Article pubs.acs.org/jpr
Metabolic Effects of Cobalt Ferrite Nanoparticles on Cervical Carcinoma Cells and Nontumorigenic Keratinocytes Ana Beatriz Bortolozo Oliveira,†,∥ Fabio Rogério de Moraes,‡,§,∥ Natalia Maria Candido,† Isabella Sampaio,‡ Alex Silva Paula,‡ Adriano de Vasconcellos,‡ Thais Cerqueira Silva,‡ Alex Henrique Miller,‡ Paula Rahal,† Jose Geraldo Nery,‡ and Marilia Freitas Calmon*,† †
Biology Department, São Paulo State University, São José do Rio Preto, 15054-000 São Paulo, Brazil Physics Department, São Paulo State University, São José do Rio Preto, 15054-000 São Paulo, Brazil § Multiuser Center for Biomolecular Innovation, São Paulo State University, São José do Rio Preto, 15054-000 São Paulo, Brazil ‡
S Supporting Information *
ABSTRACT: The cytotoxic response, cellular uptake, and metabolomic profile of HeLa and HaCaT cell lines treated with cobalt ferrite nanoparticles (CoFe2O4 NPs) were investigated in this study. Cell viability assays showed low cytotoxicity caused by the uptake of the nanoparticles at 2 mg/mL. However, metabolomics revealed that these nanoparticles impacted cell metabolism even when tested at a concentration that presented low cytotoxicity according to the cell viability assay. The two cell lines shared stress-related metabolic changes such as increase in alanine and creatine levels. A reduced level of fumarate was also observed in HeLa cells after treatment with the nanoparticles, and this alteration can inhibit tumorigenesis. Fumarate is considered to be an oncometabolite that can inhibit prolyl hydroxylase, and this inhibition stabilizes HIF1α, one of the master regulators of tumorigenesis that promotes tumor growth and development. In summary, this study showed that nanoparticle-treated HeLa cells demonstrated decreased concentrations of metabolites associated with cell proliferation and tumor growth. The results clearly indicated that treatment with these nanoparticles might cause a perturbation in cellular metabolism. KEYWORDS: cobalt ferrite nanoparticles, cell lines, cytotoxicity, cell uptake, metabolomics
1. INTRODUCTION
A promising method to achieve this goal is metabolomics screening,13 which allows an analysis of metabolite variations in the studied samples.14 Assessing the metabolite profile, for example, provides important information about the toxicological effects15 and the biological mechanisms influenced by magnetic nanoparticles.16 Because of the contribution that metabolomics represents, this method can be extremely relevant to the transition from in vitro to in vivo experiments. However, the influence of nanomaterials on the metabolite profile has not yet been widely studied.16 In this study, we attested the biocompatibility of CoFe2O4 NPs by evaluating their in vitro effects on human cervical cancer cells (HeLa) and nontumorigenic human keratinocytes (HaCaT). First, the uptake of these CoFe2O4 NPs and their intracellular localization were analyzed. Second, the cytotoxicity and apoptotic status triggered by cobalt ferrite nanoparticles were determined; finally, a metabolomic analysis was performed to compare the metabolite profiles of the two cell types before and after exposure to the CoFe2O4 NPs.
Nanotechnology involves the fabrication and use of materials on a nanoscale.1 This technology combines chemistry, engineering, and biology2 to provide materials with many uses that range from energy production to biomedical applications. 3 In this context, magnetic nanoparticles (MNPs), in particular, iron oxide nanoparticles, have been widely investigated on the basis of their chemical stability and superparamagnetism.4 Inorganic ferrite nanoparticles show considerable magnetic properties,5 and several studies of cobalt ferrite nanoparticles (CoFe2O4 NPs) are currently being performed.6,7 Among the interesting properties of cobalt ferrite are its mechanical hardness, excellent chemical stability, high anisotropy, and coercivity.8 However, in addition to these useful characteristics, several studies have reported the occurrence of some adverse effects, such as changes in cell morphology, mitochondrial function, plasma membrane permeability, and apoptosis.9−11 Consequently, understanding the interactions between nanomaterials and biological systems is crucial. Therefore, a biocompatibility investigation has become an indispensable step to be performed before testing biological and medical applications. Thus, in vitro experiments with different cell lines represent a very useful tool for assessing the behavior of nanostructured materials.12 © XXXX American Chemical Society
Received: May 5, 2016
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DOI: 10.1021/acs.jproteome.6b00411 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
2. MATERIALS AND METHODS
2.3. Prussian Blue Staining
2.1. Synthesis and Characterization of CoFe2O4 NPs
The cells were suspended in cover glass chambers at a density of 2 × 105 cells/mL in six-well plates. Medium containing CoFe2O4 NPs at concentrations of 0.5, 1, 2, or 4 mg/mL was added. The cells were incubated with the CoFe2O4 NPs for 24 h and were washed repeatedly to remove unbound particles. The cells were then fixed using Karnovsky’s solution (4% paraformaldehyde:4% glutaraldehyde) (Sigma-Aldrich) for 1 h, washed with distilled water, and treated with a 1:1 mixture of 4% potassium ferrocyanide (Sigma-Aldrich) and 4% HCl (Sigma-Aldrich) for 20 min at RT. Subsequently, the slides were rinsed with distilled water and treated with hematoxylin for 10 min and eosin for 4 min. The resulting slides were observed under 400× magnification with an Olympus BX60 light microscope. To analyze the uptake efficiency of the CoFe2O4 NPs, we quantified cells labeled with the nanoparticles. Each experiment was performed in triplicate and in two independent assays. Statistical analysis was performed using GraphPad Prism 5 Software.
A solution of iron chloride (0.4 M, 25 mL, minimum purity 99.5%) (Sigma-Aldrich, St. Louis, MO) and a solution of cobalt chloride (0.2 M, 25 mL, minimum purity 99.5%) (SigmaAldrich), both prepared in deionized water, were combined. A solution of sodium hydroxide (3 M, 25 mL) (Sigma-Aldrich) was prepared and slowly added in a dropwise manner to the salt solution. The pH of the solution was constantly monitored as the NaOH solution was added. The reactants were constantly stirred using a magnetic stirrer until a pH level of 11 to 12 was reached. A specified amount of oleic acid (0.125 mL, SigmaAldrich) was added to the solution as a surfactant and coating material. The suspension was then brought to a reaction temperature of 80 °C and stirred for 1 h. The product was then cooled to room temperature. To obtain particles free of sodium and chlorine compounds, the precipitate was then washed twice with distilled water and then with ethanol to remove the excess surfactant from the solution. To separate the supernatant liquid, the beaker contents were centrifuged for 15 min at 3000 rpm. The supernatant liquid was decanted and then centrifuged again until only a thick black precipitate remained. The precipitate was finally dried overnight at 100 °C, and the acquired substance was ground into a fine powder. In this stage, all of the remained associated water of the product (CoFe2O4 NPs) was removed, and the possible endotoxin contamination in nanomaterials was eliminated by heating the to 600 °C for 10 h.17 The phase compositions and morphologies of the product were analyzed using an X-ray diffractometer (Miniflex, Rigaku, Tokyo, Japan) and a scanning electron microscope (SEM, XL30-FEG, FEI, Eindhoven, The Netherlands). Fourier transform infrared (FT-IR) spectra for the nanoparticles was obtained in the range 4000 to 400 cm−1 with an IR-Prestige-21 (Shimadzu FT-IR spectrophotometer, Japan) using the KBr pellet method. The binding interactions of CoFe2O4 NPs with the cell medium were measured, both in aqueous solution and in the cell medium (DMEM) using a Zetasizer ZS90 (Malvern Instruments, Worcestershire, U.K.).
2.4. MTT Assay
The cytotoxicity was assessed using the MTT assay, which is a nonradioactive colorimetric assay. In 96-well plates, medium containing CoFe2O4 NPs at concentrations of 0.5, 1, 2, or 4 mg/mL was added to each well containing 1 × 104 cells. After 24, 48, 72, or 96 h of incubation with CoFe2O4 NPs, 100 μL of medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (1 mg/mL) (Sigma-Aldrich) was added to each well. After 30 min of incubation, the medium was removed and the formazan crystals were solubilized by incubation for 10 min in 100 μL of DMSO (Sigma-Aldrich). The absorbance of each well was read at 570 nm. Each experiment was performed in triplicate and in three independent assays. 2.5. Apoptosis Assay
HeLa and HaCaT cells were suspended in a chambered cover glass in six-well plates at a density of 2 × 105 cells/mL. Medium containing CoFe2O4 NPs at concentrations of 0.5, 1, 2, or 4 mg/mL was added. The cells were incubated with CoFe2O4 NPs for 24 h, then fixed with Karnovsky’s solution for 1 h and washed with distilled water. Subsequently, 4% ferric alum (Sigma-Aldrich) was added for 4 min, and the slides were rinsed with distilled water and treated with acridine orange (Sigma-Aldrich) as a counterstain for 10 min. The resulting slides were observed under 400× magnification with an Olympus BX60 light microscope. Each experiment was performed in triplicate and in two independent assays.
2.2. Cell Line and Nanoparticles Treatment
The human epithelial cervical carcinoma cell line (HeLa) and spontaneously immortalized nontumorigenic human skin keratinocytes (HaCaT) kindly provided by Luisa Lina Villa (Department of Radiology, Center on Translational Oncology Investigation, São Paulo State Cancer Institute, São Paulo University, Brazil) were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco by Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (50 U/mL), and streptomycin (0.05 mg/mL). The cells were incubated at 37 °C with 5% CO2. For the cell seeding, HeLa and HaCaT cells were cultured in serum-starved medium. After 24 h, the CoFe2O4 NPs were added to the supplemented culture medium (containing 10% FBS and antibiotics) at concentrations of 0.5, 1, 2, or 4 mg/mL. To avoid contamination in the cell culture, filter sterilization (using a Millipore filter with a pore size of 0.22 μm) of the medium containing the CoFe2O4 NPs was applied before introducing the medium to the cell culture.
2.6. Metabolite Extraction
Methanol (1.667 mL) (Sigma-Aldrich) and chloroform (0.833 mL) (Sigma-Aldrich) were added to 1.2 × 107 HeLa and HaCaT cells with and without 2 mg/mL CoFe2O4 NPs for 24 h. Subsequently, the cells were sonicated for 3 min at 130 W, 20 kHz, 50% amplitude. After sonication, 1250 mL of deionized water and 1250 mL of chloroform were added to the cells and the samples were vortexed for 1 min and centrifuged at 4000 rpm for 20 min at 4 °C. Then, the lower layer was removed and the upper layer was vacuum centrifuged. Next, samples were reconstituted in phosphate-buffered saline (PBS) for analysis by nuclear magnetic resonance (NMR) in 10% D2O (Sigma-Aldrich). The final volume was 550 μL. Each sample was vigorously vortexed for 1 min before B
DOI: 10.1021/acs.jproteome.6b00411 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research transfer to a 5 mm NMR tube, and the acquisition was performed immediately thereafter.
as well as boxplots were used to assess the relative concentration difference between control and treated samples.
2.7. Nuclear Magnetic Resonance Spectroscopy
2.9. Metabolite Identification
NMR measurements of extracted polar metabolites were performed at 298 K in Bruker AVANCE III HD operated at 600 MHz. The NOESYPR1D pulse sequence was employed for water suppression with the irradiation at the water frequency during the recycle delay of 2 s and a mixing time of 100 ms. Sixteen scans were acquired with four dummy-scans and 32k data points, and a spectral width of 20 ppm was used. All 1D free induction decays (FIDs) were multiplied by an exponential function with a 1 Hz line broadening prior to Fourier transformation. All spectra were manually phased, and automated baseline correction was performed using Topspin 3.0 software (Bruker Biospin, Germany). Lactate, specifically the methyl doublet at 1.33 ppm, was used as internal reference. The residual water region between 4.5 and 5.0 ppm was excised from the data set prior to further analysis. Total correlation spectroscopy (TOCSY) was performed to check the cross peaks that were useful for metabolite identification.
The selection of the list of important signals that were used for metabolite identification was based first on the signals that had both Welch two-sample and Mann−Whitney p values higher than 0.05. Signals having an absolute log2 value of fold change
