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Introduction

The use of antisense oligonucleotides (ASOs) to control functions in diseased cells was first recognized more than 20 years ago (1). ASOs, downregulating the expression of target molecules via mRNA silencing, exhibit tissue specificity and can be used to ameliorate the symptoms of various clinical conditions and to significantly improve the quality of life of patients (2–4). In the fight against malignant neoplasms, the strategy employing antisense therapy can block cancer genes, which produce malicious proteins; hence, ASOs have the potential to become a powerful weapon in this field (5). Moreover, it has been demonstrated that the use of a single treatment strategy against cancer is generally ineffective due to the multifactorial nature of this disease (6). Therefore, increased attention is being given to the combination of antisense therapy and chemotherapy (6,7).

Although the promising properties of ASOs have been recognized, the lack of methods applicable for ASO delivery avoiding the degradation of ASOs through the delivery routes is the major challenge in the wide utilization of these oligonucleotides in clinical practice. Thereby, the development of this new class of drugs has been delayed because of the difficulties in DNA, or RNA delivery, to cells (8), where ubiquitous nucleases may attack a cargo (9). Moreover, the transfer across membranes is also complicated due to the hydrophilic character and anionic backbone contained in the ASO structure (10).

Therefore, an effective delivery system is essential to protect a cargo against premature degradation. Regarding this issue, cationic liposomes have been demonstrated as a promising tool with which to protect ASOs from the extracellular matrix with potential to transport these molecules into cells (11–13). Liposomes can be safely administered to humans, because of their low toxicity and immunogenicity (14). However, this method still has the disadvantage of low efficacy of cargo transfer and therefore, improvement of transfer is crucial for their further utilization (15).

This study focused on the construction of cationic liposomes containing E2 in the lipid bilayer and antisense oligonucleotides designed against the human transcription factors estrogen receptor α (ERα) and β (ERβ) enclosed in their inner cavities. The E2 was employed to replace cholesterol, constituting a lipid bilayer of liposomes, to increase the efficiency of transporters. To study the effects of liposome-based transporters on proliferation, gene expression and redox status, a subclone of the well-characterized human estrogen receptor-positive breast cancer cell line MCF-7 was employed, as one of the E2-liposome transporters specifically inhibits MCF-7 cell proliferation and affects gene expression. Moreover, the levels of oxidative indicators were determined.

Materials and methods

Chemicals and pH measurement

The working solutions such as buffers or the standard solution of 17β-estradiol (E2) were prepared daily by diluting the stock solutions. Estradiol, oligonucleotides and other agents were purchased from Sigma-Aldrich (St. Louis, MO, USA) at ACS purity, unless noted otherwise. The antisense oligonucleotides against estrogen receptor α and β (Sigma-Aldrich) were designed and the sequences are shown in Table I.

Table I Antisense oligonucleotide sequences.

Table I

Antisense oligonucleotide sequences.

Antisense1α	5′-GACCATGACCATGACCCT-3′
Antisense1β	5′-TGTGCCTGGCTAGAGATC-3′

Preparation of liposomes

Liposomes were prepared following the lipidic thin-film hydration method. Briefly, 100 mg of cholesterol, 100 mg of 1,2-dioleoyl-sn-glycero-3-phosphorac-(1-glycerol) sodium salt and 100 mg of phosphatidylcholine were dissolved in chloroform (4.5 ml). A lipid film was obtained by rotary evaporation of solvent and the residual chloroform was blown out by nitrogen to remove the organic solvent until a thin film was formed. For encapsulation into liposomes, 2 ml of solution containing 17β-estradiol and ASOs was used. The samples were homogenized in ultrasonic bath Sonorex Digital 10P (Bandelin, Berlin, Germany) for 15 min. The homogenized mixtures were then heated and shaken for 15 min at 60°C using the Thermomixer comfort (Eppendorf AG, Hamburg, Germany). The samples were then washed several times with Britton-Robinson buffer (pH, 10.0) using Amicon 3K (Millipore, Billerica, MA, USA).

Determination of 17β-estradiol content in the liposomes

The hormone content in the liposomes was determined using AIA-Pack E2 sta assay (Tosoh Corp., Tokyo, Japan) according to the manufacturer’s instructions, using immunoanalyzer AIA 600 II (Tosoh).

Determination of total RNA content in the liposomes

The total amount of RNA in the liposomes was determined using an electrochemical analyzer 663 VA Stand (Metrohm, Herissau, Switzerland) using square wave voltammetry with a three-electrode system, according to our previous study (16). For each electrochemical analysis, 10 μl of antisense RNA solution was used.

MCF-7 cell culture

MCF-7 epithelial breast cancer cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY, USA), supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich), 1 mM sodium pyruvate, non-essential amino acids and 100 μg/ml penicillin and streptomycin (Sigma-Aldrich). Liposomes, containing antisense oligonucleotides at different concentrations, were applied directly into the medium to treat the cells and were labeled as L2, L3 and L4 and contained 1 μM and/or 10 μM of single or both types of oligonucleotides. L1 did not contain any ASOs and served as a control. The treatment of MCF-7 cells was conducted for 12 h.

Cell proliferation assessment using real-time cell-based assay (RTCA)

Real-time cell-based assay xCELLigence (Roche Applied Science and ACEA Biosciences, San Diego, CA, USA) consisted of four main components: RTCA analyser, RTCA station, RTCA computer with integrated software and disposable E-plate 16. First, the optimal seeding concentration for the proliferation and cytotoxic assays was determined. After seeding, the total number of cells (in 200 μl medium of each well in the E-plate 16, the attachment, proliferation and spreading of the cells) was monitored every 15 min. All the experiments were carried out for 65 h. The results are expressed as cell viability using the software of the manufacturers (Roche Applied Science and ACEA Biosciences).

Microscopy

Eclipse TS100 inverted phase contrast microscope (Nikon Instruments, Amsterdam, The Netherlands) with a ×20 magnification lens was used to view the cell cultures in cultivation flasks. The images were obtained directly using the C-5060 camera (Olympus, Tokyo, Japan).

RNA isolation and reverse transcription

High pure total-RNA isolation kit (Roche, Basel, Switzerland) was used to isolate RNA from the treated cells. The medium was removed and the samples were washed twice with 5 ml of ice-cold PBS. The cells were scraped off, transferred to clean tubes and centrifuged at 20,800 × g for 5 min at 4°C (Microcentrifuge 5417R; Eppendorf AG, Hamburg, Germany). After this step, lysis buffer was added and RNA isolation was carried out according to the manufacturer’s instructions. The isolated RNA was used for cDNA synthesis. The transcription of RNA (600 ng) was carried out using Transcriptor First Strand cDNA synthesis kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Prepared cDNA (20 μl) from total-RNA was diluted with RNase-free water to 100 μl and subsequently 5 μl was directly analyzed using the LightCycler 480 real-time PCR system (Roche Applied Science).

Quantitative polymerase chain reaction (q-PCR)

q-PCR was performed in triplicate using the TaqMan gene expression assay system with the LightCycler 480 real-time PCR system, and the amplified DNA was analyzed by the comparative Ct method using β-actin as an endogenous control for metallothionein MT1A, K-ras, TFF1 and NF-κB1 gene expression quantification. The primer and probe sets for β-actin (assay ID: Hs99999903_m1), MT1A (Hs00831826_s1), K-ras (Hs00364284_g1), NF-κB1 (Hs00765730_m1) and TTF1 (Hs00907239_m1) were selected from TaqMan gene expression assays (Life Technologies, Grand Island, NY, USA). The thermal profile for q-PCR was as follows: initial incubation at 50°C for 2 min followed by denaturation at 95°C for 10 min and then 45 cycles at 95°C for 15 sec and 60°C for 1 min. The total volume of the reaction mixture was 20 μl.

Preparation of MCF-7 cells for analyses

The harvested MCF-7 cells were frozen by liquid nitrogen to disrupt their structure. The frozen sample was further homogenized using ultrasonic homogenizer SONOPLUS mini20 (Bandelin electronic, Berlin, Germany). Subsequently, 1 ml of 0.2 M phosphate buffer (pH, 7.0) was added and the sample was homogenized for 5 min. The homogenate was further centrifuged using Microcentrifuge 5417R under the following conditions at 4°C for 15 min. Finally, the supernatant was filtered through a membrane filter (0.45-μm nylon filter disk; Millipore, Billerica, MA, USA) and analyzed.

Determination of the total protein content

The total protein content was determined for results standardization and was performed using SKALAB CBT 600T kit (Skalab, Svitavy, Czech Republic) according to the manufacturer’s instructions. For analysis, the BS-400 automated spectrophotometer (Mindray, Schenzhen, China) was used.

Determination of the metallothionein content with differential pulse voltammetry (DPV)

Differential pulse voltammetric (DPV) measurements were performed with the 747 VA Stand instrument connected to the 693 VA Processor and 695 Autosampler (Metrohm, Herissau, Switzerland), using a standard cell with three electrodes, according to our previous study (17).

High performance liquid chromatography coupled with electrochemical detector for determination of glutathiones

Contents of reduced and oxidized glutathiones (GSH and GSSG, respectively) were determined in the MCF-7 cell culture after liposome treatment using high performance liquid chromatography coupled with an electrochemical detector (HPLC-ED) system under the conditions used in our previous study (18).

Spectrophotometric determination of malondialdehyde (MDA)

Three hundred milliliters of the sample was mixed with 10 μl of 0.5 M butylated hydroxytoluene in 96% ethanol (v/v) and 310 μl of 20% trichloroacetic acid (v/v) prepared in 0.6 M HCl. After 20 min of incubation on ice, the mixture was centrifuged at 11,000 rpm for 15 min (Microcentrifuge 5417R). Subsequently, 400 μl of the supernatant was mixed with 800 μl of 30 mM thiobarbituric acid and the mixture was incubated at 90°C for 30 min. After cooling to room temperature, the absorbance of MDA was measured using the BS-400 spectrophotometer at a wavelength of 535 nm.

Descriptive statistics

Mathematical analysis of the data and their graphical interpretation were carried out using Microsoft Excel®, Word® and PowerPoint® (Microsoft, Redmond, WA, USA). The results are expressed as mean ± standard deviation (SD) unless noted otherwise. Statistical significance of the differences in gene expression was evaluated at P=0.05.

Results and Discussion

The main challenge facing the approach using the delivery of antisense oligonucleotides (ASO) to target tumor cells is how to keep them intact to ubiquitous nucleases. Moreover, the hydrophilic character and anionic backbone of antisense oligonucleotides reduce the probability of their transfer across membranes (10). Thereby, liposomes were employed to protect ASOs in our study. Zhong et al reported that, due to their properties, liposomes continue to evolve as a promising tool for the delivery of potentially useful therapeutic agents (11). Therefore, we suggested estradiol-based liposomes operating on the principle of natural affinity of hormone and estrogen receptor proteins. Cholesterol (precursor of 17β-estradiol or E2), which is commonly applied as the hydrophilic part of the lipid bilayers of liposomes (19), was replaced by E2. This complex offers similar hydrophilic properties and moreover the possibility to target the liposomes towards transcription factors (ERα and ERβ). Furthermore, it was shown that E2 enhances ASO uptake and therefore, the transfection efficiency due to the interaction with fundamental components of the plasma membrane, which causes the alteration in its physical properties (20).

The scheme of E2-based liposome interaction with cells is shown in Fig. 1. First, endocytosis occurs, since liposomes have been shown to be preferentially internalized through endocytosis (21). Acidification of endosomes causes release of ASOs into the cytoplasm and the formation of duplexes with mRNA encoding target protein due to Watson-Crick hybridization thus leading to mRNA degradation and/or inhibition of translation (10).

Figure 1 Scheme showing the antisense effect of antisense oligonucleotides encapsulated in liposomes on estrogen receptor protein formation. ASOs, antisense oligonucleotides.

17β-estradiol-based liposomes loaded with ASOs

Following the lipidic thin-film hydration method (22), we synthesized four different types of 17β-estradiol-based liposomes loaded with ASOs offering a powerful approach with which to selectively target and inhibit gene expression, critical in disease progression. As shown in Fig. 2A, liposomes L1 were prepared with no ASO cargo and were considered as the control. Other types of liposomes were prepared in the same manner, containing E2 in their lipidic envelopes, but moreover they were loaded with antisense ERα (Fig. 2B) named liposome L2, antisense ERβ (Fig. 2C) named liposome L3 and with both types of antisense sequences (Fig. 2D) named liposome L4. The antisense sequences used in our study are complementary to mRNAs encoding human transcription factors (ERα and/or β) and thus they are able to target and modulate the receptor’s responses.

Figure 2 Tested liposomes: (A) liposome with no antisense oligonucleotides, (B) liposome containing antisense oligonucleotides ERα, (C) liposome containing antisense oligonucleotides ERβ and (D) liposome containing both ERα and ERβ antisense oligonucleotides. (a) The content of E2 and antisense oligonucleotides determined by immunochemistry and electrochemistry, respectively. (b) The influence of individual liposomes on cell viability, expressing the cell growth of MCF-7 on the electrode surface under the effect of liposome treatment, where dark grey stands for control (no liposomes applied), light grey stands for a 1 μM concentration of liposomes applied and black for a 10 μM concentration of liposomes applied into the medium to obtain a final volume of 6 ml. Insets: microscopic images of (Ab) MCF-7 control cells and (Bb-Db) MCF-7 cells after treatment with the highest amount of liposomes.

To obtain the basic characteristics of liposome transporters, the hormone content was evaluated using immunochemistry and total RNA content using electrochemistry. It clearly follows from the results obtained that there was no distinct variation in 17β-estradiol content among the four types of liposomes ranging between 7–7.2 pg/ml with an average of 7.125 pg/ml (Fig. 2Aa–Da). Subsequently, electrochemistry was employed for determination of the total RNA amount in the estradiol-based liposomes and it was revealed that unlike L1 (Fig. 2Aa), the other liposomes (L2, L3 and L4, respectively) contained concentrations from 0.91 to 1.12 μM (average of 0.993 μM) of total-RNA (volume of solution, used for measurement: 100 μl). This finding approximately corresponds with the concentrations calculated prior to liposome synthesis (1 μM). Thus, we confirmed that our lipidic thin-film hydration method of synthesis works properly.

Antiproliferative effect of E2-based liposomes loaded with ASOs

The real-time cell-based assay (RTCA) was carried out to monitor the proliferation of MCF-7 epithelial breast cancer cells after application of the liposomes. The MCF-7 cells were derived from estrogen receptor-positive ductal carcinoma (23) and thus we chose this cell line for monitoring cell viability influenced by the estradiol-based liposomes. As shown in Fig. 2Ab, the application of increasing volumes of L1 (0, 90 or 900 μl) increased the proliferative effect (expressed as cell viability) of MCF-7 cells. This finding was expected due to the proliferative properties of MCF-7 cells in response to E2 as previously described by Lacroix and Leclercq (23) and Resende et al (24).

To reflect the real antiproliferative effect of ASOs, we subtracted the cell viability of the controls (L1) from the test samples (Fig. 2Bb–Db). Antisense against ERα was shown to decrease the proliferative ability of MCF-7 cells, since their proliferation is controlled by ERα-mediated gene regulation as described previously (25). While E2 stimulates proliferation via ERα, the signaling via ERβ inhibits proliferation and promotes apoptosis (26). It was shown in transfection studies in breast and colon cancer cells that the lack of ERβ proteins leads to increased cell proliferation both in culture and in vivo xenografts (27,28). Based on this information, ERβ was suggested to be a pro-apoptotic tumor suppressor (29) and thus alteration of protein translation subsequently elevated MCF-7 cell proliferation (Fig. 2Cb). Notably, the synergistic effect of both ASOs was shown to also have antiproliferative effects, but after a relatively long exposure time (Fig. 2Db). This phenomenon may be caused by a strong duplexing effect of antisense oligonucleotides complementary to mRNA encoding ERα translation and conversely only a weak complementary affinity to mRNA encoding ERβ. Therefore, despite the treatment with ASOs, the translation pathway was still able to produce ERβ proteins at threshold concentrations.

Liposomes are preferentially internalized through caveolae-dependent endocytosis (21), where the main role is played by acidification of the endocytic vesicle permitting release of antisense oligonucleotides rather than liposome membrane fusion. Therefore, in the micrographs of MCF-7 cells after liposome application, it can be observed that the cell size was increased due to endosome formation (insets in Fig. 2Ab–Db).

Influence of E2-based liposomes on MCF-7 cell selected gene expression

First, we decided to monitor the expression activity of the MT1A gene encoding metallothionein (MT) protein. MT is primarily responsible for metal sequestration and detoxification. It was previously shown that metallothionein expression is modulated also by E2 in in vivo studies using fish experimental models (30,31), where a positive correlation between MT1A expression and E2 levels was described in muscle tissue in contrast to liver and intestines. Our results revealed that the application of E2-based liposomes with no cargo elevated the MT1A expression by more than 60% (90 μl of L1 added) or 100% (900 μl of L1 added), when compared with the control (Fig. 3A), determined by expression of the gene without application of liposomes standardized to β-actin (labeled as 0 μl). The increase in gene expression may be attributed to a defense mechanism in response to oxidative stress caused by the presence of E2 (32). Importantly, after the application of liposomes containing ASOs (1 and 10 μM) against ERα protein (L2), it was shown that MT1A expression was downregulated (by >15 and/or 55%, respectively, Fig. 3B). This phenomenon points to a successful steric blockade of the ERα translational start site resulting in protein depletion and therefore downregulation of transcription related to E2-ER binding with subsequent effect on the MT1A gene. After the application of ASOs (10 μM) against ERβ (L3), a significant increase in expression of the metallothionein gene was observed (Fig. 3C). Proper translation of the ERβ protein seems to be important for the regulation of the MT1A gene. Although the role of ERβ is still under investigation in breast cancer, it was previously shown that ERβ acts as a tumor suppressor (33). Moreover, we offer evidence that ERβ downregulation directly influences the expression of the MT1A gene in MCF-7 cells. After treatment with the liposomes carrying both ASOs (L4), we observed a similar effect on MT1A (~75 and/or 85% downregulation after addition of 1 and 10 μM ASOs, respectively) as in the case of treatment with ASOs against ERα (Fig. 3D). Low threshold concentrations were required to trigger an antisense effect of ERα ASOs. On the other hand, ERβ functions were not significantly influenced when compared with ERα and thus the desired effect was observed.

Figure 3 Effect of various 17β-estradiol liposomes containing ER antisense sequences on MCF-7 cell culture. The expression levels of MT1A, NF-κB1, K-ras and TFF1 genes were observed. Schematic representations are connected to the gene expression and show the scheme of E2-based liposomes: (A) with no antisense oligonucleotide (control), (B) containing ERα ASO, (C) containing ERβ ASO and (D) containing both ERα and ERβ ASOs. Liposomes were applied at concentrations of 0, 1 and/or 10 μM into the medium. In the case of L1 (no ASOs) 0, 90 and 900 μl of the liposome solution was applied, corresponding to the amounts of E2-liposomes present in the case of 0, 1 and 10 μM ASOs in L2, L3 and L4. *Statistically significant differences between gene expression at P=0.05 level; n indicates no statistical significance. ASOs, antisense oligonucleotides.

Furthermore, the expression activity of the NF-κB1 gene was determined, as the blocking of NF-κB1 can cause tumor cells to stop proliferation, to die, or to become more sensitive to the action of chemotherapeutics (34). NF-κB1 transcription factor influences various molecules involved in oncogenesis such as matrix metalloproteinases (MMPs), their activators and/or inhibitors, cell adhesion molecules and angiogenic factors (35). In the present study, NF-κB1 expression was upregulated in MFC-7 cells after treatment with L1 (~100 and 120% after the application of 90 and 900 μl, respectively) (Fig. 3). This indicates the ability of E2 to influence MCF-7 cells undesirably. When compared with the application of ASOs, the highest effect was observed after treatment with ERα, where downregulation of ~5% was observed at 1 μM ASOs and ~20% at 10 μM ASOs (Fig. 3B). The effect of both ASOs was also shown to trigger downregulation of NF-κB1 by ~35% at 1 μM ASOs or 30% at 10 μM ASOs (Fig. 3D). The ASOs against ERα were found to influence NF-κB1, because a downregulation ~9% was observed in the case of 1 μM ASOs and further 10% in the case of 10 μM ASOs (Fig. 3C), when compared with the L1 liposome. Nevertheless, the best results were obtained using liposome L2 and L4. This phenomenon suggests that the presence of ASOs against ERα is more crucial for the downregulation of expression of NF-κB1 than ERβ. Based on these results, it can be concluded that ASOs may offer the possibility to inhibit NF-κB1 gene activity and thus increase the efficiency of chemotherapeutics applicable in breast cancer treatment.

Moreover, we determined K-ras gene expression, which was influenced in a similar way as NF-κB1. The expression of K-ras was elevated with increasings amounts of L1 applied (~70 and 125% at 1 and 10 μM of ASOs, respectively) (Fig. 3A). The application of ASOs exhibited a downregulatory influence on K-ras expression (Fig. 3B–D). The greatest effect was determined at 10 μM antisense against ERα (Fig. 3B). In the case of ASOs against ERβ, no significant differences between 0 and 1 μM application were observed at P<0.05; and moreover, a 10 μM concentration resulted in a downregulation of only 7.5% when compared to 0 μM (Fig. 3C). The ERα ASO influence on K-ras gene expression may serve as a potential tool to inhibit K-ras activity, as K-ras and ER proteins are linked in K-ras 4B-mediated cell transformation by p53-independent modulation of MDM2 functions as identified by Kato et al (36). Because of the fact that ER can be considered as one of the effectors of the Ras/Raf signaling pathway involving in tumorigenesis (37), the inhibition of estrogen receptor activity subsequently triggers the downregulation of the expression of K-ras. However, the relationship of these two factors is still not satisfactorily elucidated.

TFF1 gene encoding pS2 protein was also determined as the only one that was not downregulated by our antisense strategy. When comparing control liposome L1 (Fig. 3A) with liposomes loaded with ASOs (Fig. 3B–D), it is obvious that E2, but not ASOs, plays a crucial role in TFF1 expression due to the significant differences (P<0.05) between 0, 1 and 10 μM concentrations of ASOs observed for all liposomes including L1. The induction phenomenon of TFF1 expression was found to be primarily due to a response to estrogens and was mediated by the binding of the ER-E2 complex to a 13-bp near-palindromic ERE located 400 bases upstream of the TFF1 transcription start site (38,39). Hence, the presence of E2, which forms a complex with transcription factors, directly influences also the TFF1 transcription start site. As numerous studies have shown that TFF1 does not act as an oncogene in the mammary gland, but conversely, exerts a beneficial function during malignant processes in ER-positive breast tumors (38,40–42), our results point to the potential positive effect caused by our antisense strategy.

Oxidative stress determination after E2-based liposome application

Numerous signaling pathways, which are linked to tumorigenesis, can also regulate the metabolism of ROS through various mechanisms. As mentioned in a review by Gorrini et al, high ROS levels are generally harmful to cells, and the redox status of cancer cells is usually higher than that in healthy cells due to metabolic and signaling aberrations (43). For this reason, we assessed oxidative stress indicators, particularly glutathione, metallothionein and malondialdehyde to obtain a more detailed insight into the manner that E2-based liposomes act on MCF-7 cells at the molecular level.

It has been reported that elevated GSH levels are present in various types of tumors and thus neoplastic tissues are more resistant to chemotherapy (44). In malignant cells, an increased GSH level is associated with a proliferative response and is essential for cell cycle progression; yet, the molecular mechanism of how GSH modulates cell proliferation remains largely speculative. As shown in Fig. 4A, the addition of liposomes L1, L3 and L4 caused an elevation in GSH levels due to the presence of E2 in all cases and moreover ASOs against ERβ in the case of L3 and L4. These data also corroborate the RTCA results, where the presence of E2 and ASOs towards ERβ resulted in the increased proliferation of MCF-7 cells. On the other hand, the application of L2 (Fig. 4A) dramatically decreased the glutathione redox ratio (similar trend to the cell index of MCF-7). Hence, the application of E2-based liposomes carrying ASOs may be helpful to decrease the resistance to chemotherapy caused by high levels of GSH in tumor tissues.

Figure 4 Expression of oxidative stress markers determined in the MCF-7 cell line after treatment with the different types of liposomes. (A) GSH/GSSG ratio, (B) metallothionein and (C) malondialdehyde. Liposomes are indicated as follows: L1, liposome containing E2 with no antisense sequence; L2, liposome containing E2 with antisense sequence ERα; L3, liposome containing E2 with antisense sequence ERβ and L4, liposome containing E2 and both ERα and ERβ antisense sequences. Liposomes were applied at concentrations of 0, 1 and/or 10 μM into the medium to obtain a final volume of 6 ml. In the case of L1 (no ASOs), 0, 90 and/or 900 μl of liposome solution was applied corresponding to the amount of liposomes present in the case of 0, 1 and/or 10 μM ASOs in L2, L3 and L4. ASOs, antisense oligonucleotides.

Furthermore, metallothionein levels were assessed (Fig. 4B). Liposomes L2 and L4 were found to have a suppressive effect on protein formation. In the case of L2, an application of 0 μM ASOs resulted in 0.75 μg of metallothionein (standardized to 1 ml of total protein); 1 μM ASOs resulted in 0.72 μg of metallothionein to 1 ml of total protein and 10 μM ASOs resulted in 0.7 μg of metallothionein to 1 ml of total protein (Fig. 4B). The application of L4 at a concentration of 0 μM ASOs resulted in 0.71 μg of metallothionein to 1 ml of total protein; 1 μM ASOs resulted in 0.69 μg of metallothionein to 1 ml of total protein and 10 μM ASOs resulted in 0.46 μg of metallothionein to 1 ml of total protein. This corresponds to the results obtained from qRT-PCR. Surowiak et al reported that the elevated level of low-molecular-weight metallothionein in estrogen receptor-positive breast tumors may be explained by endoplasmic reticulum damage or by its malfunction and can be found typically in breast cancer cases with less favorable prognosis (41). It was shown that the effective application of ASOs may serve as a potential tool to mitigate the damage of endoplasmic reticulum and thus decrease the levels of metallothionein. Moreover, metallothionein was shown to be one of the potential co-factors causing the chemoresistance of tumors to platinum-based cytostatics and to anthracyclines (45–48). Hence, the downregulation of MT expression via ASO targeting of the MT1A gene may enhance tumor cell susceptibility to various chemotherapeutic agents.

Malondialdehyde (MDA) is one of the final decomposition products of lipid peroxidation and it is also formed as a product of the cyclooxygenase reaction in prostaglandin metabolism (49). It clearly follows from the results obtained that our novel strategy for antisense therapy was efficient in the case of MDA, as ASOs against ERα (10 μM application) showed a decrease in its level; >0.06 μg of MDA to 1 ml of total protein when compared to the control (Fig. 4C). Similarly, a slightly reducing effect was also observed in the case of the combination of both ASOs. As MDA reacts with double-stranded DNA to form mutagenic adducts and lipid peroxidation appears to be a major source of endogenous DNA damage in humans significantly contributing to tumor development (50), the reduction in lipid peroxidation end products may be one of the very significant effects of antisense therapy suggested by us.

In conclusion, we suggest four types of liposomes based on E2 placed in their lipid bilayer substituting commonly used cholesterol. As a cargo, antisense oligonucleotides towards estrogen receptor α and β were applied, and it was revealed that the liposomes synthesized by us showed a beneficial response in regards to a decrease in proliferation of MCF-7 cells. Moreover, it was shown that besides TTF1, the antisense strategy downregulated evaluated genes (MT1A, K-ras and NF-κB1) and also decreased the levels of oxidative stress indicators (MT, GSH/GSSG and MDA). We conclude that antisense ERα efficiently binds to ERα mRNA and thus the translation of nascent ERα protein is inhibited. Furthermore, the therapeutic strategy based on our ASOs entrapped in E2-based liposomes offers the possibility to enhance tumor susceptibility to chemotherapeutics via GSH oxidation and downregulation of MT expression through ASO targeting of the MT1A gene. Therefore, ASOs can be useful in cases where single treatment therapy does not show adequate results and chemosensitization of the tumor is required.

Acknowledgements

This study was supported by the Internal Grant Agency of the University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic (Project 4/2013/FVHE) and CEITEC CZ.1.05/1.1.00/02.0068.

References