Evaluation of the cytotoxic effect of GaFe 2 O 4 @Ag magnetic nanocomposite biosynthesized by Spirulina platensis on breast cancer cell line and evaluation of BAX, Bcl-2, CASP9 and MMP1 genes expression

Treating breast cancer is hardly achieved and �nding e�cient anticancer nanocompounds has gained great attention. In this work, the cytotoxic effect of GaFe 2 O 4 @Ag nanocomposite biosynthesized by Spirulina platensis on breast cancer cell line and expression of the BAX, Bcl-2, CASP9 and MMP1 (Matrix Metallopeptidase 1) genes were evaluated. Physicochemical features of the nanocomposite were determined using the FT-IR, XRD, SEM, TEM, EDX-mapping, VSM, Zeta potential, and DLS analyses. The cytotoxic effect of the nanocomposite for MCF-7 and HEK-293 cells was evaluated by the MTT assay. Flow cytometry analysis, Caspase-3 activation assay, and Hoechst staining were performed to evaluate the apoptosis induction potential of the nanocomposite. Further, the relative expression of the Bcl-2, BAX, and CASP9 genes was determined by quantitative PCR assay. The prepared nanocomposite was spherical with a size range of 35–60 nm. The hydrodynamic size and zeta potential of the nanocomposite were 328 nm and − 31.8 mv, respectively. GaFe 2 O 4 @Ag nanocomposite had a higher cytotoxic effect on breast cancer cells than normal human cells with the IC 50 of 18.6 and 220 µg/mL, respectively. Treating breast cancer cells with the nanocomposite induced apoptosis among 85.2% of cells, increased caspase-3 activity by 4.3 folds, and caused apoptotic nuclear changes. Also, GaFe 2 O 4 @Ag reduced the expression of the Bcl-2 and MMP1 by 1.3 and 0.6 folds and up-regulated the BAX and CASP9 genes by 2.7 and 2.65 folds, respectively. Our results revealed that GaFe 2 O 4 @Ag was highly cytotoxic for breast cancer cells via triggering apoptosis pathways and could be considered as a novel and e�cient agent against breast cancer, after further in-vivo


Introduction
With an estimated 2.3 million annual new cases and 680000 deaths, breast cancer remains the most prevalent and deathly cancer among women, worldwide [1]. Due to the high metastatic nature and drug resistance of breast cancer cells, treatment of this disease faces many di culties. Current chemotherapy approaches for treating breast cancer have some limitations and nding novel and e cient anticancer agents with fewer side effects seem to be crucially important.
In the search for nding novel anticancer agents, nanotechnology has received more and more attention. A large number of nanostructures have been investigated for the diagnosis, prediction, and treatment of several cancers [2][3]. Nanostructures not only could be used directly against cancer cells but also could be considered for the safe delivery of therapeutic agents [2]. Moreover, the fabrication of complex nanoparticles containing both therapeutic and drug delivery components could be a novel and promising approach for cancer chemotherapy.
Due to the magnetic properties, stability, and low cytotoxicity, Iron-based nanostructures, including nanoparticles (NPs) have shown promising potential to be used for site-directed drug delivery and also controlled release of therapeutic agents [4]. Iron oxide nanoparticles doped with different metal ions have shown considerable e ciency against a variety of cancer cells [5][6]. However, high e ciency, low cytotoxicity, and also easy and cost-effective synthesis of such compounds are the major challenges in this issue.
Silver has been traditionally known as a safe and e cient ion to be used in medicine. A large number of studies reported the therapeutic e cacy of silver NPs against several cancer cells [7]. In addition, Gallium, an element of the group IIIA of the periodic table, has shown several biomedical properties, including antimicrobial and anti-in ammatory features, as well as anticancer property [8][9].
Spirulina platensis is a prokaryotic microalga, order Cyanophyceae, division Cyanophyta (Cyanobacteria). It has a characteristic arrangement of multicellular cylinder-shaped trichomes in an open helix throughout its length. The helical gure of the trichomes is typical of the genus, but the size and length of the helix differ with species [10]. In 1981, the Food and Drug Administration con rmed "Spirulina is source of protein and comprises numerous vitamins and minerals. It can be legitimately marketed as a food or a food complement if it is exactly de ned and free from impurities and contaminants" and is categorized by the FDA as "Generally Recognized as Safe [11].
Green synthesis of metal NPs using the extracts of natural products is an eco-friendly, less expensive, and easy approach. Also, green synthesized metal NPs are commonly free of chemical contaminants and could be employed in medical and biological applications [12]. Algal extracts have been extensively used as biogenic and natural compounds for the stabilizing, growth termination, and reducing of metal NPs [13].
Due to the anticancer potential of Silver and Gallium and also the magnetic feature of Fe 2 O 4 , the current work aimed to fabricate GaFe 2 O 4 @Ag nanocomposite using Spirulina platensis extract and to evaluate its anticancer effect on breast cancer cells. Also, the expression of the genes involved with cell apoptosis pathways was investigated.

Preparation of Spirulina platensis extract
To prepare an aqueous extract of S. platensis, 0.5 g of algal dried powder was added to 25 mL of deionized water and maintained at a 55°C water bath for 20 min. The mixture was centrifuged for 10 min at 6000 rpm and the supernatant was passed through a Whatman paper (No. 1), and the puri ed extract was kept in the refrigerator for subsequent use [14].

Synthesis of GaFe 2 O 4 and GaFe 2 O 4 @Ag
At rst, 100 mL of Ga(NO 3 ) 3 solution (5 mM) and of FeCl 3 .6H 2 O solution (10 mM) were mixed and heated at 80°C for 60 min with continuous shaking. Then, 10 mL of NaOH (6 M) was added to the mixture to adjust the pH to 12.0. Next, the mixture was vigorously shaken at 100 rpm °C for 60 min and the resulting brownish particles were collected by centrifugation at 6000 rpm for 10 min. Obtained sediment was washed three times with distilled water and one time with ethanol to reduce the pH to 6. At last the sediment was dried at 100°C for 60 min [14].
For the synthesis of GaFe 2 O 4 @Ag nanocomposite, 50 mg of GaFe 2 O 4 NPs was added to 100 mL of distilled water and sonicated for 30 min to disperse the particles at room temperature. Then, 20 mg of AgNO 3 was added and sonicated for an additional 45 min at room temperature. Next, 10 mL of S. platensis extract was added to reduce Ag ions and the mixture was shaken for 24 h at room temperature in light. The fabricated GaFe 2 O 4 @Ag was harvested at 6000 rpm for 10 min. The sediment was washed three times with distilled water and one time with ethanol. The nal obtained sediment was dried at 100°C for 60 min. Cytotoxic effect of GaFe 2 O 4 @Ag for breast cancer and normal human cells The cytotoxic effect of GaFe 2 O 4 @Ag for MCF-7 (breast cancer cell line) and HEK-293 (normal human embryonic kidney cell line) was investigated using the 2-(4,5-dimethythiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. For culture maintenance, the cells were cultured on standard Dulbecco's modi ed Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 µg/ml streptomycin, and 100 units/ml penicillin solution at 37°C in the presence of 5% humidi ed CO 2 in air. After the preparation of the cell monolayers in the DMEM in 96-well plates (1×10 4 cell/well), the cells were exposed to different concentration of the nanocomposite including 12.5, 25, 50, 100, 200 and 400 µg/mL for 24h. The negative control cells were treated with Phosphate buffered saline (PBS) and positive control wells received cisplatin (12.5, 25, 50, 100, 200 and 400 µg/mL), as a standard anticancer drug. Next, 200 µL of MTT solution (0.5 mg/mL) was added to the wells, incubated for 4 h, and then, the medium was replaced by 200 µL of dimethyl sulfoxide (DMSO). The plates were shaken for 30 min and then, the optical density of each well was measured at 570 nm (UV-1601, Shimadzu, Kyoto, Japan). Finally, the 50% inhibitory concentration was calculated as follows [15]: (1)

Apoptosis induction
The frequency of apoptotic and necrotic cells in GaFe 2 O 4 @Ag treated and untreated MCF-7 cells was evaluated using the ow cytometry assay. The MCF-7 cells (about 5000 cells) were treated with the nanocomposite (at IC 50 concentration), incubated for 24 h, washed with PBS, and nally the annexin V-FITC PI [propidium iodide and Annexin V (Roche, Germany)] staining was then added to the cells and incubated for 15 min in dark. After staining, ow cytometry was employed to determine viable, early apoptotic, late apoptotic, and necrotic cells(Cy ow, UK).

Gene expression assay
The relative expression of the Bcl-2, BAX, CASP9 and MMP1, the regulatory genes associated with cell apoptosis and metastasis, in the GaFe 2 O 4 @Ag treated and control MCF-7 cells was investigated. After the preparation of the cell monolayer, the cells (about 5000 cells) were treated with the nanocomposite at IC 50 concentration for 24 h. Then, the cells were harvested, washed and their total RNA content was extracted using the TriZol reagent (Sigma-Aldrich). The cDNA was synthesized by the SinaClone cDNA synthesis kit (Iran), and the relative expression of the genes was measured using the SYBR green quantitative polymerization chain reaction (q-PCR). The temperature program for the q-PCR was as follows: 95°C for 30s, 45 cycles were followed at 95°C for 5 s, and 60°C for 30 s. The GAPDH gene was used as an internal control gene and the relative expression of the genes in treated and control cells was measured using the 2 −ΔΔCT method [16]. The assay was performed in triplicates and the sequence of the primers that were used in this study was presented in Table 1. The effect of GaFe 2 O 4 @Ag on the activity of caspase-3 was evaluated using the method described previously [14]. In Brief, the MCF-7 cells (about 5000 cells) were treated with the IC 50 concentration of nanocomposite for 24h, washed with PBS, and lysed using a cell lysis buffer. Then, DEVD-pNA was added to the cell supernatant (Sigma-Aldrich, CASP3C) and incubated for 2 h in dark room. Finally, the absorbance was measured at 405 nm using a microplate reader (Bio-Tek Instruments, Inc., USA).

Hoechst staining
The Hoechst staining was performed using the method described previously [15]. To perform the assay,

Statistical analyses
The assays were performed in triplicates and one-way ANOVA analysis was used to determine the signi cant difference between the nanocomposite treated and control groups. A p-value less than 0.05 was considered statistically signi cant.
The morphology and size distribution of the nanocomposite was evaluated using scanning and electron microscopy. The SEM images revealed that the majority of the particles were almost spherical and were in the size range of 35-60 nm. The TEM images of GaFe 2 O 4 @Ag revealed that the fabricated nanocomposite was in the nano-scale size range. Also, low aggregation of the particles was observed in the SEM and TEM images. The results were displayed in Fig. 3.
The VSM analysis of the nanocomposite showed that the maximum saturation magnetization of GaFe 2 O 4 @Ag was − 1emu/g, suggesting the proper magnetic potential of the synthesized nanocomposite (Fig. 4).
Moreover, the EDX-mapping analysis indicated that the synthesized nanocomposite contained Fe, Ga, O, and Ag atoms that con rms the purity of the GaFe 2 O 4 @Ag (Fig. 5). Further, the zeta potential and DLS analysis revealed that the zeta potential of GaFe 2 O 4 @Ag was − 31.8 mV and the hydrodynamic size of 328 nm. The results suggest the proper stability and low aggregation of the fabricated nanocomposite. The results were provided in Fig. 6.

Anticancer potential of GaFe 2 O 4 @Ag
The cytotoxic potential of GaFe 2 O 4 @Ag in human breast cancer cells and in normal human cells was compared. Also, the cytotoxicity of the nanocomposite in comparison with cisplatin was evaluated in MCF-7 cells. Our results revealed that the GaFe 2 O 4 @Ag was considerably more toxic for MCF-7 cells than HEK-293 cells. We observed that at concentrations of 12.5 µg/mL and higher, GaFe 2 O 4 @Ag signi cantly reduced the population of viable MCF-7 cells. In other words, at this concentration, less than 50% of MCF-7 cells survived that indicates the high toxicity of the nanocomposite for human breast cancer cells. The IC 50 of GaFe 2 O 4 @Ag in MCF-7 cell line was calculated18.68 µg/mL. In contrast, the IC 50 of GaFe 2 O 4 @Ag in HEK-293 cells was determined 200µg/mL which was signi cantly higher than MCF-7 cells. Moreover, the IC 50 of cisplatin for breast cancer cells was measured 41µg/mL (Fig. 7).

Effect of GaFe 2 O 4 @Ag on the expression of apoptosis genes
The expression of Bcl-2, BAX, and CASP9 in the MCF-7 cells treated with GaFe 2 O 4 @Ag and control cells was investigated. The results showed that treating with the nanocomposite reduced the expression of Bcl-2 cells by 1.3 folds that was signi cantly lower than control cells. In contrast, the expression of the BAX and CASP9 genes were signi cantly increased in GaFe 2 O 4 @Ag treated cells compared with the control. Our results showed that the expression of the BAX was up-regulated by 2.7 folds, CASP9 gene showed an increased expression of 2.65 folds and MMP1 gene expression levels decreased to 0.6 compared with the control group. The results were presented in Fig. 8.

Flow cytometry assay
A ow cytometry assay on GaFe 2 O 4 @Ag treated and control MCF-7 cells revealed that treating with the nanocomposite considerably induced apoptosis in breast cancer cells. We observed that the frequency of early and late apoptosis in control cells was 1.95 and 1.54%, respectively. In contrast, treating the cells with the nanocomposite signi cantly increased the frequency of late and early apoptosis to 12.7 and 72.5%. Overall, 3.49% of control cells showed apoptosis while treating with the nanocomposite induced apoptosis in 85.2% of breast cancer cells. Figure 9 shows ow cytometry analysis of the treated and control cells.

Caspase-3 activity
Evaluation of the activity of caspase-3 in GaFe 2 O 4 @Ag treated and control cells showed that exposure to the nanocomposite increased the activity of caspase-3 by 4.3 folds, which was considerably higher than control cells (Fig. 10).

Nuclear damages
To investigate the possible nuclear damages in nanocomposite treated cells, Hoechst staining was conducted. The results revealed obvious nuclear damages in the breast cancer cells following the exposure to GaFe 2 O 4 @Ag. The nuclear damages include chromatin fragmentation, the appearance of apoptotic bodies, and also chromatin condensation. The Hoechst staining images of the treated and control cells were displayed in Fig. 11.

Discussion
Breast cancer is considered the most prevalent and deathly cancer among women, worldwide and de nitive treatment of this disease is hardly achieved [1]. Treating breast cancer faces many di culties which is contributed to the poor prognosis, late diagnosis, metastasis, and drug resistance of cancer cells [19]. Therefore, nding novel anticancer drugs to treat breast cancer has been the aim of many studies. The use of nanocompounds has attracted many interests for disease diagnosis, drug delivery and cancer chemotherapy. However, the toxicity of these compounds is a major limitation of them to be used in biomedical applications. Biosynthesis of metal nanocomposites using the extracts of natural products could be considered an easy and eco-friendly approach to prepare less toxic nanocompounds [10]. Thus, in the current study, GaFe 2 O 4 @Ag nanocomposite was fabricated by S. platensis extract and its cytotoxic effect on breast cancer cells was evaluated. Further, the effect of the nanocomposite on the expression of Bcl-2, BAX, and CASP9 genes was investigated.
Physicochemical characterization of GaFe 2 O 4 @Ag revealed the proper synthesis of the nanocomposite, proper magnetic property, and lack of impurities. Moreover, the nanocomposite was almost spherical, in the nano-scale size range, and with good stability in an aqueous solution. The prepared nanocomposite contained Fe, O, Ga and Ag atoms. Silver is considered a safe and e cient metal that has been used in several biomedical applications, including cancer treatment. The anticancer potential of silver ions is mainly contributed to the direct interaction of the metal with cell components and also generation of radical oxygen species (ROS) that damage cell structures [20]. The antitumor activity of Gallium has also been reported. It was shown that Gallium has Iron mimicking activity and could disrupt cellular functions that are dependent on Iron metabolism [21].
Evaluation of the cytotoxic effect of GaFe 2 O 4 @Ag showed that the nanocomposite was highly cytotoxic for breast cancer cells with an IC 50 of 18.6µg/mL. Comparing the cytotoxicity of GaFe 2 O 4 @Ag with cisplatin showed that the nanocomposite was even more e cient than cisplatin against breast cancer cells. In contrast, normal human cells showed considerably lower susceptibility to the nanocomposite. These ndings revealed the e ciency of the nanocomposite to eradicate breast cancer cells and possibly lower toxicity and side effects for normal cells and human organs. Cancer cells have normally higher cell proliferation rate and higher membrane permeability and nutrient uptake than normal cells. Therefore, the higher toxicity of GaFe 2 O 4 @Ag for breast cancer cells seems to be contributed to the higher endocytosis of nanocomposite into the cancer cells [22]. As explained above, exposure to metal NPs could increase the generation of ROS molecules that damage cell structures and interfere with cell viability and proliferation [23][24].
Treating the MCF-7 cells with GaFe 2 O 4 @Ag considerably increased the population of apoptotic cells, compared with the control cells. Therefore, our results suggest that apoptosis induction is the main outcome of the exposure to the GaFe 2 O 4 @Ag. As a major outcome of the exposure to metal NPs, the generation of oxidative stress could imbalance cell redox hemostasis that results in the irreversible oxidative modi cation of cell lipids, nucleic acids, and proteins. Damages to cell components could initiate cell apoptosis pathway [25].
To evaluate the mechanism of apoptosis induction in breast cancer cells that were treated with GaFe 2 O 4 @Ag, the relative expression of Bcl-2, BAX, and CASP9 genes was investigated. We found that treating with GaFe 2 O 4 @Ag signi cantly attenuated the expression of Bcl-2 gene, while the BAX and CASP9 were considerably overexpressed. Bcl-2 codes for a protein that inactivates the BAX, a main proapoptotic protein in eukaryote cells. Also, Bcl-2 is associated with the inhibition of BAX/BAK oligomerization and thus, inhibits the activation of several apoptogenic molecules [26]. The Caspase-9 protein is associated with the cleaving and activation of other caspases that initiate the cell apoptosis pathway [27]. The decreased expression of Bcl-2 and overexpression of BAX and CASP9 in nanocomposite treated cells indicates the apoptosis induction activity of the fabricated nanocomposite.
In agreement with the molecular nding, a considerably increased activity of Caspase-3 protein in nanocomposite treated cells was observed that could be contributed to the increased expression of caspase genes and apoptosis initiation in the exposed MCF-7 cells. In accordance with the present results, previous studies have demonstrated that the expression of MMP1 was decreased under treatment silver nanoparticles on HaCaT keratinocyte cells [29], gallic acid-coated gold nanoparticles on dermal broblast cells [30] and gold nanoparticles on human dermal broblasts [31].
In agreement with other ndings, the Hoechst staining assay revealed the proapoptotic morphological characteristics, including DNA fragmentation and the appearance of apoptotic bodies. This nding complies well with the ow cytometry and gene expression analyses that suggest apoptosis induction, as the major outcome of exposure to GaFe 2 O 4 @Ag in breast cancer cells.   Viability of (a) HEK293 and (b) MCF-7 cells after treating with the GaFe 2 O 4 @Ag nanocomposite. c)