Functionalisation of Fe₃ O₄ nanoparticles by 2‐((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide enhances the apoptosis of human breast cancer MCF‐7 cells

Abstract

Cancer is a major cause of death. Thus, the incidence and mortality rate of cancer is globally important. Regarding vast problems caused by chemotherapy drugs, efforts have progressed to find new anti‐cancer drugs. Pyrazole derivatives are known as components with anti‐cancer properties. In here, Fe₃ O₄ nanoparticles were first functionalized with (3‐chloropropyl) trimethoxysilane, then 2‐((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide (P) was anchored on the surface of magnetic nanoparticles (PL). The synthesized nano‐compounds were characterized using Fourier transform infrared spectroscopy, X‐ray diffraction, scanning electron microscopy, Zeta potential, dynamic light scattering, and energy‐dispersive x‐ray spectrometry analyses. The cytotoxicity effect was evaluated using MTT assay, apoptosis test by Flow cytometry, cell cycle analysis, Caspase‐3 activity assay and Hoechst staining on MCF‐7 cell line. The high toxicity for tumor cells and low toxicity on normal cells (MCF10A) was considered as an important feature (selectivity index, 10.9). Based on results, the IC50 for P and PL compounds were 157.80 and 131.84 μM/ml respectively. Moreover, apoptosis inducing, nuclear fragmentation, Caspase 3 activity and induction of cell rest in sub‐G1 and S phases, were also observed. The inhibitory effect of PL was significantly higher than P, which could be due to the high penetrability of Fe₃ O₄ nanoparticles.

1 Introduction

Cancer is one of the most important causes of mortality and breast cancer is the second cause of mortality [1]. Breast cancer is originated from the breast tissue and could be invasive or non‐invasive. This cancer has an increasing incidence trend in developing countries and is the most common cancer type among women, accounting for nearly 30% of all cancers [2]. Different factors including, lifestyle and genetic mutations have role in the development of breast cancer. In developed countries, breast cancer is mainly observed in women over 63 years, however, in developing countries, women with breast cancer are mostly under 50 years. So, finding efficient methods to treatment of breast cancer is very important [3].

Pyrazole derivatives are compounds that are used in cancer treatment. They are heterocyclic compounds that are known as hopeful scaffolds in chemotherapy. They have a wide range of biological properties such as anti‐inflammatory, anti‐microbial and anti‐cancer. Today, the use of these compounds as anticancer agents is widely extended [4, 5, 6]. These derivatives are highly effective in inactivation of aurora kinase [7], as well as cyclin‐dependent kinase [8]. Structural inhibition caused by these compounds occurs due to their affinity to binding the ATP binding site of these enzymes. Since the central role of protein kinases in cell signalling and its outcomes in malignant lesions are well recognised, extensive efforts have been performed to use them in treatment of different cancers [9]. Inhibition of aurora kinase in cancer cells results in increased apoptosis [10], which is the most important non‐surgical approach for the treatment of cancer [11, 12]. The potential of different forms of pyrazole in design of aurora kinase inhibitors has been proved [13]. For example, the importance of Pyrazol‐4‐yl‐urea in inhibition of AUR A/B that leads to the inhibition of cancer cells growth is well shown [14].

The effect of different derivatives of pyrazole on inactivation of kinases such as B‐Raf, VEGR, PDGFR and AKT has also been reported [15, 16]. In addition, a study by Nossier et al. [17] demonstrated the effect of 1,3,4‐triarylpyrazole compound on inactivation of AKT1, AKT2, DRAF, EGFR and P38α kinases.

Thiosemicarbazones and their metal complexes have extensive applications such as anti‐fungal, anti‐viral, anti‐bacterial and anti‐cancer effects. Due to the presence of amide, imine and thione groups in thiosemicarbazones, these compounds are known as polydentate ligands [18, 19, 20].

Today, the design of some heterocyclic compounds as anti‐cancer agents is accessible and extensive efforts have been devoted to find new anti‐cancer drugs [21]. Metal complexes of thiosemicarbazone can affect different cancers via inhibition of ribonucleotide diphosphate reductase, a vital enzyme for DNA biosynthesis and cell division [22].

Reduction of normal cells sensitivity toward drugs is considered as one of the key problems in tumour treatment. Therefore, several nanoparticles have been used in drug‐delivery systems to increase the efficiency of the drugs [23]. In this case, magnetic nanoparticles including, Fe₃ O₄ are regarded as one of promising agents. Due to their high environmental compatibility, high paramagnetic characteristics with acceptable stability and targeted cytotoxicity, Fe₃ O₄ nanoparticles are widely used for targeted drug delivery [24]. They can also cause easier drug solubility [25], and exert their cytotoxicity via the production of reactive oxygen species (ROS) on cancer cells [26]. Fe₃ O₄ magnetic nanoparticles like other nanoparticulates such as liposomes, polymeric micelles, dendrimers and colloidal have been used in targeted cancer therapies. Magnetic nanoparticles based on their active ligand, bind to the receptors expressed on surface of tumour cells [27].

The aim of this study is to assess the cytotoxic effect of 2 ‐ ((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide (P) alone, and in conjugation with Fe₃ O₄ – (3‐chloropropyl) trimethoxysilane (PL) on the MCF‐7 cells.

2 Materials and methods

2.1 Synthesis of 2‐((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide derivatives (thiosemicarbazone) (P)

Pyrazole carbaldehyde (2 mmol) and thiosemicarbazide (2 mmol) were dissolved in 96% ethanol (20 ml) and were reflexed in 80–90°C. Progression of the reaction was followed using thin‐layer chromatography (petroleum ether: ethyl acetate 3:5). After completion of the reaction, the reaction mixture was added to ice powder. The produced sediment was washed with ethanol and dried at room temperature. The schematic illustration for the preparation of thiosemicarbazone derivative during the reaction of pyrazole carbaldehyde and thiosemicarbazide is shown in Fig. 1.

Fig. 1.

Reaction of pyrazole carbaldehyde and thiosemicarbazide to produce 2‐((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide

2.2 Preparation of Fe₃ O₄ magnetic nanoparticles

Fe₃ O₄ magnetic nanoparticles were synthesised by chemical co‐precipitation method as reported previously [28].

Typically, FeCl₂. 4H₂ O (3.174 g, 16 mmol) and FeCl₃. 6H₂ O (7.568 g, 28 mmol) were dissolved in 300 ml of degassed water. The solution was stirred vigorously under a N₂ atmosphere at 80°C for 1 h. Subsequently, NH₃. H₂ O (40 ml) was rapidly added into the reaction mixture to precipitate ferrous and ferric hydroxides, 2Fe(OH)₃. Fe(OH)₂. Then, the reaction mixture was stirred at 80°C for another 1 h under N₂ atmosphere leading to the formation of Fe₃ O₄ via elimination of four water molecules from mixed metal hydroxide [29, 30]. The chemical reaction is as follows:

$$\mathrm{F}(II)+2Fe(III)+8O{H}^{-}\to \mathrm{F}{\mathrm{e}}_3{\mathrm{O}}_4+4{\mathrm{H}}_2\mathrm{O}$$

Finally, the black product was separated by an external magnet, washed several times with hot water and dried at 80°C.

2.3 Preparation of chloropropyltriethoxysilane‐coated magnetite nanoparticles (Fe₃ O₄ @SiRCl)

At first, 2 g of the prepared Fe₃ O₄ and 2 ml of ammonium hydroxide 25% solution were added to dry ethanol (100 ml). Then 3‐chloropropyltriethoxysilane (5 ml) was added dropwise to the reaction mixture under vigorous stirring at 50°C. After 48 h, the product was magnetically separated, washed 2–3 times with ethanol and dried in an oven at 70°C for 8 h.

2.4 Immobilisation of drugs on Fe₃ O₄ @SiRCl (Fe₃ O₄ @SiRCl/P)

Fe₃ O₄ @SiRC l (1 g) was added into a solution of 2‐((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide in 50 ml of dimethylformamide (DMF). The mixture was sonicated for 20 min and vigorously stirred at 60°C for 48 h. Then, the solid material was magnetically separated, washed with DMF and ethanol and, dried at 70°C for 8 h.

2.5 Characterisation of Fe₃ O₄ @SiRCl/P nanoparticles (PL)

2.5.1 Scanning electron microscopy (SEM)

For assessing the morphology and particle size of the PL component, SEM instrument model VEGA\\TESCAN‐LMU (Czech Republic) was used. For sample preparation, first the sample was placed on a foundation and then a gold cover was added to it.

2.5.2 Transmission electron microscopy (TEM)

A carbon‐coated formvar film was used to prepare Fe₃ O₄ @SiRCl/P nanoparticle samples for TEM analysis. The samples deposited on formvar film were air‐dried and placed on a copper grid for imaging under a Zeiss‐EM10C TEM at 100 kV.

2.5.3 Energy‐dispersive X‐ray (EDX) spectrometry

EDX test was performed for the determination of chemical composition of PL and point analysis of elements using a VEGA\\TESCAN‐LMU instrument (Czech Republic). For this purpose, after magnification and recording the picture using the SEM instrument, a SEM‐EDX system was used to perform EDX analysis for determination of PL components.

2.5.4 X‐ray diffraction (XRD)

Crystal structure of the synthesised nanoparticle was determined via X‐ray powder diffraction pattern analyses using the Philips X'Pert MPD diffractometer (Co‐Ka X‐radiation, k  = 1.79 A°). The dried sample was subjected to an applied current of 20 mA and an accelerating voltage of 45 kV with Cu Kα radiation. Finally, the mean crystalline size of the nanoparticles was calculated by Scherrer's formula after applying the necessary corrections.

2.5.5 Fourier transform infrared spectroscopy (FTIR)

FTIR spectrophotometer (ABB Bomem MB‐100‐series) was used for assessing the functional group of P and PL compounds. For this purpose, the dried samples were mixed with potassium bromide (KBr) to produce pellet, at a ratio of 1:100, which were examined in a wavelength range of 500–4000 cm⁻¹.

2.5.6 Dynamic light scattering (DLS) and Zeta potential analysis

A Zetasizer Nano ZS90 instrument (Malvern Panalytical, UK) was used for measuring the size distribution of the nanoparticles by DLS and zeta potential to evaluate the stability of the nanoparticles.

2.6 Cell culture

In this study, the MCF‐7 and MCF‐10A cell lines were purchased from the Pasteur institute of Iran. MCF‐7 Cells were cultured in RPMI 1640 (Dacell, South Korea) culture medium containing NaHCO₃ 0.2 g, 1 ml pen/strep antibiotics (0.05 mg/ml penicillin G and 0.08 mg/ml streptomycin) and 10% fetal bovine serum (FBS) for each 100 ml of culture medium. The 25 cm² flasks were used for culture propagation and cultured cells were incubated at 37°C with 5% CO₂ and 95% humidity. After preparation of the cells with 80–90% confluency, the cells were harvested from the surface of the flask using 1 mL of Ethylenediaminetetraacetic acid (EDTA)/trypsin solution (0.25% trypsin and 0.001% EDTA), and after making sure that the cells are completely isolated, the culture medium containing 10% FBS was added. MCF10A cells were cultured in DMEM/Ham's F‐12 supplemented with 100 ng/ml cholera toxin, 20 ng/ml epidermal growth factor, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone and 5% chelex‐treated horse serum. The cells were cultured in a humidified incubator with 5% CO₂ at 37°C. After at least five passages and entering the cells in the exponential phase, they were stained with trypan blue and live cells were counted using a light microscope.

2.7 MTT aasay

The cytotoxicity effect of Fe₃ O₄ nanoparticles, P and PL compounds against cancer (MCF‐7) and normal cell lines (MCF10A) was assessed using MTT (3‐(4,5‐di‐methylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. To calculate the selectivity index (SI) of the PL compound, the equation: CC₅₀ (no cancer cells)/IC₅₀ (cancer cells) was used [31]. In brief, the cells with a population of 1 × 10⁵ were cultured in 96‐well plates and incubated at 37°C with 5% CO₂. After reaching appropriate confluency, different compounds with concentrations of 15.625, 31.25, 62.5, 125, 250 and 500 µM/ml were used to determine the cytotoxicity effect and IC₅₀ value during 24 h. Untreated cells were used as control. Then, the MTT solution was added and incubated for 4 h. After discharging compounds from the culture medium, 100 µL of Dimethyl Sulfoxide (DMSO) was added to each well to dissolve insoluble crystals of formazan. Finally, after 10 min, the optical density of the wells was measured at 570 nm using an Enzyme‐linked Immunosorbent Assay (ELISA) reader instrument (Biotech, USA).

2.8 Apoptosis assay by flow cytometer

Cell staining was performed using Annexin‐V/PI apoptosis detection kit (Roche, Germany) to determine the percentage of apoptotic cells in drug‐treated cells and compared with negative control. After treatment of cells with IC₅₀ of P and PL compounds for 24 h, the cells were trypsinated and centrifugation was performed at 15,000 rpm for 4 min (twice). After washing the obtained pellet using PBS, 100 µl of binding buffer was added to the collected pellets in a microtube. Afterward, 10 µl of PI and 5 µl of Annexin‐V were added to the microtube. Then, the content of the microtube was gently mixed and incubated for 10 min at room temperature. Finally, cell analysis was performed using BD FACSCalibur flow cytometer (BD Biosciences, USA).

2.9 Cell cycle analysis

After culturing the cells in 24‐well plates, the cells were treated with IC₅₀ concentration of P and PL for 24 h. Then, the cells were washed using PBS solution and fixed with 70% ethanol (45 min at 4°C). Cells were washed using PBS solution and centrifuged at 2000 rpm for 2 min and the supernatant was discharged. After that, the cells were treated with 50 µl of PBS, 100 µg/mL RNase and 200 µl of PI (50 µg/ml), and were investigated using a flow cytometer instrument (Biotech). The percentage of the cells in G1, S, G2, and sub‐G1 phases was assessed.

2.10 Caspase‐3 activity assay

The activity of caspase‐3 protease was determined based on the detection and breakage of the caspase specific substrate using a DEVD‐P‐nitroaniline (pNA) kit (St. Louis, MO, USA). Finally, released pNA was quantified using an ELISA reader (Biotech) instrument at 450 nm. For this aim, after treating the cells with IC₅₀ concentration of P and PL compounds for 24 h, the cells were treated with 30 μl of cell lysate, 150 μl of protease reaction buffer (50 mM HEPES, 1 mM EDTA and 1 mM Dithiothreito (DTT)) with pH = 7.2, and 20 μl of DEVD‐pNA (Caspase‐3 substrate). Then the microtubes were incubated at 37°C for 1 h. The component of each microtube was transferred to a 96‐well plates and the optical density of each well was read at 450 nm. Negative control cells were treated with 900 µl of DEME culture medium in addition to 100 µl of the FBS.

2.11 Hoechst staining

Hoechst staining (33242) was used to characterisation of cell morphology. At first, a 1 mg/ml stock solution of the Hoechst stain was prepared. MCF‐7 cells were treated with P and PL at IC₅₀ concentration and incubated at 37°C for 24 h. Then, the cells were washed with PBS (twice) and treated with trypsin. Centrifugation was performed at 12,000 rpm for 1 min. After that, 80% acetone was used to fix cells on slides, and the slides were kept in a refrigerator for 30 min. The cells were extracted and washed with PBS. In the final step, the cells were stained with Hoechst stain in darkness and evaluated using a fluorescence microscope.

2.12 Statistical analysis

One way analysis of variance (ANOVA) was used for statistical analysis using Graph Pad Prism 5.0 software. P <0.05 was considered as the statistical significance. All measurements in the present study were done in triplicates.

3 Results and discussion

3.1 Scanning electron microscopy

Fig. 2 illustrates the SEM image of the Fe₃ O₄ @SiRCl/P nanoparticles. Results showed that the prepared particles have similar morphologies and exhibit a spherical shape with nano‐dimensions of about 15 nm. The nanoparticle agglomeration is also visible in the SEM image. The use of coating agents such as polyphenols in nanoparticle synthesis can prevent the nanoparticle agglomeration. Therefore, the use of methods such as plant extract mediated synthesis in the synthesis of magnetic nanoparticles can be very important [32].

Fig. 2.

SEM image of Fe₃ O₄ @SiRCl/P nanoparticles

3.2 Transmission electron microscopy

Fig. 3 shows the TEM image of the synthesised Fe₃ O₄ @SiRCl/P nanoparticles. The image showed the nanoparticles had a spherical shape with nano‐dimensions of about 14–30 nm.

Fig. 3.

TEM image of Fe₃ O₄ @SiRCl/P nanoparticles

3.3 Energy‐dispersive x‐ray

Fig. 4 illustrates the EDX image of the Fe₃ O₄ @SiRCl/P nanoparticles.

Fig. 4.

EDX image of Fe₃ O₄ @SiRCl/P nanoparticles

The presence of Fe, Si, Cl, S, C, and O elements confirmed the success of Fe₃ O₄ @SiRCl/P compound.

3.4 X‐ray diffraction

The XRD pattern of Fe₃ O₄ @SiRCl/P nanoparticles is presented in Fig. 5. XRD pattern of the synthesised materials shows the typical reflection plans (220), (311), (222), (400), (422), (511) and (440) at 2θ  = 30.0, 35.5, 43.1, 52.9, 57.1 and 62.6° which indicate the face‐centered cubic phase of Fe₃ O₄ [33]. The broad peak at around 20° indicates the amorphous structure of silicate layers. The size of the synthesised Fe₃ O₄ @SiRCl/P nanoparticles were calculated from the most intense peak (311) by the Scherrer's equation: D  = kλ /β cosθ, where, D is the average crystalline size, k is the Scherrer constant (0.89), λ is the X‐ray wavelength used, β is the angular line width at half maximum intensity and θ is the Bragg's angle in degrees unit [32, 34]. The calculated crystalline size was found to be 9 nm.

Fig. 5.

XRD pattern of the synthesised Fe₃ O₄ @SiRCl/P sample

3.5 Fourier transform infrared spectroscopy

The FTIR spectra of Fe₃ O₄, 2‐((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide (P) and Fe₃ O₄ @SiRCl/P (PL) nanoparticles have been displayed in Fig. 6. The spectra exhibit two absorption peaks at 580 and 628 cm⁻¹ which were assigned to the stretching vibrations of the Fe–O bonds at the tetrahedral and octahedral sites in spinel structures, respectively [35]. The FT‐IR spectrum of the prepared nanoparticles reveals a peak at 792 cm^–1 which was assigned to the stretching vibrations of the C  = S bond [36]. The sharp band at 1068 cm^‒1 was assigned to the stretching frequency of Si–O [28]. The band at around 3427 cm^‒1 can be attributed to the N‐H vibrational mode of the pyrazole ring [37]. The results indicate the successful anchoring of 2‐((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide on the surface of nano‐sized Fe₃ O₄ @SiRCl.

Fig. 6.

FT‐IR spectrum of

(a) Fe3O4 nanoparticles, (b) 2‐((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide, (c) Fe₃ O₄ @SiRCl/P

3.6 Dynamic light scattering

Fig. 7 shows the evolution in the size of Fe₃ O₄ @SiRCl/P nanoparticles, assessed by DLS. DLS analysis proved a considerable change in the hydrodynamic size (d _H) of the Fe₃ O₄ @SiRCl/P nanoparticle (186.5 nm). The measurement of nanoparticle size using TEM images showed an average size of about 14–30 nm for Fe₃ O₄ @SiRCl/P nanoparticles (Fig. 3). The particle size measured by DLS was generally higher than the size of nanoparticles observed using TEM imaging. This difference in size, was due to the hydration layer surrounding the nanoparticles during the preparation for DLS analysis [38].

Fig. 7.

DLS of Fe₃ O₄ @SiRCl/P component

3.7 Zeta potential

The zeta potential value of Fe₃ O₄ @SiRCl/P nanoparticle was about −30/3 mV (Fig. 8). In general, nanoparticles with zeta potential values greater than positive 30 mV or less than negative 30 mV have a good physical stability [39].

Fig. 8.

Surface charge (zeta potential) of Fe₃ O₄ @SiRCl/P compound

3.8 MTT assay

Based on the results of [3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide] (MTT) assay, the IC₅₀ values for P and PL compounds against MCF‐7 cells were determined 157.80 and 131.84 μM/ml, respectively. The CC₅₀ for MCF10A cells was 1440.4 μM/ml. Results of MTT assay for the cells treated with Fe₃ O₄ nanoparticles, P and PL compounds are shown in Fig. 9. The rate of cells growth inhibition for both P and PL compounds was dose‐dependent and the inhibition enhanced as the concentration of P and PL was increased. The IC₅₀ value of PL significantly was lower than the P compound (p ≤ 0.005). Furthermore, an increased survival rate was observed in MCF10A cells at a low concentration of the compounds after treatment for 24 h (Fig. 10). The calculated SI against the MCF‐7 cell line was 10.9, while the SI values >2 can be recommended for developing anticancer drugs [40]. In a study performed by Sever et al. [41], the SI for four different compounds of thiosemicarbazone on the A549 cell line, were calculated 1.64, 1.65, 4.24 and 4.65 which were lower than the SI calculated in the present study. In the case of P compound, a significant decrease was observed in cellular proliferation rate at concentration of 125 μM/ml, while a significant decrease in cell proliferation rate for PL compound was observed at concentration of 62.5 μM/ml. The high toxicity of Fe₃ O₄ /thiosemicarbazone compared to thiosemicarbazone alone, on A549 cancer cells was demonstrated in a study by Habibi et al. [42]. The high toxicity of different pyrazole derivatives on MCF‐7, MB‐231 and MDA breast cancer cells lines has also been observed in the study by Czarnomysy et al. [43]. The cause of lower IC₅₀ observed for PL in comparison to P compound, could be the higher penetrability of PL compound and production of ROS by iron oxide nanoparticles in the structure of this compound [44].

Fig. 9.

Survival percentage of MCF‐7 cells after treating with different concentrations of Fe₃ O₄ nanoparticles, P and PL compounds based on μM/mL during 24 h. ***P < 0.005 means a significant difference in the IC₅₀ rate between MCF‐7 cells treated with P and PL compounds. IC₅₀ for P and PL compounds were observed 157.80 and 131.84 μM/mL, respectively. Furthermore, Fe₃ O₄ nanoparticulates present minor inhibitory effects against breast cancer cells

Fig. 10.

Dose–response of MCF10A after 24 h treatment with different concentrations of PL compound (CC₅₀  = 1440.4). Thus, an increased survival rate can be observed in MCF10A cells at low concentrations. Finally, the cytotoxic effect of higher concentrations of the PL compound was observed

3.9 Apoptosis test results

Figs. 11 and 12 show the apoptosis and necrosis in MCF‐7 cells in treatment with P and PL compounds after 24 h. In the present study, the late apoptosis rate (Annexin V⁺ PI⁺) among the cells treated with P compound was measured 15.6% and with PL compound 20.7%. Moreover, necrotic alterations were significant in treated cells; so that, necrosis rate (Annexin V⁻ PI⁺) for the cells treated with P and PL were 11.4 and 14.7%, respectively. It should be mentioned that the healthy cells are shown in the Q4 area, early apoptotic cells in Q3, late apoptotic cells in Q2, and necrotic cells in Q1 area (Fig. 11). Dot plots are representations of logarithmic Annexin V fluorescence versus PI fluorescence. Apoptosis is one of the main pathways that lead to cell death and is accompanied by nuclear fragmentation and chromatin condensation. Cancers cells often have defective apoptotic pathways and apoptosis induction is considered as an effective method for cancer treatment [45, 46]. Apoptosis induction using pyrazolic derivatives has been reported in several cell lines [47, 48]. In a research performed by Nitulescu et al. [14], the apoptotic effect of eight different derivatives of pyrazole was evaluated on HT‐29 cancer cell line, of which, the most early apoptosis rate was shown in 4e compound (5.69%). Moreover, the necrosis rate for the cells treated by this agent was 8.43%, which was more than the apoptosis rate in treated cells. It should be mentioned that no significant change was reported in the secondary apoptosis rate among the cells treated with different derivatives of pyrazole. In the mentioned study, apoptosis and necrosis rate were lower than the present study. In another study performed by Czarnomysy et al. [43], the apoptotic effect of different derivatives of pyrazole was evaluated on MCF‐7 cell line and the early and late apoptosis rate in treated cells were 17.2 and 64.9%, respectively. Moreover, no significant change was reported for the early apoptosis and necrosis rates in treated cells. In contrast, in the present study, an increase was observed in necrosis and late apoptosis rates. Increase in necrosis was not consistent with the mentioned study. The necrotic factors include: excessive production of ROS, decrease in glucose level, decrease in ATP and post‐translation modifications [44]. Apoptosis and necrosis mostly occur via similar stimulators and their early regulation also is performed by the same pathways. Apoptosis and necrosis together and in a complementary manner consequently facilitate cell degradation [49, 50]. Some causes of necrosis in studied MCF‐7 cells are the presence of caspase inhibitors, which have increased activity due to increase in caspase activity [44]. In addition, increasing ROS, which is an effective mechanism for nanoparticles and chemotherapy drugs in treating cancer, can prompt to ATP release from the cell. This release causes a change in the cellular death pathway from apoptosis to necrosis [51]. It should be mentioned that the low levels of ROS in cells will result in apoptosis, and its excessive result in necrosis via ATP releasing from the cell [44]. Maybe another cause of necrosis in the studied cells was the release of HMGB1 (Box1 Group Mobility High) from necrotic cells in the early stages of treatment with used compounds. Released HMGB1 had the ability to bind different types of kinases such as ERK, and AKT [52, 53, 54]. Probably one of the action mechanisms of studied compound, performed via affection of kinases and their inactivation. Binding of HMGB1 to the mentioned kinases can result in disruption in apoptosis pathway. It is recommended to use agents such as N‐acetyl‐L‐cysteine to reduce ROS and switch necrosis to apoptosis [55].

Fig. 11.

Effect of P and PL compounds in inducing apoptosis in MCF‐7 cells after 24 h

(a) Control cells, (b) Cells treated by IC₅₀ concentration of P component, (c) Cells treated by IC₅₀ concentration of PL component

Fig. 12.

Flow cytometry diagram of double‐staining with Annexin V and PI after treatment with IC₅₀ concentration of P and PL components (24 h)

3.10 Cell cycle analysis results

The results of cell cycle analysis were illustrated in Figs. 13 and 14. In MCF‐7 cells treated with P compound, cell population increased in G1 and sub‐G1 phases in comparison to untreated cells. This means that the cell cycle was stopped in P‐treated cells. In MCF‐7 cells treated with PL compound, cell population significantly increased in S and sub‐G1 phases (p ≤ 0.05), and cell population decreased in G1 phase in comparison to untreated cells. Results of cell cycle analysis showed that both compounds lead to an increase in cell arrest in sub‐G1 phase. However, increase in cell population in S phase was only observed in the cells treated with PL. Also the population of cells in the S phase reduced after treatment with P compound.

Fig. 13.

Cell cycle analysis of MCF‐7 cells treated with P and PL compounds after 24 h. Cells were fixed with ethanol, stained with propidium iodide and then cell cycle distribution was analysed by flow cytometry

(a) Control cells, (b) Cells treated with P compound, (c) Cells treated with PL compound

Fig. 14.

Percentage of the cell population in sub‐G1, G1, S and G2 phases after treatment with IC₅₀ concentration of P and PL compounds (24 h)

The research performed by Czarnomysy et al. [43], investigated the effect of different pyrazole derivatives on different phases of cell cycle in MCF‐7 cells. They reported an increase in cells population in S phase of the cell cycle. This finding was in concordance with the results of PL treatment. Moreover, by analysing the effect of three pyrazole derivatives on different phases of cell cycle in MCF‐7 cells, an increase in G0/G1 cell population and a reduction in S and G2 cell population due to using of all three studied derivatives was reported [56]. Stopping cell cycle in sub‐G1 and S phases is a result of nuclear fragmentation and loosing DNA, which could be occurred via apoptosis or necrosis route. However, nuclear fragmentation is mostly considered for apoptotic cell death and is a feature of apoptosis. In PL‐treated cells, the cell arrest in S and sub‐G 1 phases was higher than control and P‐treated cells. Presumably due to increase in penetrability and toxicity of PL compound via binding to iron oxide nanoparticles. It should be mentioned that the results of cells cycle analysis are completely in accordance with the results of PI Annexin V test.

3.11 Caspase‐3 activity assay

After treatment of MCF‐7 cells with IC₅₀ concentration of P and PL, a significant increase in caspase‐3 activity was observed in comparison to untreated cells (p ≤ 0.05). Caspase‐3 activity for cell treated with P and PL was 1.29 and 1.33 folds, respectively (Fig. 15). However, no significant difference was observed between P and PL compound. Caspases have central role in apoptosis. They are considered as suitable targets for treatment of diseases associated with uncontrolled cell proliferation, such as cancer and autoimmune diseases. Low transcription of caspase‐3 has been reported in breast cancer [57]. In the present study, a significant increase in activity of caspase‐3 was observed in cells treated with P and PL compounds. In a study conducted by Cazarnomysy and co‐workers the impact of various derivatives of pyrazole on the MDA‐MB‐231 breast cancer cell line was investigated. The least and the most caspase‐3 activity in treated cells was 11 and 30.6%. The highest level of caspase activity in the study of Cazarnomysy et al. [43] was consistent with the observed changes in the present study.‏ A 1.6‐fold increase in caspase‐3 activity was also observed in A549 cells treated with pyridine derivative of thiosemicarbazone/Fe₃ O₄ [42].‏

Fig. 15.

The activity of caspase‐3 which was evaluated by colorimetric method. Activity increased 1.29‐ and 1.33‐fold after treatment with P and PL compounds, respectively, (24 h)

Caspase‐3 plays its role in a wide range of pH that is slightly higher than the range activity of other caspases. This wide range shows that the caspase‐3 has a complete function in apoptotic situation of cells [58]. Both intrinsic and extrinsic apoptosis pathways can activate caspase‐3 that results in autoproteolysis, as well as cleavage and activation of other series of caspases family. As a result, quick and irreversible apoptosis will be started [37]. This function is often observed along with nuclear fragmentation that has also been observed in the present study.

3.12 Hoechst staining results

Hoechst staining was performed to confirm the cytotoxicity effect of P and PL compounds on MCF‐7 cells, and evaluation of morphology and structure of cells. Treated cells had fragmented nuclei and the nuclear fragmentation rate was slightly more in the PL‐treated cells than P‐treated. Results of Hoechst staining are presented in Fig. 16. Some properties such as chromatin condensation and appearing fragmented nuclei were obvious in staining. The present findings are consistent with those from a study by Habibi et al. [42] on nuclear fragmentation and chromatin condensation in A549 cells treated with pyridine derivative of thiosemicarbazone. Similar results were also observed in MCF‐7 cells treated with different derivatives of thiosemicarbazone in a study by Bai et al. [59].

Fig. 16.

Nuclear morphologic changes of MCF‐7 breast cancer cells after treatments for 24 h

(a) Untreated cells as control, (b) MCF‐7 cells after treatment with IC₅₀ concentration of P component, (c) MCF‐7 cells after treatment with IC₅₀ concentration of PL compound. The concentrations of P and PL were 157.80 and 131.84 μM/ml, respectively. Magnification folds × 400. Arrows indicate cells with nuclear condensation and fragmentation

4 Conclusion

The results of this study demonstrated an acceptable cytotoxicity of Fe₃ O₄ @SiRCl/P nanoparticles (PL) compound compared to the 2‐((pyrazol‐4‐yl) methylene) hydrazinecarbothioamide alone (P). This means that the Fe₃ O₄ nanoparticle increases the cytotoxicity of pyrazole derivative of thiosemicarbazone. Inducing of apoptosis, increasing of caspase‐3 activity and the cell cycle arrest in sub‐G1 phase was also observed in MCF‐7 cells treated with both P and PL compounds, which was more significant in using PL compound. This implies the importance of Fe₃ O₄ nanoparticle in PL structure.