Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism

Chekhun V.F., Todor I.N., Lukianova N.Yu.*, Horbyk D.M., Lozovskaya Yu.V., Burlaka A.P., Borikun T.V.

Summary. Aim: To study the effect of Ferroplat (FrP) on the indexes of pro/antioxidant balance and energy metabolism in breast cancer cells of different malignancy degree and different sensitivity to drug therapy. Materials and Methods: The study was carried out on breast cancer cells of low (T47D, MCF-7) and high malignancy degree (MCF-7/DDP (cisplatin-resistant), MDA-MB-231, MDA-MB-468) using cell culture techniques, immunocytochemical, biochemical, biophysical methods, flow cytometry and polarography. Results: We established that the addition of FrP to the culture medium reduces the activity of glucose-6-phosphate dehydrogenase (G6PDH), superoxide dismutase (SOD) and the level of non-protein thiols by 32–41% (p < 0.05). At the same time, there was an increase of the total level of ROS and the rate of NO generation by inducible NO synthase by 1.7–2.5 times (< 0.05). This testifies that FrP disturbs the antioxidant balance in cells, resulting in their death. Also, the use of FrP led to a decrease in the rate of oxygen absorption in MCF-7 and T47D cells by 26% and 25%, respectively (p < 0.05). In cells of high malignancy degree this index decreased by 38–40% under the influence of FrP. Incubation of MCF-7 and T47D cells with the indicated agent also reduced the content of phospholipid cardiolipin by 15–16% (p < 0.05), and in MDA-MB-231, MCF-7/DDP, MDA-MB-468 cells — by 29%, 30% and 32%, respectively. In addition, the effect of FrP caused a decrease in the levels of Mg2+ and lactate in MCF-7 and T47D cells by 21–29% and 14–24%, respectively, whereas in MDA-MB-231, MDA-MB-468, MCF-7/DDP cells — by 34–38% and 32–35%, respectively. In this case, the agent raised the level of glucose in the cells of low malignancy degree by 20–23% (p < 0.05), and in the cells of high malignancy degree and with the phenotype of drug resistance — by 31–36%. However, the nanocomposite did not affect the activity of lactate dehydrogenase in all studied breast cancer cells. Conclusion: The study has shown that FrP has an effect on the pro/antioxidant balance and energy metabolism of cancer cells. In addition, the denoted effect of FrP was more pronounced in the breast cancer cells with a high malignancy degree and the phenotype of drug resistance.

Submitted: November 22, 2018.
*Сorrespondence: E-mail: nataluk10@gmail.com
Abbrevіations used: BC — breast cancer; FrP — Ferroplat; G6PDH — glucose-6-phosphate dehydrogenase; PBS — phosphate buffered saline; ROS — reactive oxygen species; SOD — superoxide dismutase.

The introduction of modern advances in nanotechnology plays an important role among the new promising non-standard approaches to the treatment of cancer patients. In the field of oncology, research on the creation of nanocomposites for controlled transport of antitumor preparations based on ferromagnetic is conducted. It is known that ferromagnetics can affect the exchange of endogenous iron in tumor cells [1, 2]. According to the literature, the development of tumors of various geneses is accompanied by the reprogramming of iron metabolism in different ways, which ultimately leads to its accumulation directly in the tumor tissue and cells of tumor microenvironment [3]. Intracellular iron ions in the “free” state are toxic and catalyze the formation of reactive oxygen species (ROS), including hydroxyl radicals, which cause DNA damage and initiate carcinogenesis [4, 5]. A significant increase in the level of iron in tumor cells is necessary to support their enhanced metabolic and proliferative needs [6]. On the other hand, the content of “free iron” in tumor cells may be reduced due to its exhaustion, which also indicates an increased need for this ion in rapidly growing and dividing cancer cells. Numerous studies of recent years have shown the role of the endogenous iron exchange disorders and ROS formation in the implementation of the cytotoxic effects of such antitumor drugs as paclitaxel [7], etoposide [8] and doxorubicin [9]. At the same time, there is no evidence on the role of endogenous iron exchange disorders in the formation of the phenotype of drug resistance to cisplatin [1, 10–12].

Up to date, the considerable interest of experimental and clinical oncologists is focused on the use of chelators and donators of iron ions in tumor growth [13]. The most promising exogenous modifiers of the endogenous iron exchange in the form of nanomaterials are ferromagnetics [14, 15]. It has been established that due to their properties, ferromagnetic nanoparticles are capable to conduct intercellular and intracellular transport of antitumor drugs, to perform the controlled release of the substances from their medicinal form, and to deliver the drug to biological targets [16, 17]. Current needs of oncology have intensified the use of modern methods for the creation of nanocomposites based on antitumor drugs and ferromagnetics [18, 19]. However, despite the active search for ways to optimize the cytotoxic therapy of cancer patients, especially those with drug-resistant cancer, so far there are no standardized antitumor nanocomposites in Ukraine. The existing results of experimental studies also did not provide satisfactory information about the mechanism of nanomaterial’s action and the feasibility of their usage to overcome the drug resistance of malignant tumors.

The participation of the ROS in the mechanisms of the action of many antitumor drugs, including those based on ferromagnetics, allowed to create a separate branch of anticancer therapy — redox-directed therapy of malignant neoplasms [20–22]. However, the influence of nanocomposites on the basis of ferromagne­tics on the energy metabolism of tumor cells, in particular, the processes of glycolysis and mitochondrial oxidative phosphorylation, is not determined. It is also unknown how ferromagnetic nanocomposites affect the level of Mg2+ in the cells, which is a necessary factor for most of the phosphorylation reactions. Particularly relevant is the answer to the question of how under the influence of nanocomposites, these indexes will change in transformed cells of varying malignancy degree and sensitivity to cytostatics.

Therefore, the aim of this work was to study the effect of ferromagnetic nanocomposite Ferroplat (FrP), which includes cisplatin, on pro/antioxidant balance and energy metabolism of human breast cancer (BC) cells of varying malignancy degree and different sensitivity to antitumor drugs.

Materials and methods

Ferromagnetic nanocomposite (FrP) characteristics. The technology for obtaining antitumor nanocomposite FrP which includes cisplatin and ferromagnet was designed in the collaboration with colleagues from the Chuiko Institute of Surface Chemistry, National Academy of Sciences of Ukraine under the supervision of the doctor of physical and mathematical sciences, professor P.P. Gorbyk.

Synthesis of magnetite was carried out by a liquid-phase method, based on the co-precipitation of two- and trivalent iron salts by aqueous ammonia solution. The magnetite particles were stabilized with sodium oleate (С8Н17СН = СН(СН2)7СО ОNa) while stirring continuously at a temperature of 80 °C for 1 h. Stabilization is carried out due to chemisorption of sodium oleate molecules on the surface of magnetite.

The DDP adsorption on the surface of the nanoparticles of the magnetic fluid was carried out for the Fe3O4/oleate Na (magnetic fluid) system. In order to increase stability and reduce aggregation of particles, the resulting magnetic fluid with adsorbed cytostatics was additionally modified with polyethylene glycol (PEG-2000).

At each stage of FrP synthesis, the physical, chemical and magnetic properties of the developed nanocomposite were monitored using the wide range of methods (X-ray diffraction, thermogravimetric analysis, X-ray photoelectron spectroscopy, transmission electron microscopy, vibration magnetometry, dynamic light scattering, atomic emission spectroscopy). The obtained nanoparticle size distributions made it possible to characterize the synthesized nanocomposite as a monodispersion with the size of 32.7 nm. The shape of the particles was close to the globular. The results of electron microscopy studies indicate that this colloidal system is monodisperse and has a low predisposition to aggregation. By the method of atomic emission spectrometry, we established that the concentration of Fe3O4 in nanocomposites FrP is 3 mg/ml, and the concentration of cisplatin is 0.4 mg/ml.

Cell lines. The study was conducted on 5 human BC cell lines T47D, MCF-7, MCF-7/DDP, MDA-MB-231, and MDA-MB-468 [23]. The malignancy degree of the cells was determined by the indexes of receptor status, proliferative and invasive activity [23].

Analysis of the cytotoxic activity of FrP and its components. The cytotoxic activity of FrP toward T47D, MCF-7, MCF-7/DDP, MDA-MB-231, and MDA-MB-468 cells was investigated using trypan blue and the MTT test [22].

To evaluate the cytotoxicity, 20 μl of MTT solution (Sigma, USA) (5 mg/ml phosphate buffered saline (PBS)) was added into the wells of a 96-well plate with the examined cells and incubated for 3 h. After centrifugation of the plate (1500 rpm, 5 min), the supernatant was removed. To dissolve the formazan crystals in each well, 100 μl of dimethyl sulfoxide (Serva, Germany) was added. The optical absorption of the solution was measured using a plate reader (“STAT FAX 2100”) at a wavelength of 540 nm, and the number of living cells was determined relative to the control.

The evaluation of the viability of the cells exposed to FrP was carried out using a hemocytometer. For this purpose, the cells were stained with a vital dye Trypan blue. The number of cells was determined according to the formula:

А/80 ×2 = Х 106 cells per ml,

where A is the number of cells observed in a hemocytometer (in 5 squares), ×2 — trypan blue dilution (1:1).

To study the biological effects of FrP, a dose corresponding to IC30 (1.5 μg/ml for cisplatin and 12 μg/ml for Fe) was used. All studies were conducted taking into account the control parameters of cells that were cultured for 24 h only in the culture medium and in the medium with the addition of cisplatin or FrP at concentrations corresponding to IC30.

Biophysical methods (low-temperature EPR). The rate of NO generation by inducible NO synthase was determined by EPR spectroscopy using spin traps DETK (Sigma) at room temperature [19]. After 24 h of cultivation in the culture medium, the cells were separated from the substrate (on ice), washed in PBS, centrifuged at 4000 g for 10 min at 40 °C, and the precipitate was resuspended in PBS. A suspension of 2•106 cells was transferred to the EPR tubes and frozen in liquid nitrogen. During the recording of the spectra, the samples were kept at –196 ˚C. Low-temperature EPR was carried out with the following parameters: band width 1525 G; frequency 9.15 GHz; microwave power 40 mW; amplitude modulation 10.0 G; modulation frequency 100 kHz. The value of g was calculated using the standard formula:

G = H / βH,

where A is Plank constant; V is the frequency; β is the Bohr magneton; and H is the external.

Superoxide dismutase (SOD) activity was studied according to Ogura et al. [24]. A suspension of 2•106 cells with 2 ml of 0.1 M PBS was homoge­nized in a glass homogenizer. After this, the cells were centrifuged at 3000 rpm for 20 min. SOD acti­vity was measured in the resulting supernatant with the following composition of the reaction components: 50 μl of 2 mM hypoxanthine in 0.1 M PBS (pH 7.8); 15 μl DMPO (spin trap 5.5-dimethyl-1-pyrroline-N-oxide); 35 μl 10 mM DETAPAC in PBS (diethylene glycol triamino-pentacetylacetate); 50 μl of supernatant (~ 20 μl of protein in PBS); 50 μl xanthine oxidase (0.2 mg/ml). The analysis of SOD activity was carried out on a spectrometer EPP-1307 at room temperature.

The consumption of oxygen was determined using a closed platinum electrode and a polarograph LP-7E (Czech Republic). Cells were introduced into a polarographic chamber of a volume of 1 ml. The polarographic chamber contained PBS saturated with oxygen. The voltage applied to the platinum electrode was 700 mV. Determination of the oxygen consumption rate by the cells was carried out at 37 °C for 7–10 min. The rate of oxygen consumption was expressed in nanoatoms О2/min/106 cells.

Flow cytometry. Determination of ROS content, analysis of the content of SH-group, analysis of mitochondrial transmembrane potential, and measurement of cardiolipin content were performed using specific dyes on the cytometer Beckman Coulter EPICS® XL Flow Cytometer (USA) as previously described [25]. Graphs of the distribution density of the studied parameters obtained on the flow cytometer were analyzed using the program for cytometric data processing FCS Express V3.

Biochemical method. The levels of Mg2+, lactate, glucose, and lactate dehydrogenase activity were determined on the ChemWell 2900 biochemical analyzer (GBG, USA) according to the protocols for the corresponding kits.

Statistical processing of the obtained results was carried out with the help of the mathematical program for medical-biological statistics STATISTIСA (V. 6.0). Calculations and comparisons of the significance of the differences between mean values were made using Student’s t-criterion. Differences with a probability of not less than 95% were considered significant (p < 0.05).

RESULTS AND DISCUSSION

Morphocytological characteristics of human BC cell lines. The morphocytological analysis of the cells of the investigated BC cell lines has established various cytological features, in particular, the diffe­rences in the shape and size of the cells (Fig. 1–3). During cultivating, the cells of a low malignancy degree form colonies, in which the cells adhere to each other. Only a small number of cells go beyond the boundaries of the colonies and change the shape, forming small cytoplasm evaginations (Fig. 1).

 Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism
Fig. 1. Microphotography of living cells of low malignancy degree, × 400

When cultured, cells of T47D line form colonies of irregular shape. The nucleus occupies 35–40% of the cell volume. In the culture of MCF-7, the cells grow individually and have a spindle-shaped or crested-shaped form. The volume of nucleus reaches 40–45% of the cell volume (Fig. 1, 2).

High malignant MDA-MB-468 cells grow without the formation of colonies, significant distances are observed between the cells, and the cells themselves are in contact with each other through cytoplasm evaginations, which in some cells are quite long. The high malignant MDA-MB-231cells grow tightly in groups and look like polyhedra. Mostly, the volume of the nucleus in MDA-MB-231 and MDA-MB-468 cells occupies almost 45–50% and 55–60% of cell volume, respectively (Fig. 2, 3).

 Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism
Fig. 2. Cytomorphological features of cells of different malignancy degree and cells resistant to cisplatin (according to the morphological shape of cells and the size of cells and nuclei). Hematoxylin and eosin, × 400
 Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism
Fig. 3. Microphotography of living cells of high malignancy degree, × 400

The MCF-7/DDP cells resistant to cisplatin, are mostly round-shaped (only some are spindle-shaped, single, grow more flattened. The nuclei occupy about 45–50% of the cell volume (Fig. 2, 3). Thus, the morphocytological analysis of human BC cells showed their significant variability in growth characteristics.

Effect of FrP on pro/antioxidant balance in BC cells. According to the goal, the effect of FrP on the pro/antioxidant balance of BC cells of various malignancy level was investigated by the indexes of the activity of glucose-6-phosphate dehydrogenase (G6PDH) and SOD, the total level of non-protein thiols, ROS and the rate of NO generation.

Adding FrP to the culture medium of cells of various malignancy degree increased the level of free radicals, namely ROS and NO. On the one hand, there are exo­genous and endogenous factors for the transformation of cells, and on the other hand, the cell provides the formation of mechanisms for protection against such transformation. It should be noted that the results of the research demonstrated an increased ROS content under the influence of FrP in all investigated BC cell lines (Table 1).

Table 1. Effect of FrP on the ROS content in BC cell lines, a.u. (n = 10)
Cell line Control FrP
T47D 2.71 ± 0.35 4.60 ± 0.25**
MCF-7 3.10 ± 0.20 5.89 ± 0.33**
MCF-7/DDP 6.95 ± 0.27* 17.37 ± 0.70**
MDA-MB-231 11.30 ± 0.60* 26.00 ± 0.95**
MDA-MB-468 7.27 ± 0.27* 15.99 ± 0.73**
Note: *р < 0.05 compared with cells of low malignancy degree; **р < 0.05 compared with the control.

Thus, in cells of low malignancy degree, 1.7–1.9-fold increase in the level of ROS was observed, and in cells of high malignancy degree, this index increased by 2.2–2.3 times. An even greater increase (2.5 times) of the ROS level was observed in cells resistant to cisplatin.

Treatment of the sensitive and resistant to cisplatin BC cell lines with FrP resulted in increase of ROS levels by 1.9 and 2.5 times, respectively.

The addition of FrP to the culture medium of the cells increased the rate of NO generation in cells of low malignancy degree — by 1.7–1.8 times, which reached up to 288.3 ± 17.3 nmol/2.5•105 cells/min and 306.8 ± 15.4 nmol/2.5•105 cells/min in Т47D and MCF-7 cells, respectively, and in the cells of high malignancy degree — by 2.3–2.5 times. The above data testify to the effectiveness of FrP usage in BC cells (Table 2).

Table 2. Influence of FrP on the rate of NO generation by inducible NO-synthase in BC cells of various malignancy degrees, nmol/2.5•105 cells/min (n = 10)
Cell line Control FrP
T47D 160.00 ± 13.30 288.30 ± 17.30**
MCF-7 180.50 ± 10.00 306.80 ± 15.40**
MCF-7/DDP 240.30 ± 9.50* 528.60 ± 20.00**
MDA-MB-231 255.70 ± 10.50* 639.30 ± 23.50**
MDA-MB-468 275.00 ± 12.70* 632.50 ± 21.90**
Note: *р < 0.05 compared with low-degree malignat cells; **р < 0.05 compared with the control.

When FrP was added to the sensitive and resistant to cisplatin cell lines, the rate of NO generation was by 2.0 and 2.2 times higher than in the control.

After the addition of FrP, the activity of SOD in the BC cells decreased regardless of the degree of malignancy, which suggests a positive effect of the nanocomposite. Thus, in cells of low malignancy degree, the activity of this antioxidant enzyme decreased by 33–34%, and in cells of high malignancy degree, the activity of the enzyme was reduced by 36–40% (Table 3). Reduction of SOD activity was observed in MCF-7 and MCF-7/DDP cell lines after cultivation with FrP (by 1.5- and 1.6-fold respectively).

Table 3. Influence of FrP on the activity of SOD in BC cells of different malignancy degrees, a.u. (n = 10)
Cell line Control FrP
T47D 5.80 ± 0.18 3.82 ± 0.20**
MCF-7 6.90 ± 0.13 4.65 ± 0.17**
MCF-7/DDP 8.33 ± 0.21* 4.91 ± 0.20**
MDA-MB-231 9.41 ± 0.24* 5.65 ± 0.23**
MDA-MB-468 8.57 ± 0.18* 5.50 ± 0.17**
Note: *р < 0.05 compared with low-degree malignat cells; **р < 0.05 compared with the control.

Treatment of the cells with FrP caused the decrease of the level of non-protein thiols that protect the cell from free radicals. In particular, in BC cells of low malignancy degree, the total level of non-protein thiols decreased by 33–35% and amounted to 3.24 ± 0.22 a.u. and 3.67 ± 0.25 a.u. in T47D and MCF-7, respectively, and in BC cells of high malignancy degree it decreased by 39–41% (Table 4).

Addition of FrP to the culture medium of the cisplatin-resistant and cisplatin-sensitive cells (MCF-7/DDP and MCF-7) resulted in the decrease of the level of non-protein thiols by 1.7 and 1.5 times, respectively (Table 4).

So, these data indicate the effect of FrP on the pro/antioxidant balance of human BC cells. At the same time, its effect was more pronounced in the cells of high malignancy degree and with the phenotype of drug resistance.

Table 4. Influence of FrP on the level of non-protein thiols in cells of different malignancy degrees, a.u. (n = 10)
Cell line Control FrP
T47D 4.83 ± 0.30 3.24 ± 0.22**
MCF-7 5.65 ± 0.25 3.67 ± 0.25**
MCF-7/DDP 1.10 ± 0.15* 0.64 ± 0.30**
MDA-MB-231 0.95 ± 0.09* 0.58 ± 0.07**
MDA-MB-468 0.78 ± 0.08* 0.46 ± 0.06**
Note: *р < 0.05 compared with low-degree malignat cells; **р < 0.05 compared with the control.

Effect of FrP on the indexes of energy metabolism in BC cells. It is known that 90–97% of oxygen that enters the cell is absorbed by mitochondria [25]. On the internal membrane of these organelles is located the respiratory chain, the functioning of which leads to the formation of ATP molecules necessary for the life of cells. It was found that in MCF-7 cells, the rate of oxygen consumption was 7.2 ± 0.6 nАО2/min/106, whereas in resistant cells (MCF-7/DDP) — 13.0 ± 0.6 nАО2/min/106 cells (Fig. 4).

 Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism
Fig. 4. Effect of FrP on the rate of oxygen consumption by human cancer cells of different malignancy degree and sensitivity to cisplatin. *р < 0.05 compared with the control

However, when comparing other cells of low malignancy degree (T47D) with cells of high malignancy degree (MDA-MB-231 and MDA-MB-468), the rate of oxygen consumption was also significantly lower.

As known, oxygen is mainly absorbed by mitochondria, and changes in transmembrane mitochondrial potential, so the content of phospholipid cardiolipin have been studied. It is localized on the inner and outer sides of the mitochondrial membrane, as well as in the places of eukaryotic cells contacts. It is necessary to provide catalytic activity of a number of enzymes involved in energy metabolism [25]. For example, cardiolipin is involved in supporting of the process of oxidative phosphorylation in mitochondria, and its deficiency is associated with the development of mitochondrial dysfunction. The interaction of cardiolipin with mitochondrial proteins is specific, since its replacement with other phospholipids makes it impossible, at least in vitro, to completely restore the activity of the respiratory chain. The acyl composition of cardiolipin is an important factor that provides the functional activity of mitochondrial enzymes. In most studies, the idea that cardiolipin directly initiates apoptosis is supported [26]. That is, before the start of apoptosis, the interaction of cardiolipin with cytochrome C is disturbed, which leads to permeabilization of the mitochondrial membrane.

The data obtained in our study indicated that the content of cardiolipin in the cells of low malignancy degree was lower by 10–17% (p < 0.05) compared with the cells of high malignancy degree (Fig. 5).

 Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism
Fig. 5. Influence of FrP on the level of cardiolipin in human breast cancer cells of different malignancy degree. *р < 0.05 compared with the control

The use of FrP led to a decrease in the studied parameters in cells of all human BC lines. However, a more pronounced effect of the nanocomposite was detected in high malignant cells (MDA-MB-231, MDA-MB-468 and MCF-7/DDP). The rate of oxygen consumption by T47D and MCF-7 cells decreased by 25–26% while in MDA-MB-231, MDA-MB-468 and MCF-7/DDP cells — by 38–40%. Similarly, the content of cardiolipin under the action of FrP decreased by 15–16% in T47D and MCF-7 cells, whereas in the cells of high malignancy degree — by 29–32%.

Investigation of the transmembrane mitochondrial potential using the JC-1 dye showed that FrP exerted more significant effect on the potential in cisplatin-resistant cells (MCF-7/DDP) than in MCF-7 cells.

It was found that under the action of FrP the activity of G6PDH decreased in all studied cells. In cells of low malignancy degree, the addition of FrP to the culture medium reduced the activity of G6PDH by 32–33%, while in cells of high malignancy degree — by 37–40% (Table 5). Along with this, under the influence of FrP, in the MCF-7 and MCF-7/DDP cells the activity of G6PDH decreased by 1.5 and 1.6 times, respectively.

Table 5. Influence of FrP on the activity of G6PDH in the BC cells of different malignancy degrees, a.u. (n = 11)
Cell line Control FrP
T47D 380 ± 25 258 ± 20**
MCF-7 400 ± 33 268 ± 30**
MCF-7/DDP 580 ± 35* 360 ± 25**
MDA-MB-231 510 ± 30* 321 ± 33**
MDA-MB-468 530 ± 35* 318 ± 27**
Note: *р < 0.05 compared with low-degree malignat cells; **р < 0.05 compared with the control.

In the conducted studies, it was also found that in BC cells of low malignancy degree the levels of Mg2+ and lactate were lower than in highly malignant cells (MDA-MB-231, MDA-MB-468, and MCF-7/DDP, Fig. 6, 7). Glucose splitting by glycolysis gives little energy, but pyruvic acid can enter the mitochondria and enter the Krebs cycle. Therefore, the above indicators reflect the nature of bioenergetic reactions.

The use of FrP led to a decrease in levels of magnesium ions and lactate, and increased glucose content (Fig. 6–8). However, the nanocomposite did not affect the activity of lactate dehydrogenase in all investigated BC cell lines (Fig. 9).

 Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism
Fig. 6. Effect of FrP on the level of Mg2+ in human breast cancer cells of different malignancy degree. *p < 0.05 compared with control
 Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism
Fig. 7. Effect of FrP on the lactate level in human breast cancer cells of different malignancy degree. *р < 0.05 compared with the control
 Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism
Fig. 8. Effect of FrP on the glucose content in human breast cancer cells of different malignancy degree. *р < 0.05 compared with the control
 Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism
Fig. 9. Effect of FrP on the activity of lactate dehydrogenase in human breast cancer cells of different malignancy degree

Probably, FrP caused a decrease in lactate level by reducing the number of pyruvate, and inhibited phosphorylation reactions, for which Mg2+ ions are required. The indicated effect of FrP was more pronounced in human BC cells of high malignancy degree and with the phenotype of drug resistance. The levels of magnesium ions and lactate in MCF-7 and T47D cells decreased by 21–29% and 14–24%, respectively, whereas in MDA-MB-231, MDA-MB-468 and MCF-7/DDP cells — by 34–38% and 32–35%, respectively.

FrP increased glucose levels in cells of low malignancy degree by 20–23% (p < 0.05), and in cells of high malignancy degree and with a phenotype of drug resistance — by 31–36% (Fig. 8).

Thus, the research data showed that FrP (ferromagnetic nanocomposite) directly affects the pro/antioxidant balance and energy metabolism of human BC cells of different malignancy degree. The indicated effect was more pronounced in cells of high malignancy degree and with the phenotype of drug resistance.

Due to the affinity for iron ions, transformed cells of high malignancy degree and resistant cancer cells accumulate more nanoparticles of ferromagnet nanocomposite than T47D and MCF-7 cells. The high level of “free iron” and ROS in cells of high malignancy degree and resistant cells on the background of significant accumulation of ferromagnetic nanoparticles causes increased activation of the ROS formation and oxidative stress (Fenton reaction). The consequence of oxidative stress under the influence of nanocomposite in transformed cells is the initiation of apoptosis by mitochondrial pathway, mediated by cisplatin. Oxidative damage of transformed cells under the action of nanocomposite also leads to an increase of lipid peroxidation, which causes structural and functional rearrangement of their membranes and contributes to the reduction of invasive properties. That is, due to a violation of the exchange of endogenous iron and a system of antioxidant protection, cells with high malignancy degree and resistant to cytostatics cells become more susceptible to oxidative damage. Consequently, the nanocomposite has the ability of redox-regulation in the cells of high malignancy degree and resistant to drug therapy.

ACKNOWLEDGMENT

The work is carried out within the framework of the integrated target program for fundamental research of the National Academy of Sciences of Ukraine “Fundamental Problems of Development of New Nanomaterials and Nanotechnologies”, 2015–2019 years and the Presidium of the National Academy of Sciences of Ukraine order 27.07.2018 № 419 “Financing of Scientific Projects of the Target Integrated Program of Fundamental Researches of the National Academy of Sciences of Ukraine “Fundamental Problems of Development of New Nanomaterials and Nanotechnologies” in 2018 for CPCEL 6541230 (CPCEL is a program code of classification of expenditures and lending)”.

REFERENCES

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