Introduction

Ovarian cancer is one of the three major gynecologic malignancies and the most lethal among women worldwide (1). Surgery and chemotherapy based on platinum-based agents are the most commonly available protocols for the treatment of ovarian malignancies but the occurrence of drug-resistance and relapse of the malignancy in many cases could result in short-term survival of affected patients (2). Recently, there are several new drugs that have been tested in the clinical trials to evaluate their efficacy for the treatment of ovarian cancers (2). Furthermore, evaluation of the effect of various kinds of targeted therapy for the cure of epithelial ovarian cancer (EOC) is under active investigation (3). Among several markers that have been defined for EOC, DNA damage and repair system factors, molecules involved in the G1 cell cycle, PI3K pathway, MAPK pathway, and also growth factors attracted more attention for targeted therapy (3). Higher expression of epidermal growth factor receptors (EGFRs) in human ovarian cancers was found to be associated with shorter disease-free and overall survival. None of the designed EGFR inhibitors to date have shown promising results in clinical trials for successful treatment of patients with ovarian cancer. Unsatisfactory results were also obtained with the recombinant humanized monoclonal antibody, pertuzumab (Perjeta), that target specifically the extracellular domain of HER2 (4). The average 5-year relative survival rate for all types of ovarian cancer is about 47% (5). Thus, in the absence of a satisfactory ovary cancer cure protocol, the development of new drugs to improve the efficacy of cancer treatment and to increase the patient’s survival rate seems to be necessary.

One of the most substantial targets in human malignancy targeted therapy is EGFRs (6). This tyrosine kinase family group includes four closely related receptors: HER1/ErbB1, HER2/ ErbB2, HER3/ErbB3, and HER4/ErbB4, which have several important roles in the growth and development of human normal cells (6). The HER2 receptor is highly expressed in various types of malignancies and its overexpression is associated with poor prognosis, cell transformation, tumor progression, angiogenesis, and metastasis of malignant cells (7). Therefore, HER2 has been used as a target for specific therapy in several solid tumors including breast, gastric, ovarian, lung, prostate, and bladder cancers (8-12). Overexpression frequency of HER2 in invasive epithelial ovarian cancer was reported 1.8% to 76% (13). There are some reports about HER2-targeted therapy of human ovarian cancer and several possible benefits of HER2-targeted immunotherapy have been discussed (13). Conjugation of monoclonal antibodies with a cytotoxic drug (antibody-drug conjugates or ADCs) by reducing the dose of chemotherapeutic agents is a promising approach in the treatment of patients with cancer (14, 15). These chimeric structures can deliver conjugated drugs to the targeted cells more selectively and efficiently (16). Alternatively, the antibody moiety of ADCs could be replaced with a low molecular weight affibody molecule. Affibodies are a new class of engineered affinity proteins that originated from the B-domain of the immunoglobulin binding region of staphylococcal protein A (17). Due to several advantages including low molecular weight, robustness, and thermostability, affibody is a valuable peptide molecule for targeted drug therapy and also tumor imaging (18-20). Affibody Z_HER2 has a great affinity for the HER2 receptor and previously has been used for the delivery of various anti-cancer drugs to HER2-overexpressed cancerous cell lines (20-22). Our previous in vitro study indicated that idarubicin-Z_HER2 affibody conjugate could be successfully used for specific ablation of HER2-overexpressed HN-5 cells originated from head and neck squamous cell carcinoma (HNSCC) (23). In the current study, the cytotoxic effects of idarubicin and idarubicin-Z_HER2 affibody conjugate on two different HER2-overexpressed cell lines, ovarian malignant cells (SK-OV-3) and breast cancer cells (SK-BR-3), were compared to MCF-7 as a low expressing HER2 cell line. 

Chemicals

Idarubicin was purchased from Selleckchem (Houston, United States). Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC), HisPur™ Ni-NTA resin, imidazole, and β-D-1-thiogalactopyranoside (IPTG) were obtained from Thermo Fisher Scientific (Massachusetts, United States). Ethylenediaminetetraacetic acid (EDTA), ampicillin, phenylmethanesulfonyl fluoride(PMSF), 3-[4, 5, dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT), and Dulbecco's modified Eagle's medium (DMEM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The human HER2-positive cell lines including ovarian carcinoma cell line (SK-OV-3), and breast cancer cell lines (SK-BR-3, and MCF-7) were purchased from the National Cell Bank of Iran (Tehran, Iran). All other chemicals were obtained from Merck (Darmstadt, Germany).

Expression and Purification of Z_her2 Affibody

Affibody Z_HER2 gene was synthesized by de novo gene synthesis and one cysteine codon was also added to the 5΄ end of the gene (24). The recombinant Z_HER2 affibody gene was inserted into a Champion™ pET302/NT-His plasmid from Invitrogen™ (Thermo Fisher Scientific) and cloned in competent E. coli BL21 cells. Z_HER2 Affibody was expressed, purified, and qualified as previously described elsewhere (23). Briefly, the cultured cells that contained recombinant plasmid were centrifuged and the bacterial pellet was sonicated in lysis buffer (25 mM Tris-HCl, 1.0 mM EDTA and 1.0 mM PMSF, pH 6.8) by using a probe sonicator (UP50H, Hielscher, Teltow, Germany). After centrifugation of the cell lysate, the supernatant was collected for affibody purification. Purification of recombinant Z_HER2 affibody was performed on the HisPur™ Ni-NTA resin (Thermo Fisher Scientific) according to the manufacturer's protocol. Finally, protein fractions were examined using coomassie brilliant blue (CBB) staining following SDS-PAGE to confirm the purity of the prepared affibody. Finally, affibody-containing fractions were pooled and protein concentration was determined using Bradford reagent (25). 

Construction of Idarubicin-Z_her2 Affibody Conjugate

The idarubicin-Z_HER2 affibody conjugate was constructed using sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) as a heterobifunctional crosslinker as previously described (23). The Sulfo-SMCC allows covalent conjugation of amine- and sulfhydryl-containing molecules through the formation of stable thioether bonds. Briefly, DMSO-dissolved Sulfo-SMCC diluted in conjugation buffer (PBS, 2.0 mM EDTA, pH 7.2) and added to the idarubicin solution (1.0 mg mL^-1) at a 2-fold molar excess. The reaction mixture was incubated for 30 min at room temperature and then affibody solution was added to the mixture in a 1:1 molar ratio. After 30 min, unreacted cross-linker and unconjugated idarubicin molecules were separated from Idarubicin-Z_HER2 affibody conjugate using a desalting column equilibrated with conjugation buffer. The product was concentrated using Amicon Ultra-15 centrifugal filter unit. Construction of idarubicin-Z_HER2 affibody conjugate was confirmed using a UV-Vis spectrophotometer (SPECORD 210 plus, Analytik Jena, Germany) and fluorescence spectrophotometer (LS-45 Fluorescence Spectrometer, Perkin-Elmer, USA) as previously described (23). 

Cell Viability Assay

The SK-BR-3 and MCF-7 cell lines were cultured in complete DMEM with high glucose, and the SK-OV-3 cell line was cultured in complete RPMI-1640 (with high glucose) under a humid atmosphere (37°C, 5% CO₂). The cell viability was measured by MTT colorimetric assay. Briefly, the cells in the logarithmic growth phase were seeded at approximately 1×10⁴ cells well⁻¹ into flat-bottom 96-well plates (Nunc, Roskilde, Denmark). The cells were treated with various concentrations of idarubicin (0.001-0.1 µM), Z_HER2 affibody (0.1-100 µM), and idarubicin-Z_HER2 affibody conjugate (0.01-50 µM) for 48 hours in triplicate. Then, the culture medium was replaced with fresh medium and MTT solution was added and incubation continued for another 4.0 hours. After washing the cells three times with PBS the pure DMSO (200 μL) was added. The absorbance of each well was read at 570 nm using a microplate reader (BioTek ELX800, USA).

Determination of Her2 Expression Level in Cancerous Cell Lines

Western blot analysis was performed for comparison of HER2 expression in three investigated cancerous cell lines. The cells at a density of 5×10⁵ were plated into the 10-cm dishes, incubated at 37°C overnight, and then homogenized in RIPA buffer (10 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 3% SDS, 1.0 mM EDTA and 150 mM NaCl) supplemented with protease inhibitors. The cell lysate was centrifuged at 10,000 g for 15 min at 4°C and protein concentration of the supernatant was determined using a bicinchoninic acid (BCA) protein quantitation kit. The samples (50 μg each) were electrophoresed on 12% SDS-PAGE, and then were electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, US). After blocking with 5% skimmed milk-containing TBS-T solution (Tris-buffered saline with 0.1% Tween 20) for 2 hr at room temperature, primary anti-HER2 antibody (Abcam, Cambridge, UK) was added and incubation was processed overnight at 4°C. After washing the blotted membrane three times with TBS-T and incubation with HRP-conjugated secondary antibody for 1 hr, protein bands were visualized with a Chemi-Doc gel documentation system (Bio-Rad, Hercules, CA, USA) by enhanced chemiluminescence (ECL) kit (Bio-Rad, Hercules, CA, USA). The intensity of bands was quantified by densitometry analysis of blots using Image J software and described as a ratio to β-actin expression.

 Statistical Analysis

All experiments were performed in triplicate. All data were processed using SPSS software version 15 (SPSS Inc., Chicago, IL., USA) and expressed as mean ± SEM. Data were analyzed by ANOVA to determine differences between experimental groups. A P-value