Abstract
ASP was used to synthesize FeNPA. They were characterized by UV-vis spectroscopy, FT-IR, TEM, SEM, XRD and ZP. The aim of this study was to evaluate in vitro cytotoxic activity and antioxidant acitivities of FeNPA and ASP. The antioxidant properties were evaluated using DPPH, ABTS+ and H2O2 assays. FeNPA had higher antioxidant activity comparing to ASP according to DPPH (IC50: 3.48 μg/mL) and ABTS+ (60.52%) assays. Anti-cancer activities of FeNPA and ASP were investigated in breast cancer, melanoma and control cell lines. FeNPA was more cytotoxic than ASP in MCF-7, MeWo, CHL-1, and HEL 299 cells. FeNPA had shown that mitochondria induce apoptosis through stress in MDA-MB-231, and cells MeWo. ASP also induced apoptosis 2.23-fold in MCF-7 cells. Progesterone receptor gene expression showed a 10-fold increase in a hormone-dependent MCF-7 cell line in ASP, and FeNPA treatment. Expressions of BCL6, CXCL12, DNAJC15, RB1 and TPM1 in melanoma cancer cell lines were significantly increased in ASP and FeNPA administration. It had been shown that FeNPA regulates gene expressions that may be considered important in terms of prognosis in breast cancer and melanoma cell lines and it is suggested that gene expressions regulated by FeNPA are also evaluated in animal models in vivo.
1 Introduction
Nanotechnology allows to work scale in the 1-100 nm range and takes advantage of small structures for new properties and activities [1, 2, 3]. Since 1990s iron nanoparticles (FeNPS) draw much attention in nanotechnology, because of their high intrinsic reactivity of surface sites [4], biocompatibility, surface modifiability and high surface to volume ratio [5]. Iron nanoparticles can be synthesized by different chemical and physical methods such as sol-gel reactions, hydrothermal methods, polyol methods, microemulsion process, radiolysis, aerosol pyrolysis and laser pyrolysis [6]. Recently synthesizing iron oxide nanoparticles using green chemistry attract scientists because of their non toxic and eco friendly properties [7].
Iron oxide nanoparticles are widely used in various fields of science. Their usage in biomedical field such as anticancer, antimicrobial, larvicidial, antiplasmodial, targeted drug delivery and cancer therapy are remarkable. Besides, iron nanoparticles are used in lithium ion batteries, high performance CO gas sensors, biosensors and jet printing make use of iron nanoparticles [6,7]. However, in recent years the most prominent features of them are their antitumoral effects [8].
Asphodelus aestivus is a species from Liliaceae family. A. aestivus shows a wide spread in Turkey. In the previous study, our research group obtained, characterized and investigated antioxidant activity of silver nanoparticles from A. aestivus [9,10].
Cancer is one of the most important fatal disease. It is specified as the second most common disease which causes death. Millions of people are reported to die because of cancer [11, 12, 13]. Cancer treatment has emerged as a seperate issue in recent years due to the side effects [14]. Nano-technology may take care and companse some of the problems, so scientist are trying to use less toxic and useful natural compounds in nano dimensions [15, 16, 17].
In the whole world lung, breast, colorectal, stomach and liver cancers are most common ones. Breast cancer is the most common malignancy in women and is one of the three most prevalant cancers worldwide with lung and colon cancers [18]. A variety of methods and drugs have been discovered and used for their treatment [19, 20, 21]. MDA-MB-231 and MCF-7 are breast cancer cell lines derived from mammary gland tissue derived from the pleural effusion metastatic region (ATCC, 2016). MDA-MB-231 represents a basal-like subtype with a triple negative feature [22, 23, 24, 25].
Melagnoma is caused by malignant transformation of melonocyte cells responsible for the formation of melanin pigment [26]. MeWo is a human tissue-derived melanoma cell line of the skin tissue initiated by primer culture from the lymph node of the metastatic region. CHL-1, RPMI 7932 is a human malignant melanoma cell line derived from the skin tissue created by transefection of the human melanoma cell line. While MeWo cells morphologically have displayed fibroblast structure, CHL-1 cells have showed epithelial structure (ATCC, 2016). In the present study iron oxide nanoparticles of A. aestivus were prepared and FeNPA were characterized. The aim of the present research was to evaluate the in vitro cytotoxic and antioxidant activities both FeNPA and aqueous extract of A. aestivus and compare the differences caused by nanoparticles.
2 Materials and methods
2.1 Plant material
A. aestivus Brot. (Asphodelaceae) was collected from Kusadası (Aydın) on January 2014. The plant was identified by Prof. Dr. Bijen KIVCAK from Ege University Faculty of Pharmacy, Department of Pharmacognosy, Izmir, Turkey and a voucher specimen (No. 1520) is deposited in Herbarium of the Ege University, Faculty of Pharmacy.
2.2 Preparation of the plant extract
The aerial plant extract was prepared by infusion in the ratio of 5%. It was then filtered and the filtrate was concentrated using vacuum evaporator. A. aestivus aqueous extract (ASP) was obtained.
2.3 Synthesis of FeNPA
5 mL of A. aestivus water extract was mixed with 5 mL of 0.001 M aqueous FeCl3 solution in a 1 : 1 ratio. The mixture was incubated at 50°C-60°C for 20 min with shaking. The color development, which turns black from orange, had shown the formation of iron oxide nanoparticles. The UV spectrum of Fe particles solution was drawn. Therefore, mixture centrifuged at 5000 rpm for 30 min. The material was then stored at -80°C for 24 h and then lyophilized.
2.4 Characterization of nanoparticles
2.4.1 Ultraviolet-vis (UV-vis) spectroscopy
UV-vis spectra was recorded using Thermo Evalution Array UV-vis spectrophotometer (Evolution Array; Thermo Scientific, Waltham, MA, USA) in the range of 200-800 nm at room temperature. In order to carry out the experiment quartz cuvettes were used. The purpose of UV-vis spectra was to observe the formation of iron nanoparticles [7].
2.4.2 Fourier transform infrared spectroscopy (FT-IR)
To perform FTIR spectroscopy potassium bromide pellets were prepared for each experiment. This method was used to determine the stability and the attachment of biomolecules to the iron oxide ions. A Perkin-Elmer, Spectrum 100 spectrometer was used for the analysis in the range of 4000-280 cm−1 (Perkin Elmer Inc., Wellesley, MA, USA). According to FT-IR analysis of FeNPA and ASP, the bonds responsible from formation of iron oxide nanoparticles are detected. Among these analyses, we would determine the bonds responsible from formation of nanoparticles by comparison FT-IR results.
2.4.3 Transmission electron microscopy analysis (TEM)
Transmission electron microscopy (TEM) analysis was performed to get a priliminary idea about particle size and take a microscope image of nanoparticles [29]. The FeNPA images were taken by a Libra 120 transmission electron microscope (Carl Zeiss, Oberkochen, Germany). Electronic radiation was used to take images and it detects the electrons transported through the sample. 80-120 kV accelaration volt and in column OMEGA filter was used for the device.
2.4.4 Scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX)
This characterization method shows the size and shape of the nanoparticles. The morphology was determined by SEM. A SEM 250 FEG Quanta instrument was used for the determination. SEM 250 FEG Quanta instrument was used with an EDX detector (Oxford Azteck) to identify the composition of nanoparticles.
2.4.5 Thermal gravimetric analysis (TGA)
TGA experiments indicated the thermal stability of iron oxide nanoparticles [30]. A Perkin-Elmer Diamond TG/DTA (Thermogravimetric and Differential Thermal Analysis) was used for the experiments. Device was operated with a heating rate of 20°C/min and achiewed between 20°C-500°C temperature rate.
2.4.6 X-ray diffraction analysis (XRD)
The analyses were done at room temperature by the device Rigaku Ultima IV X-ray diffractometer (Rigaku Corp., Tokyo, Japan) to examine the differactions of nanoparticles. The device was operated at a voltage of 45 kV and copper potassium alpha radiation (40 mA) was used.
2.4.7 Particle size distribution and zeta potential
Nano-zs Zetasizer (Zetasizer-Nano-ZS, Malvern, UK) was used for particle size distribution and zeta potential. Particle size distribution and zeta potential are important to understand the homogeneity, stability and functionality of particles. High zeta potential shows how stable nanoparticle is [5]. Samples were diluted in distilled water. Particle size distribution and polydispersity index were examined at room temperature. A polydispersity index higher than 0.5 showed that the formation was not homogenous. Zeta potential was also evaluated at room 25°C using the angle 17°, with 78.5 dielectric constant. Electrical field was operated to 15 v/ cm.
2.5 Determination of antioxidant activities
2.5.1 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS+) assay
FeNPA and ASP were determined for their ABTS cation radical scavenging activity. 245 mM potassium persulphate solution and 7 mM ABTS were mixed. The mixture was incubated for 16 h. After incubation the solution was diluted until the absorbance at 743 nm becomes 0.7. The prepared solution and 100 μL sample were stirred and incubated at room temperature for 6 min. The absorbance was measured at 734 nm using UNIQCAM 8625 UV/Vis spectrophotometer [31]. Ethanol was used as blank whereas α-tocopherol was used as a standard.
2.5.2 1-1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay
The free radical scavenging activity was determined using the 1-1-diphenyl-2-picryl-hydrazyl (DPPH) 0.1 mL of 0.1 mM solution of DPPH was added to 3 mL of ASP and FeNPA of different concentration. The mixture was incubated at room temperature for 30 min and absorbance was measured at 517 nm using UNIQCAM 8625 UV/Vis spectrophotometer [31]. The percentages of scavenging activity values was calculated using the following formula:
where:
A0 = Absorbance of control
A1 = Absorbance of sample.
α-Tocopherol was used as a positive control.
2.5.3 Hydrogen peroxide scavenging assay (H2O2)
0.1 M, pH 7.4 phosophate buffered saline was added in hydrogen peroxide (40 mM) solution. 600 μL of hydrogen peroxide solution and 1 mL of the ASP and FeNPA containing samples of different concentration were rapidly mixed. The mixture was incubated at room temperature for 10 min. The absorbance was measured at 230 nm by UNIQCAM 8625 UV/Vis spectrophotometer against a blank (without hydrogen peroxide). The percentage of scavenging of hydrogen peroxide was calculated using the following formula. Quercetin was used as a positive control [32].
where:
A0 = Absorbance of control
A1 = Absorbance of sample
2.6 Cell lines
Cancer cell models were selected as breast cancer models MCF-7 and MDA-MB-231 and melanoma cancer models MeWo and CHL-1 and fibroblast cell models WI-38 and HEL 299 (control cell models). Cell lines were cultured and maintained in mediums (EMEM for MCF-7, MeWo, HEL-299, WI-38 cell lines, DMEM for CHL-1 cell line and L15 for MDA-MB-231 cell line) containing 1% L-glutamine supplemented with 10% inactivated FBS and 1% penicillin/ streptomycin and were maintained in a humidified incubator at 37°C with 5% CO2. Analysis of cell viability was carried with trypan blue test.
2.7 Cytotoxicity assay
To determine the cytotoxic effect of FeNPA and ASP on the MCF-7, MDA-MB-231, MeWo, CHL-1, WI-38 ve HEL 299 cell lines, cells were seeded in 96-well E-plate and treated with variable concentrations (3.13-100.00 μg/mL) of FeNPA and ASP. Cytotoxicity activity was achieved by normalize cell index using xCELLIGENCE software and IC50 values and analysis were determined by sigmoidal dose-response analysis.
2.8 Analysis of apoptosis
Apoptotic effects of the ASP and FeNPA on the cells were evaluated by AnnexinV-EGFP Apoptosis Detection Kit Biovision. Initially, 5 x 105 cells were treated with their IC50 doses of each for 72 h. 72 h later, cells were collected. Then washed and resuspended. AnnexinV-EGFP and propidium iodide (PI) were added, and the cells were incubated. After incubation, cells were examined by flow cytometry named BD Accuri C6 Flow Cytometer.
2.9 Gene expression analyzes
In breast cancer and melanoma cell models, effects of the FeNPA and ASP on gene expressions were assessed by real time online RT-PCR (LightCycler 480 II) by using RT2Profiler PCR Array Human Breast Cancer (Table 2) and Human Melanoma (Table 3) PCR Array, Qiagen Kits. Total RNA extraction and cDNA were performed according to the kit manual, by using RNeasy Mini Kit (Qiagen) and RT2 First Strand Kit cDNA Synthesis kit (Qiagen), respectively.
Determination of antioxidant activity, total phenolic and flavonoid content of FeNPA and ASP.
| DPPH(IC50) (μg/mL) | ABTS+(%) (μg/mL) | H2O2(%) (μg/mL) | |
|---|---|---|---|
| ASP | 31.65 ± 0.01a | 39.62 ± 0.02 | 55.86 ± 0.14 |
| FeNPA | 3.48 ± 0.01 | 60.52 ± 0.01 | 16.83 ± 0.06 |
a Results are mean ± SD of three replicate analysis
“PCR Array Human Breast Cancer” 96-well microplate gene design.
| ABCB1 | ABCG2 | ADAM23 | AKT1 | APC | AR | ATM | BAD | BCL2 | BIRC5 | BRCA1 | BRCA2 |
| A01 | A02 | A03 | A04 | A05 | A06 | A07 | A08 | A09 | A10 | A11 | A12 |
| CCNA1 | CCND1 | CCND2 | CCNE1 | CDH1 | CDH13 | CDK2 | CDKN1A | CDKN1C | CDKN2A | CSF1 | CST6 |
| B01 | B02 | B03 | B04 | B05 | B06 | B07 | B08 | B09 | B10 | B11 | B12 |
| CTNNB1 | CTSD | EGF | EGFR | ERBB2 | ESR1 | ESR2 | FOXA1 | GATA3 | GLI1 | GRB7 | GSTP1 |
| C01 | C02 | C03 | C04 | C05 | C06 | C07 | C08 | C09 | C10 | C11 | C12 |
| HIC1 | ID1 | IGF1 | IGF1R | IGFBP3 | IL6 | JUN | KRT18 | KRT19 | KRT5 | KRT8 | MAPK1 |
| D01 | D02 | D03 | D04 | D05 | D06 | D07 | D08 | D09 | D10 | D11 | D12 |
| MAPK3 | MAPK8 | MGMT | MKI67 | MLH1 | MMP2 | MMP9 | MUC1 | MYC | NME1 | NOTCH1 | NR3C1 |
| E01 | E02 | E03 | E04 | E05 | E06 | E07 | E08 | E09 | E10 | E11 | E12 |
| PGR | PLAU | PRDM2 | PTEN | PTGS2 | PYCARD | RARB | RASSF1 | RB1 | SERPINE1 | SFN | SFRP1 |
| F01 | F02 | F03 | F04 | F05 | F06 | F07 | F08 | F09 | F10 | F11 | F12 |
| SLC39A6 | SLIT2 | SNAI2 | SRC | TFF3 | TGFB1 | THBS1 | TP53 | TP73 | TWIST1 | VEGFA | XBP1 |
| G01 | G02 | G03 | G04 | G05 | G06 | G07 | G08 | G09 | G10 | G11 | G12 |
| ACTB | B2M | GAPDH | HPRT1 | RPLP0 | HGDC | RTC | RTC | RTC | PPC | PPC | PPC |
| H01 | H02 | H03 | H04 | H05 | H06 | H07 | H08 | H09 | H10 | H11 | H12 |
Melanoma PCR Array 48 well microplate gene design.
| ADAMTS18 | CLEC3B | LRP2 | NRP1 | RB1 | HPRT1 |
| A01 | A02 | A03 | A04 | A05 | A06 |
| AKAP12 | CXCL12 | LRRC2 | PCNA | SOCS2 | ACTB |
| B01 | B02 | B03 | B04 | B05 | B06 |
| ARPC2 | DNAJC15 | MME | PPP1R3C | SOCS3 | GAPDH |
| C01 | C02 | C03 | C04 | C05 | C06 |
| BCL6 | DPP4 | MMP1 | PPP2R4 | THBD | B2M |
| D01 | D02 | D03 | D04 | D05 | D06 |
| BIRC5 | ENC1 | MMP12 | PRDX2 | TNFRSF10A | HGDC |
| E01 | E02 | E03 | E04 | E05 | E06 |
| BRAF | GDF15 | MMP14 | PTEN | TP53INP1 | RTC |
| F01 | F02 | F03 | F04 | F05 | F06 |
| CDH3 | IRF8 | MMP2 | QPCT | TPM1 | RTC |
| G01 | G02 | G03 | G04 | G05 | G06 |
| CDH8 | LAMC2 | MMP9 | RASEF | RPL13A | PPC |
| H01 | H02 | H03 | H04 | H05 | H06 |
3 Results and discussion
Green synthesized FeNPA had been characterized by various methods. These methods are: ultraviolet-vis (UV-vis) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy analysis (TEM), scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX), thermal gravimetric analysis (TGA), X-ray diffraction analysis (XRD) and particle size distribution and zeta potential.
Basically, the characteristic color change during process from yellow to reddish brown showed the formation of FeNPA. In the next step UV spectrums for FeNPA and ASP were evaluated [33]. After the color change a peak occured at 250 nm and 450 nm which proves iron oxide nanoparticles presence (Figure 1) [30,34,35]. The FTIR spectrum was performed by A Perkin-Elmer Spectrum 100 Spectrometer. ASP and FeNPA spectrums were compared in Figure 2. ASP spectrum gave absorbance bands at 3397 cm-1, 2917.24 cm-1, 1618.74 cm-1, 1384.47 cm-1, 1076.66 cm-1 and 824.97 cm-1. Subsequent to formation spectrum had changed and shifts occured. The absorbance band at 1618.74 cm-1 which is responsible from C=C aromatic chain streching vibration of phenolic compounds shifted to 1611.75 cm-1. The bending vibration of phenols of –OH which gave band at 1384.47 cm-1 shifted to 1366.54 cm-1. C–O or C=O streching vibration formed by polysachharides which gave band at 1076.66 cm-1 shifted to 1041.78 cm-1. All of these shifts were due to a new formation: FeNPA. In addition to the shifts new absorption bands around 1000-1400 cm-1 (1449.67 cm-1, 1212.24 cm-1, 1041.78 cm-1) occured because of C=C, OH, C–O, C=O or C–O streching vibration [36]. One of the most important band was at 612.98 cm-1 occured beacuse of the Fe–O streching proved FeNPA transformation from ASP. [37]. In the next step TEM analysis was evaluated. TEM analysis provided information about general morphology. According to results, the particle size of FeNPA were about 20-25 nm (Figure 3). In order to acquire more detailed information about morphology, surface properties and elemental composition SEM/EDX analysis were used. Results indicated FeNPA had rough surfaces. They were cavity like structures as shown in Figure 4. As it was occured in Figure 5 analysis confirmed the presence of Fe and O [7]. An other characterization method TGA analysis was presented in Figure 6. The initial weight loss till 193°C was due to removal of moisture. Second step between 193.12°C-383.26°C may due to combustion of organic consituents, elimination of carbon groups. Last weight loss was because of the crystal structure of FeNPA [30,38,39]. The XRD pattern of FeNPA were shown in Figure 7. The 2θ peaks at 18.106°, 35.4° and 40.48° were attributed to crystal planes at (111), (220) and (311) respectively. The position and the relative intensity of the diffraction peaks match well with the standard XRD data for iron oxide nanoparticles (JCPDS file No. 19-0629) [3,30]. Finally, zeta potential analysis was carried out to prove the stability of FeNPA (Figure 8) The value (-21.2 mV) guides the stability [40,41]. ABTS+, DPPH and H2O2 scavenging activities were performed for both ASP and FeNPA. According to results FeNPA was more active than ASP by DPPH and ABTS+ methods. On the other hand results of H2O2 scavenging assay showed that ASP was more active than FeNPA (Table 1). Mostly the FeNPA showed more activity than ASP which gave rise to thought that FeNP s could used in many diseases caused by oxidative stress.
UV–visible spectra of the iron nanoparticles synthesized from Asphodelus aestivus extract and the aqueous extract of Asphodelus aestivus (FeNPA:Fe nanoparticles prepared by Asphodelus aestivus aqueous extract; ASP: the aqueous extract of the aerial part of Asphodelus aestivus).
FT-IR spectra of A. aestivus aqueous extract (ASP) and FeNPA synthesized from ASP.
Transmission electron microscopy (TEM) images of FeNPA synthesized by ASP.
Scanning electron microscopy (SEM) images of FeNPA synthesized by ASP.
EDX spectrum of synthesized FeNPA using ASP.
TGA analysis of synthesized FeNPA using ASP.
X-ray diffraction (XRD) patterns of the FeNPA synthesized by ASP.
Zeta potential stabilization pattern analysis of synthesized FeNPA using ASP.
3.1 Cytotoxicity of FeNPA and ASP
Breast cancer models MCF-7 and MDA-MB-231 and melanoma cancer models MeWo and CHL-1 and fibroblast cell models WI-38 and HEL 299 (control cell models) were treated with FeNPA and ASP 72 h to analyze the cytotoxic effects. The IC50 values of FeNPA were determined respectively as 139.40 μg/mL, 47.00 μg/mL, 38.48 μg/mL, 5.26 μg/mL, 179.12 μg/mL, 32.70 μg/mL and ASP were 4,39 mg/mL, 25,56 μg/mL, 74,18 μg/mL, 29,44 μg/mL, 98,98 μg/mL, and 146,10 μg/mL, respectively (Table 4). FeNPA were more cytotoxic than ASP in MCF-7, MeWo, CHL-1, and HEL 299 cells (>31-, >1.9-, >5-, and >4-fold change in IC50 values, respectively). When nanoparticle cytotoxicity was assessed, FeNPA showed cytotoxicity in all cell lines. Aslanturk and Çelik used aqueous, diethylether, ethyl acetate and methanol extracts of A. aestivus. Aqueous extracts gave results 185.92 μg/mL at 24 h and 89.68 μg/mL for 72 h in MCF-7 cells (40). This difference in cytotoxicity is due to the use of different parts of the A. aestivus plant. In the present study, the upper parts of A. aestivus plant were used, however Aslanturk and Çelik were used ground tubers.
IC50 values of FeNPA and ASP in cell lines (μg/mL).
| Cancer model | Cell lines | FeNPA | Water extract | Fold change |
|---|---|---|---|---|
| Breast | MCF-7 | 139.40 | 4390.00 | 31.49 |
| MDA-MB-231 | 47.00 | 25.56 | –1.84 | |
| Melanoma | MeWo | 38.48 | 74.18 | 1.93 |
| CHL-1 | 5.26 | 29.44 | 5.60 | |
| Control | WI-38 | 179.12 | 98.98 | –1.81 |
| HEL 299 | 32.70 | 146.10 | 4.47 |
3.2 Apoptotic effect of FeNPA and ASP
The apoptotic effect of FeNPA IC50 values was determined and apoptosis was increased 2.21 fold in MCF-7 cells, 2.31 fold in MDA-MB-231, 1.29 fold in CHL-1 cells, 1.72 fold in MeWo cells, 19.85 fold in WI-38 cells and 3.71 fold in HEL 299 cells compared to the control group and ASP induced 4.93 fold, 1.21 fold, -1.03 fold, -2 fold, 1.55 fold, and 31.85 fold, respectively. FeNPA had shown that mitochondria induce apoptosis through stress in MDA-MB-231, and cells MeWo (>1.9-, and >1.7-fold). ASP also induced apoptosis 2.23-fold in MCF-7 cells (Table 4). Aslanturk and Çelik were observed similar apoptosis for ASP. They had suggested that the apoptotic effect of A. aestivus plant extract was due to oxidative DNA damage resulting from proximal flavonoids present in the extract [42]. In the present study, results indicated that FeNPA was caused by oxidative DNA damage in apoptotic effect in MCF-7 and MDA-MB-231. However, it has been shown that FeNPA did not have apoptotic effects on melanoma cell lines, CHL-1 and MeWo it was referred to the fact that the antioxidant systems worked well in these cell lines so that no oxidative DNA damage could occur. Austin et al. had suggested that synthesized nanoparticles increase reactive oxygen species (ROS) levels, damage mitochondrial membranes, decrease ATP levels, and induce caspase-independent apoptosis in the nucleus loci of p38 MAPK and apoptosis inducing factor (AIF) proteins [43]. In the course of these findings, it was also been considering the apoptosis induction through the caspase independent pathway of FeNPA synthesized in the present research.
3.3 Effect of FeNPA on gene expression
FeNPA reduced 2 to 6 times of the 14 gene’s expression and increased the expression of 5 genes 2 to 9 times in MCF-7 cells, reduced 7 gene expression by 2 to 9 fold, and increased 3 gene expressions by 2 to 10 fold in MDA-MB-231 cells. ASP reduced the expression of 11 genes by 2 to 7 fold and increased the expression of 4 genes by 2-11 fold compared to the control group in MCF-7 cells and increased expression of 4 genes by 2-9 fold in MDA-MB-231 cells (Figure 9). In breast cancer cells, Keratin 5 (KRT5) gene expression was detected 4-fold increase and a 2-fold reduction in BIRC5, ESR1, IGF1 and TWIST1 gene expressions by FeNPA and ASP treatment.
IC50 value of FeNPA were determined. (a) IC50 was 139.40 μg/mL in MCF-7, (b) 47.00 μg/mL in MDA-MB-231, (c) in MeWo38.48 μg/mL, (d) 5.26 μg/mL in CHL-1, (e) 179.12 μg/mL in WI-38 and (f) 32.70 μg/mL in HEL 299.
It had been shown that FeNPA treatment in breast cancer cell lines (MCF-7 and MDA-MB-231) affects the expression of many genes in mRNA. Kabos et al. observed a decrease in ESR1 protein and an increase in KRT5 protein in the combined administration of 17β-estradiol and antiestrogen tamoxifen or fulvestrant in MCF-7 cell line [44]. The same effect was found in ER+ tumors of neoadjuvant endocrine therapy patients and they suggested that it may be related to resistance to standard endocrine and chemotherapeutic agents. In the present study, FeNPA treatment showed an increase in KRT5 expression and a decrease in ESR1 expression in breast cancer cell lines. The similar response in two gene expressions without positive endocrine and/or chemotherapy was evaluated positively. Insulin-like growth factor (IGF) signaling was an important regulator of breast development and breast cancer. IGF1 and IGF2 were ligands of the IGF signaling pathway. It had been shown that IGF1 and IGF2 overexpression increase cell proliferation and tumor formation in mouse xenografts and do not alter resistance to chemotherapeutic drugs such as taxol. Overexpression of IGF1 hd also been shown to cause overexpression of 9 genes in the IGF signaling pathway. It had been suggested that upregulation of these genes has stimulating effects on the IGF signaling pathway on global protein synthesis [45]. In this study, FeNPA administration reduced IGF1 expression about 3 fold in the MCF-7 cell line and about 9 fold in the MDA-MB-231 cell line. The present research supposed that the downregulation of IGF1 expression and the decrease of cell proliferation in FeNPA cytotoxicity were effective and and apoptosis was induced by a decrease in global protein synthesis (Figure 11).
In MeWo cells, FeNPA reduced the expression of 14 genes by 2 to 7 folds and increased the expression of 19 genes by 2 to 13 times and in CHL-1 cells, increased the expression of 17 genes by 2 to 8 times and decreased the expression of 7 genes by 3 to 9 times compared to the control group. ASP reduced the expression of 12 genes by 2 to 13 fold and increased the expression of 21 genes by 2-14 fold compared to the control group in CHL-1 cells and increased expression of 28 genes by 3-15 fold and reduced the expression of 8 genes by 2 to 11 fold in MeWo cells (Figure 10). BCL6, CXCL12, DNAJC15, RB1 and TPM1 gene expressions were increased in melanoma cancer cell. Bai et al. demonstrated that overexpression of BCL6 induces apoptosis by downregulation of BCL2 and BCL-XL in the diffuse large B cell lymphoma [46]. In CHL-1 and MeWo cell lines, FeNPA and ASP treatment had been shown to increase BCL6 gene expression and FeNPA and ASP administration in apoptosis induction has been assessed as providing BCL2 and BCL-XL suppression (Figure 12).
Apoptotic effect of FeNPA. Apoptosis was increased (b) 2.21 fold in MCF-7 cells, (d) 2.31 fold in MDA-MB-231, (f) 1.29 fold in CHL-1 cells, (h) 1.72 fold in MeWo cells, (j) 19.85 fold in WI-38 cells and (l) 3.71 fold in HEL 299 cells compared to the control group (a, c, e, g, i and k are untreated control groups of the cell lines, respectively).
Effect of FeNPA and ASP on gene expression in breast cancer model.
Effect of FeNPA and ASP on gene expression in melanoma model
4 Conclusion
This is the first report on the antioxidant activity, cytotoxic activity and characterization of iron oxide nanoparticles synthesized from the aerial part of A. aestivus. Iron oxide nanoparticles were characterized by UV-visible, FT-IR, TEM, SEM/EDX, TGA, XRD and Zeta potential characterization methods. Biological activities of the extract and nanoparticles were compared. FeNPA showed higher antioxidant and cytotoxic activities than ASP. FeNPA was highly effective in breast cancer and melanoma cell lines, induced apoptosis and altered gene expression regulation that is important for prognosis. It was suggested that gene expressions regulated by FeNPA should be evaluated in further in vivo studies.
Acknowledgements
This research was supported by TUBITAK (project number SBAG-114S730). We are thankful to FABAL (Ege University, Faculty of Pharmacy Pharmaceutical Sciences Research Centre) and Izmir Institute of Technology, Center for Materials Research.
List of abbreviations
- FeNPa
Iron nanoparticles
- FeNPA
Iron oxide nanoparticles of Asphodelus aestivus aqueous extract
- ASP
Asphodelus aestivus aqueous extract
- UV-vis
Ultraviolet-vis spectroscopy
- FT-IR
Fourier transform infrared spectroscopy
- TEM
Transmission electron microscopy analysis
- SEM/EDX
Scanning electron miroscopy/ energy disperse X-ray spectroscopy
- TGA
Thermal gravimetric analysis
- XRD
X-ray diffraction analysis
- ABTS+
2,2’-azino-bis (3-ethyl benzo-thiazoline-6-sulphonic acid)
- DPPH
1-1-diphenyl-2-picryl-hydrazyl
- H2O2
Hydrogen peroxide
- EMEM
Eagle’s Minimum Essential Medium
- DMEM
Dulbecco’s Modified Eagle Medium
- PI
Propidium iodide
- ROS
Reactive oxygen species
- AIF
Apoptosis inducing factor
- KRT 5
Keratin 5
- IGF
Insulin-like growth factor
Conflict of interest: The authors of this work declare that they have no conflict of interest.
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