Comparison of the effect of rhodium citrate-associated iron oxide nanoparticles on metastatic and non-metastatic breast cancer cells

Abstract

Background

Nanocarriers have the potential to improve the therapeutic index of currently available drugs by increasing drug efficacy, lowering drug toxicity and achieving steady-state therapeutic levels of drugs over an extended period. The association of maghemite nanoparticles (NPs) with rhodium citrate (forming the complex hereafter referred to as MRC) has the potential to increase the specificity of the cytotoxic action of the latter compound, since this nanocomposite can be guided or transported to a target by the use of an external magnetic field. However, the behavior of these nanoparticles for an extended time of exposure to breast cancer cells has not yet been explored, and nor has MRC cytotoxicity comparison in different cell lines been performed until now. In this work, the effects of MRC NPs on these cells were analyzed for up to 72 h of exposure, and we focused on comparing NPs’ therapeutic effectiveness in different cell lines to elect the most responsive model, while elucidating the underlying action mechanism.

Results

MRC complexes exhibited broad cytotoxicity on human tumor cells, mainly in the first 24 h. However, while MRC induced cytotoxicity in MDA-MB-231 in a time-dependent manner, progressively decreasing the required dose for significant reduction in cell viability at 48 and 72 h, MCF-7 appears to recover its viability after 48 h of exposure. The recovery of MCF-7 is possibly explained by a resistance mechanism mediated by PGP (P-glycoprotein) proteins, which increase in these cells after MRC treatment. Remaining viable tumor metastatic cells had the migration capacity reduced after treatment with MRC (24 h). Moreover, MRC treatment induced S phase arrest of the cell cycle.

Conclusion

MRC act at the nucleus, inhibiting DNA synthesis and proliferation and inducing cell death. These effects were verified in both tumor lines, but MDA-MB-231 cells seem to be more responsive to the effects of NPs. In addition, NPs may also disrupt the metastatic activity of remaining cells, by reducing their migratory capacity. Our results suggest that MRC nanoparticles are a promising nanomaterial that can provide a convenient route for tumor targeting and treatment, mainly in metastatic cells.

Background

Over the past few years, the nanotechnology field has emerged as a promising approach with the potential to produce diagnostic and therapeutic applications (Paris and Vallet-Regí 2018). The nanoscale of nanomaterials allows better access to biological sites. Among other applications, cancer cell targeting would benefit greatly from highly specific and localized drug delivery. In this context, iron oxide nanoparticles hold great promise as diagnostic and therapeutic agents in oncology. Their intrinsic physical properties are particularly interesting for simultaneous drug delivery, molecular imaging and applications such as localized hyperthermia (Bender et al. 2018; Iv et al. 2015). These technical features provide special perspectives for breast cancer treatment and diagnosis, which is especially important because of the high incidence, drug resistance and recurrence risk related to this disease (Low et al. 2018; Harmon et al. 2015; Karakatsanis et al. 2016). Current studies with maghemite nanoparticles, an iron oxide compound, have demonstrated in vitro and in vivo specific cytotoxic action for target cells, and these results have indicated this nanoparticle as a promising option for drug delivery (Manikandan et al. 2018; Chaves et al. 2017; Magro et al. 2018). Maghemite nanoparticles associated with rhodium citrate (MRC) have showed colloidal stability and antitumor activity in breast cancer cells (Carneiro et al. 2011; Peixoto et al. 2015; Chaves et al. 2015; da Silva Nunes et al. 2013; Rocha et al. 2017).

The unique structural features of many solid tumors, including hypervasculature with defective architecture, and impaired lymphatic drainage, allow for the well-characterized enhanced permeability and retention effect (EPR) and are key factors for advancing this technology platform (Maeda et al. 2013). However, nanomedicine activity is not only related to tumor EPR effect. It is also influenced by the intra-tumoral distribution of the delivery system, the extent and kinetics of drug release within the tumor and the exposure to drug released in circulation (Wicki et al. 2015). Recent works (Cook et al. 2014; Hare et al. 2017) suggest that efforts should be directed toward understanding the correlations between the tumor cell biology and the nanoparticles’ behavior. Since cancer is a heterogeneous disease, designing nanomedicines to overcome a well-defined challenge with a particular tumor cell type has a greater chance of success (Shi et al. 2017).

In our previous work, MRC nanoparticles induced apoptosis in MCF-7 cells after 24 h of exposure (Chaves et al. 2015). The different uptake mechanism of MRC in metastatic and non-metastatic breast cancer cell models also was identified (Chaves et al. 2017). However, to our knowledge, the behavior of these nanodevices in human tumor cells over time has not yet been explored, and nor has MRC cytotoxicity and therapeutic effectiveness comparison in different cancer cells been performed until the present moment. And there was no evaluation aiming to verify antimetastatic action of the MRC nanoparticles. Drug-loaded NPs may increase accumulation of intracellular drugs by inhibiting the functions of PGP and exhibit a multidrug resistance (MDR)-reversing effect (Cheng et al. 2017; Liang et al. 2018; Peng et al. 2018). More prolonged investigation allows for monitoring the development of resistance mechanisms, such as the expression of proteins that mediate the efflux of drugs such as PGP (Chen et al. 2018). Thus, in this work we evaluate the actions of the nanoparticles against tumor cells for up to 72 h of exposure connecting with possible mechanisms of resistance and the migratory capacity after the treatment with focus on the comparison of the effectiveness of NPs in different cell types, aiming to elect the most responsive.

Results

In vitro cytotoxicity study

The in vitro cytotoxicity of MRC in two selected breast human cancer cell lines (MDA-MB-231 and MCF-7) and human non-tumor mesenchymal cells (HNTMC) was determined by MTT assay. The metal compound chemotherapeutic agent rhodium citrate (RC) was used as a positive control. Non-tumor cells (HNTMC) have their viability statistically altered by treatment for up to 72 h with MRC, and RC at concentrations of up to 200 μM (Fig. 1a–c). As shown in Fig. 1, MRC complexes exhibited a broad viability inhibition on human cancer cells, mainly in the first 24 h (Fig. 1a). However, while MRC induced cytotoxicity in MDA-MB-231 in a time-dependent manner, progressively decreasing the required dose for significant reduction in cell viability after 48 and 72 h, MCF-7 appears to recover its viability from 48 h of exposure (Fig. 1b, c). The MRC half-maximal inhibitory concentration (IC₅₀) calculated for 24 h of treatment was 125 for MDA-MB-231 and 162 μM for MCF-7.

Fig. 1

Viability of metastatic breast tumor cells (MDA-MB-231), non-metastatic mammary carcinoma cells (MCF-7) and human non-tumor mesenchymal cells (HNTMC). The assay was performed after 24- (a), 48- (b) and 72-h (c) exposure to 50, 100 and 200 μM rhodium citrate free (RC) or associated with maghemite nanoparticles (MRC). *p < 0.05, **p < 0.01, ***p < 0.005

Cell migration and cell cycle analysis

The different effects of MRC on metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer cells observed in the viability assay over time drove us to explore the effect of MRC on migration capacity in metastatic cancer cells. MDA-MB-231 cells treated with MRC had their migration capacity reduced (Fig. 2). After treatment with the NPs, the number of cells that were able to transpose the in vitro-simulating extracellular matrix (mean = 844 cells) was statistically lower than in the untreated control (mean = 1500 cells).

Fig. 2

Analysis of migration capacity of MDA-MB-231 cells after treatment with 100 mM rhodium citrate associated with maghemite nanoparticles (MRC). Micrographs of migrant untreated (control) or treated MDA-MB-231 cells. The graph represents the cell counts. (**) p ≤ 0.01

Evaluation of P-glycoprotein (pgp) in tumor cells treated with MRC

The PGP level was not altered in MDA-MB-231 cells after MRC treatment for 24 and 48 h of exposure. However, in MCF-7 cells, a significant increase in this protein stimulated by MRC nanoparticles (24 h) was detected (Fig. 3).

Fig. 3

Immunoblot of P-glycoprotein production in MCF-7 and MDA-MB-231 cells. The bands related to the control (untreated) and MRC (100 μM, 24 and 48 h) groups of MCF-7 and MDA-MB-231 cells were quantified. Samples loading was adjusted to equalize total protein. The graphs represent the results of the quantification of PGP production which reveals a significant increase in the production of PGP in the MCF-7 cells; (*) p ≤ 0.05

MRC nanoparticles effects on DNA replication and cell cycle

Treatment with NPs for 24 h reduced DNA synthesis in MDA-MB-231 and MCF-7 cells. After 24 h, there was an increase in thymidine incorporation in untreated cells, while in treated cells (MRC), the detection of bromodeoxyuridine (BrdU) was reduced (compared to untreated control cells after 24 h), especially in MDA-MB-231 cells (Fig. 4a). Figure 4b shows the cell cycle changes induced by MRC. In both cell lines, the treatment induced an increase in the S phase, reflecting a stop at that point in the cycle and consequent reduction in the gap 2 phase (G2), mainly in MDA-MB-231 (Fig. 4b).

Fig. 4

Effects of exposure to MRC for 24 h on DNA synthesis and cell cycle of MDA-MB-231 and MCF-7 cells. a Number of cells treated with NPs (24 h) in DNA synthesis (S phase) compared to the untreated control (0 h and 24 h). (*) p ≤ 0.05; (**) p ≤ 0.01. b Cell cycle assessment. Percentage of cells (treated with NPs with 100 μM MRC by 24 h) in gap 1 (G1), DNA synthesis (S) or gap 2 (G2) phase

Discussion

Studies of viability after MRC treatment of non-tumoral cell types (MFC-10A) have already demonstrated a low toxicity of this nanocomposite in these cells (Chaves et al. 2015; da Silva Nunes et al. 2013). However, it is known that cells from established strains undergo immortalization processes that modify and make them more resistant (Bana and Bagrel 2011). The use of primary culture cells as used in the present work was essential to complement the in vitro elucidation of a possible passive specific effect of NPs. The different behaviors regarding viability after MRC treatment over time observed in two cell models should be considered for designing the most appropriate administration schedule. In tumors with the same characteristics as MCF-7 cells, repetitive exposure to NPs appears to be more appropriate, while in tumors that reassemble MDA-MB-231 cells, a continuous infusion may be sufficient (Eastman 2017).

Not only cytotoxicity in tumor cells is an important target for the development of antitumor drugs. One of the major problems in cancer control is the metastatic ability of the cells. Therefore, the synthesis of drugs and nanomedicines that control this process should be stimulated (Jin et al. 2019). Although the involved mechanism is not yet understood, the reduction in migration of MDA-MB-231 cells treated with ruthenium-based metal complexes has been reported (Cao et al. 2015). In vitro reduction in the migration of metastatic breast cancer cells after treatment with NPs (Fig. 2) suggests an important action of MRC in the control of metastasis in remaining cells.

MCF-7 cells were less sensitive to the NP treatment. Studies have already demonstrated the induction of resistance in MCF-7 cells (Tsou et al. 2015). This feature in these cells can be related to data from the viability assay, which demonstrated a milder action on cytotoxicity over time. Studies indicate that increased expression of PGP protein is related to cell resistance (Nath et al. 2013; Jain 2008). It is also known that the high basal expression of this protein, as demonstrated in this study in MCF-7 compared to MDA-MB-231 (Fig. 4), means a greater predisposition to multidrug resistance (Wishart et al. 1990). The high half-inhibitory concentration (IC₅₀) has also been related to a higher resistance index (Wu et al. 2014). In the present study, the MRC IC50 calculated for 24 h of treatment was 125 for MDA-MB-231 and 162 μM for MCF-7. It has already been suggested that the efflux mechanism of NPs can be avoided by maintaining a high concentration of nanoparticles at the treatment site in a shorter incubation time (Panyam and Labhasetwar 2003). This relationship makes sense for MCF-7 cells, in which a reduction in sensitivity, NPs accumulation (Chaves et al. 2017) and an increase in expression of PGP proteins after 24 h of MRC exposure (Fig. 4). However, experiments focused on the molecular mechanisms involved in PGP transport are needed to better understand this process and to confirm the MDR effects related to MRC exposition in MCF-7 cells.

Many metal compounds reach the nuclei of cells causing changes that lead to cellular inviability (Zhang and Lippard 2003). This action, which may occur by means of inhibition of essential enzymes for cellular replication, or by impairment of DNA synthesis, has already been demonstrated in studies with free rhodium citrate (Erck et al. 1974; Zyngier et al. 1989). Our previous findings suggested that the action of RC in the nuclei of tumor cells seems to be kept after its association with maghemite nanoparticles (MRC) (Chaves et al. 2017). The occurrence of S phase arrest with the use of MRC (Fig. 4), as observed in the present work, corroborates previous studies with free rhodium citrate.

The progression of the cell cycle is regulated by multiple checkpoints at different stages of the cell cycle. The three main ones are G1/S, G2/M and at the transition metaphase/anaphase during mitosis. Failure of this regulation can lead to abnormal growth or apoptosis. The G1/S checkpoint is the most critical for the control of cell proliferation by intracellular and extracellular signals related to the transport and integration of molecules in the nucleus (Skotheim et al. 2008). A positive relationship between the amount of cell cycle arrest at the S phase and apoptosis was established (Zhu et al. 2014). In accordance, a previous study conducted by our group reported that MRC are capable of inducing MCF-7 cells to apoptosis (Chaves et al. 2015). Taken together, our data suggest that MRC nanoparticles accumulate in the nucleus of tumor cells, inhibiting DNA synthesis and cell proliferation and inducing cell death.

Materials and method

Materials

The magnetic fluids used were synthesized by the co-precipitation method of Fe²⁺ and Fe³⁺ ions in alkaline medium, subsequently oxidized by bubbling oxygen. The functionalized fluids of rhodium citrate associated with maghemite nanoparticles (MRC) and citrate-loaded maghemite nanoparticles (MC) were obtained by adsorption. MRC with 59.6 µM γ-Fe₂O₃ and 2.85 µM rhodium citrate, MC with 64.7 µM γ-Fe₂O₃ and 2.55 µM [citrate], and 2.513 mM [Rh₂(C₆H₇O₇)₄(H₂O)₂ [rhodium (II) citrate] were synthesized at the Institute of Chemistry of the Goias Federal University (Goiania, Brazil). For maintenance of cell cultures, DMEM, RPMI and L15 mediums, fetal bovine serum (FBS) and 0.25% trypsin EDTA were used, and all were obtained from GIBCO Company-Life Technologies, Carlsbad, CA, USA, except Leibovitz’s L15 (Sigma-Aldrich, St. Louis, MO, USA). The MRC solution diluted in H₂O had a mean hydrodynamic diameter of 119.5 nm with a polydispersity index (PDI) of 0.17, ζ-potential of − 35 mV, analyzed by Zetasizer Nano ZS machine (Malvern, Malvern, UK) using a technology called dynamic light scattering (DLS), and similar values were maintained after subsequent measurements at different time intervals. The antibodies used in this study were: anti-P-glycoprotein (monoclonal) produced in mice (Abcam, Cambridge, MA); anti-BrdU conjugated to Alexa Fluor 488 (Invitrogen Life Technologies, Carlsbad, CA, USA); HRP-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). The reagents used throughout the work were: MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide), DNAse (Sigma-Aldrich, St. Louis, MO, USA); bicinchoninic acid assay (BCA, Thermo Scientific, Rockford, IL); ATP (Invitrogen, San Diego, CA, USA). Inserts for invasion assay and basement membrane matrix (Matrigel™) were purchased from BD Biosciences, San Jose, CA, USA. For Western blot analyses, we used a protease inhibitor (Hoffman-La Roche, Basel, Switzerland). The development was made with Amersham ECL prime Western blotting detection reagent, and image acquisition and analyses were performed with an Image Quant LAS 4000 (GE Healthcare, Little Chalfont, UK); further image processing and analysis were done with the ImageJ program (NIH, USA). For analysis of cell migration, an Axiovert light microscope (5× magnification) was used and the images were captured with the AxioVision 100 software, both from Zeiss, Germany. The Spectramax M5 equipment and the data analyzed by the SoftMax Pro 5.2 program, both from Molecular Devices, LLC, USA, were used to read absorbances. The flow cytometer used was a FACS Calibur, BD Biosciences, Inc., San Jose, CA, and data analysis was performed on the Flow Jo v.5.2.7 program, Tree Star, Inc.

Cell culture

The human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium and Leibovitz’s L15 medium without CO₂, respectively, containing 1% (v/v) penicillin–streptomycin and 10% (v/v) heat-inactivated fetal bovine serum (FBS). Primary cultures of human non-tumor mesenchymal cells from dental pulp (HNTMC) were obtained from health volunteers under approval of the Human Ethics Committee of the University of Brasilia (UnB). HNTMC were cultured with DMEM, supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin–streptomycin. HNTMC isolation was performed according to the following protocol: Extracted dental pulp was placed directly to a sterile tube containing DMEM, supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin–streptomycin until it was transferred to a plate (6 well) in the biological safety cabinet. To keep the cells fixes and facilitate adhesion to the bottom of the plate, a sterile pipette folded in half was used as a hook. The dental pulp received culture medium DMEM and was maintained at 37 °C, 5% CO₂ and 80% of humidity. A half of the culture medium was replaced twice a week, until the cells reach confluency (80–90%). Then, the adherent cells were dissociated with trypsin and were frozen as seed stock in the presence of a cryoprotective agent dimethylsulfoxide (DMSO).

In vitro cytotoxicity study

In order to understand how the cells respond to MRC nanoparticles, MDA-MB-231, MCF-7 and HNTMC cells were incubated for 24, 48 and 72 h with various concentrations of this compound. Furthermore, the tumor cells were also exposed to free rhodium citrate (RC) at the same concentrations as those in MRC. Viability of the cells after exposure to NPs and RC was evaluated by use of the MTT assay according to the manufacturer’s recommendations (Invitrogen Life Technologies, Carlsbad, CA, USA). Briefly, 1 × 10⁴ cells/well were seeded in 96-well plates and exposed to the treatments at the concentrations of 50 μM, 100 μM, 200 μM (rhodium citrate). All experiments were repeated at least three times in triplicates and were preceded by kinetic tests to adjust the initial cells amount for each exposure time and to avoid the control group confluency at later time of exposition. Results were expressed as percent of viability (%V) according to the following formula (blank discounted): %V = absorbance (cells + medium + NPs) − absorbance (medium + NPs). After cell treatment, the medium was removed from each well and replaced with new medium containing MTT (15 μl of MTT solution at 5 mg/ml and 135 μl of culture medium) for two and a half hours at 37 °C in a humidified atmosphere with 5% CO₂. The resulting formazan product was dissolved in 200 μl of dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA). Afterward, the supernatants of each sample were transferred to a fresh 96-well plate (to avoid interference of nanoparticles that did not enter the cells and accumulate on the bottom of wells), and absorbance was measured by a spectrophotometer (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA) at the wavelength of 595 nm.

Cell migration test

To assess the migration/invasion capacity of MDA-MB-231, the cells were treated with MRC and plated (1 × 10⁶) in 25-cm² flasks for 24 h. Treatment with 200 μM nanoparticles was performed in the culture flasks for 24 h. Cell viability was evaluated by means of trypan blue assay. The cells were trypsinized, resuspended (2 × 10⁴) in L15 medium without fetal bovine serum (FBS) and added to the top of inserts (Transwell for 24-well plates, BD Biosciences) with 8-μm pores coated with a thin layer (30 μl) of Matrigel™ (BD Biosciences) diluted 5× in frozen fetal bovine serum-free L15 medium. The plate was left for 1 h at 37 °C for solidification of the Matrigel layer. In the wells of the plate, below the inserts, the L15 medium was supplemented with 10% SFB to attract a migration of the cells through the pores. After 48 h, the inserts were removed from the plate and the cells that adhered to the top of the Transwell (which did not migrate) were removed with a cotton swab. Cells remaining in the lower part of the insert were fixed (3.7% paraformaldehyde, 15 min) and stained with 0.1% crystal violet for 5 min. All stained cells were photographed (5× magnification), and an invasiveness rate was given by automated counting of cells processed with the ImageJ software.

The effect of MRC nanoparticles on DNA synthesis

For analysis of DNA replication and cell cycle, 5 × 10⁵ MDA-MB-231 and MCF-7 cells were plated in 6-well plates (10 cm²) in appropriate medium. After adherence, medium was exchanged for another containing 10 μM bromodeoxyuridine (BrdU) and incubated for 2 h at 37 °C for incorporation of BrdU into the DNA. As a negative control, one group did not receive BrdU. Subsequently, the cells received new medium, and in the experimental group, nanoparticles containing 200 μM MRC were added, and the treatment lasted for 24 h. The group which did not receive the treatment was considered the zero point and was collected immediately after incorporation of BrdU, and processed until the overnight fixation step at 4 °C. The cells were trypsinized and centrifuged at 1500 rpm for 5 min. The medium was discarded, and MDA-MB-231 cells were washed with ice-cold phosphate-buffered saline (PBS) and fixed with 70% ice-cold ethanol for 30 min. The fixative solution was removed, and DNAse (0.3 mg/ml) was added for 10 to 15 min for denaturation of the DNA at 37 °C. After discarding the solution containing DNAse, the pellet was resuspended in blocking solution (30 min) containing 50 μl of Tween 20 and 1% serum albumin (BSA)/PBS. Samples were incubated for 1 h with primary anti-BrdU antibody (1:100), and after 45 min with FITC-conjugated secondary antibody (1:10,000) in a solution containing 5 μl of RNAse (10 mg/ml). Subsequently, the pellet was resuspended in 1 ml with 5 μg/ml propidium iodide (PI) for 10 min. The cells (10,000 of each group in triplicate) were analyzed on a flow cytometer.

Immunoblotting analysis

For western blot, MDA-MB-231, MCF-7 and HNTMC cells (1x10⁶) were grown in flasks. Fresh medium with 100 µM MRC was added to the flasks, which were incubated for 6 h at 37 °C. Cells were rinsed three times in ice-cold PBS, and protein extraction was performed with lysis buffer (50 mM Tris pH 7,4; 150 mM NaCl; 5 mM ethylenediamine tetraacetic acid (EDTA); and 1% Triton X100 and protease inhibitor cocktail). Lysates were centrifuged (10 min, 4 °C). Protein concentrations were determined by the bicinchoninic acid assay. Protein total lysates (20 µg) were separated by 4–12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS)—0.1% Tween, followed by incubation with primary (1:1000 anti-P-glycoprotein) and secondary (goat anti-mouse HRP—1:5000) antibodies. Development was performed by ECL chemiluminescence kit. The experiment was repeated three times.

Statistical analysis

The quantitative data were submitted to the ‘t’ test for unpaired samples (between two groups) or simple variance (ANOVA) (between three or more groups) followed by the Bonferroni post-test. Statistical analyses were conducted using the GraphPad Prism software, and the results were expressed as mean ± SEM. Values of p < 0.05 were considered statistically significant.

Conclusions

The MRC cytotoxicity effect was dependent on concentration and time of exposure in MDA-MB-23 cells, whereas prolonged exposure was not effective in MCF-7 cells. Together, the data suggest that MRC act at the nucleus, inhibiting DNA synthesis and proliferation and inducing cell death. These effects were verified in both tumor lines, but MDA-MB-231 (metastatic cells) appears to be more susceptible to the effects of MRC. In addition, NPs may also act on the metastatic activity of remaining cells, reducing their migratory capacity. Our results suggest that MRC nanoparticles are a promising nanomaterial that can provide a convenient route for tumor targeting and treatment.