akt/Snail1 signaling; 3) Akt/Snail1 signaling is involved into progression of metformin resistance. The results presented may be considered as the first evidence of the progression of cross-resistance to metformin and tamoxifen in breast cancer cells. Importantly, the acquired resistance to both drugs is based on the constitutive activation of Akt/Snail1/E-cadherin signaling that opens new perspectives to overcome the metformin/tamoxifen resistance of breast cancer.

Snail/beta-catenin signaling protects breast cancer cells from hypoxia attack.

The tolerance of cancer cells to hypoxia depends on the combination of different factors--from increase of glycolysis (Warburg Effect) to activation of intracellular growth/apoptotic pathways. Less is known about the influence of epithelial-mesenchymal transition (EMT) and EMT-associated pathways on the cell sensitivity to hypoxia. The aim of this study was to explore the role of Snail signaling, one of the key EMT pathways, in the mediating of hypoxia response and regulation of cell sensitivity to hypoxia, using as a model in vitro cultured breast cancer cells. Earlier we have shown that estrogen-independent HBL-100 breast cancer cells differ from estrogen-dependent MCF-7 cells with increased expression of Snail1, and demonstrated Snail1 involvement into formation of hormone-resistant phenotype. Because Snail1 belongs to hypoxia-activated proteins, here we studied the influence of Snail1 signaling on the cell tolerance to hypoxia. We found that Snail1-enriched HBL-100 cells were less sensitive to hypoxia-induced growth suppression if compared with MCF-7 line (31% MCF-7 vs. 71% HBL-100 cell viability after 1% O2 atmosphere for 3 days). Snail1 knock-down enhanced the hypoxia-induced inhibition of cell proliferation giving the direct evidence of Snail1 involvement into cell protection from hypoxia attack. The protective effect of Snail1 was shown to be mediated, at least in a part, via beta-catenin which positively regulated expression of HIF-1-dependent genes. Finally, we found that cell tolerance to hypoxia was accompanied with the failure in the phosphorylation of AMPK - the key energy sensor, and demonstrated an inverse relationship between AMPK and Snail/beta-catenin signaling. Totally, our data show that Snail1 and beta-catenin, besides association with loss of hormone dependence, protect cancer cells from hypoxia and may serve as an important target in the treatment of breast cancer. Moreover, we suggest that the level of these proteins as well the level of AMPK phosphorylation may be considered as predictors of the tumor sensitivity to anti-angiogenic drugs.