Propolis nanoparticles synthesis and characterization with cytotoxic and apoptotic effects on breast cancer cells

Abstract

The aim of this work was to investigate the antiproliferative and pro-apoptotic effects of propolis on human breast cancer cells using a simple and inexpensive nanoformulation. Various methods were used to characterize the surface and morphology of propolis nanoparticles (NP-Pro), including UV-VIS, FTIR, DLS, and EDX-SEM. The cytotoxic activity of MCF-7, MDA-MB-231 breast cancer cells was measured by XTT technique and compared with healthy cells (MCF-10A). The apoptotic effect of NP-Pro was assessed by flow cytometry using Annexin/PI. The IC₅₀ values of NP-Pro on MCF-7, MDA-MB-231 and MCF-10A cells were 13.67 ± 0.89, 17.89 ± 0.6, 29.9 ± 0.56 µg, respectively. According to these findings, the NP-Pro form we prepared was more cytotoxic on cancer cells than propolis. Cancer cells had much stronger apoptotic activity than healthy cells (p < 0.0001). In this work, NP-Pro was produced through a distinct, straightforward, and low-cost method. In summary, the NP-Pro we synthesized may be a promising potential agent for breast cancer treatment due to its specific cytotoxicity via apoptosis.

KEYWORDS:

• Breast cancer
• propolis
• nanoparticle
• cytotoxicity

1. Introduction

Cancer has a high mortality rate for men and women throughout the world. Although the life expectancy of patients can be prolonged by new treatments and methods of early cancer detection, the treatment successes are not at the desired level. Therefore, cancer has been a major public health problem for decades [1]. Therefore, new ways to treat or prevent cancer are needed. In particular, new agents that are effective on cancer cells but have fewer chemotherapeutic side effects in healthy cells have been explored [2, 3].

Propolis is a promising natural therapeutic agent for cancer treatment. Propolis, a bee product, is widely used as a dietary supplement because it contains several bioactive ingredients. Flavonoids, polyphenols, and terpenes are the main components of propolis [4]. Propolis is a natural substance that has antiseptic, anti-inflammatory, antifungal, spasmolytic, anesthetic and antioxidant properties [5, 6]. It has also been used as an anti-inflammatory and antimicrobial agent for decades [7]. With its wide range of activities and a variety of possible pharmacological effects, propolis has an important structure for the development of new derivatives with potential therapeutic applications [8].

Propolis has an anticarcinogenic effect, which is one of its most important biological effects. The main active element in propolis responsible for the anticancer effect has been identified as caffeic acid phenethyl ester (CAPE). In a study, it was found to slow down the proliferation of transformed cells by causing apoptosis in human cells [9]. While CAPE selectively exerts cytostatic effects on tumour-transformed cells, it has been observed to be less effective or ineffective on normal fibroblasts [10, 11]. It can also inhibit the proliferation of many human tumour cells, and is thought to achieve this effect by inhibiting the oxidative process triggered by the mitogenic stimulus [12, 13]. In a study conducted on the human leukemic HL-60 cell line, the apoptosis-inducing mechanism was investigated, and it was reported that treatment with CAPE caused a time-dependent increase in the number of apoptotic cells in this cell line [14]. The protective effect of caffeic acid esters such as methyl cafeate, phenethyl cafeate, and phenylethyldimethyl cafeate in propolis against cancer has been studied. The inhibitory effect of azoxymetal on colon cancer was found for all three of these compounds. In human colon adenocarcinoma, all three caffeic acid esters inhibited the action of the enzyme tyrosine kinase [15]. In another study, water-soluble phenolic compounds in propolis were shown to have an antimetastatic effect in mouse tumours [16]. Cinnamic acid, one of the active components of propolis, has been shown to have an antitumour effect on human malignant tumours such as prostate, lung, melanoma, glioblastoma and adenocarcinoma [17]. Another active component of propolis is chrysin, which has anti-inflammatory, anti-cancer, and antioxidant effects. Chrysin can decrease the levels of COX −2 proteins and mRNA produced by lipopolysaccharide (LPS). It has been suggested that the nuclear factor for IL-6 is responsible for the COX −2 inhibition mediated by chrysin [18]. Therefore, much of the research has focused on studying the molecular-level effects and antitumour activity of propolis to uncover its bioactivity.

With the developments in nanotechnology, new and more complex nanomaterials are being used, especially in the fields of biology and medicine, and attempts are being made to produce multifunctional nanocarrier systems, especially for the diagnosis and treatment of common cancers and complex diseases, and interest in this field is increasing. With the development of nanocarriers, new treatment options are being sought that have both reduced side effects in humans and targeted effects on cancer cells [19]. In recent years, nanodrug delivery systems have offered significant advantages in terms of increased permeability and retention (EPR) in the targeted treatment of tumours and may reduce the side effects of anticancer drugs [20]. In particular, nanoparticles smaller than 100 nm are more successful in passively targeting the tumour and are promising carriers to release the therapeutic potential of the drug and reduce its side effects. Nanocarriers can extend the half-life of drugs in therapy, have an improved pharmacokinetic profile, and improve patient compliance. To this end, various nanocarrier systems are being investigated [21].

It is important to obtain nanoparticles with size less than 100 nanometers by optimizing the physical properties of propolis nanoparticles such as particle size, polydispersity index, zeta potential, encapsulation efficiency, and surface morphology [22–25]. Moreover, the synthesis of propolis in nanoparticle size offers advantages in many aspects. Since propolis is hydrophobic, it is not very well absorbed by the body, so other technologies are needed to solve this problem. It is possible to increase the effectiveness of propolis in the body by making it nanosized. Moreover, antitumour studies have shown that the antiproliferative effect of propolis prepared in the form of nanoparticles was much better than that of propolis [26]. Considering all these studies, it is believed that the nanoparticle structure of propolis, which is known to have anticancer activity, could be more effective.

Although there are many studies on nanoformulations of propolis in the literature, the size of nanoparticles is very large [27–29] and the study of their in vitro anticancer activities is limited. In this study, we aimed to determine the effects of propolis on breast cancer cells using a different, easy-to-use, and inexpensive nanoformulation. To this end, we investigated the anti-proliferative and pro-apoptotic effects of propolis on human breast cancer cell lines (MCF-7 and MDA-MB-231) by preparing novel nano-propolis (NP-Pro). The cytotoxic activity was determined by XTT method on cancer cells and compared with healthy cells (MCF-10A). Apoptotic effects and cell cycle analysis were assessed by flow cytometry using Annexin / PI.

2. Material and metot

Ethanol with dimethyl sulfoxide (DMSO) from Sigma’s (St. Louis, USA). Sigma’s (St. Louis, MO, USA) “Cell Viability test kit (XTT), Dulbecco’s Modified Eagle’s Medium (DMEM)” were used. Phosphate buffer saline (PBS) from Medicago (Uppsala, Sweden); Eagle’s Minimum and FBS from Biochrom (Berlin, Germany); penicillin, streptomycin and trypsin from Gibco (Paisley, England) were used. Commercially purchased bee’o up propolis drops (180 mg/mL) (Turkey) were used to prepare nano propolis.

2.1. Preparation of nano propolis

First of all, commercially available liquid propolis was dried in the evaporator at 40 °C and 1 g of powdered propolis was weighed and the nano synthesis procedure was continued. We mixed 1 g of commercially purchased propolis with 50 mL of pure ethanol. A shaking water bath at 30 °C was used to incubate the mixture for five days. As soon as the extract had been cooled, it was filtered through ordinary filter paper [30, 31]. After that, the filtrate was diluted 1:10 with distilled water. An ultrasonic bath was used for 1 h to sonicate the aqueous extract. Lastly, the sample was filtered through a 220 nm filter and stored in a dark environment at room temperature (Figure 1).

Figure 1. Preparation of nanopropolis.

2.2. Characterization of nanopropolis

2.2.1. Zetasizer measurements

By measuring dynamic light scattering (DLS) with the Zetasizer Nano ZS, which employs a 4 mW He–Ne laser at a wavelength of 633 nm and a detection angle of 173°, the size analysis of Nanopropolis samples was assessed. Zetasizer data were reviewed at room temperature. Each sample was measured three times, and the average of the three readings was taken.

2.2.2. FTIR analysis

Fourier Transform Infrared Spectroscopy (FTIR) with the BRUKER ALPHA instrument with an ATR-crystal prism attachment, was used to investigate in diffuse reflection mode at a resolution of 4 cm x 1 Ten times scans were captured in order to have a satisfactory signal to noise ratio. The peak intensity of absorbance for distinct bands was determined in order to examine the quantitative changes in the samples.

2.2.3. EDX-SEM analysis

For SEM and EDX analysis, propolis samples were dropped on a glass substrate and coated with Au/Pd at 45 angstrom thickness by Polaron sc 7620 mini sputter coater device. Regarding liquid samples’ analysis, imaging was performed by dropping on glass substrates and putting on the device after drying.

2.3. Cell cultures

Human breast cancer cell lines (MCF-7 and MDA-MB-231) and healthy breast fibroblast cell lines (MCF-10A) obtained from ATCC were used in cell culture investigations. Dulbecco’s Modified Eagle’s Medium (DMEM) was utilized, which contains 10% fetal bovine serum (FBS), 1% L-Glutamine, and 100 IU/ml penicillin–streptomycin for MCF-7 and MDA-MB-231 and HUVEC cells. Then, cell lines were multiplied at 37 °C, 95% humidity, and 5% in a CO₂ incubator.

2.3.1. Cytotoxicity analysis

The cytotoxicity of NP-Pro (50-60 nm-sized) on MCF-7 (human breast cancer) cells, MDA-MB-231 (human breast cancer) cells, and MCF-10A (normal human breast fibroblast) cells was investigated using XTT (2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)−2H-tetrazolium-5-carboxyanilide) assay [32]. We treated MCF-7, MDA-MB-231 and MCF-10A cells with increasing concentrations of propolis (5, 10, 20, 100 and 200 µg/mL) and NP-Pro (1.25, 5, 10, 20 and 100 µg/mL) for 24 h. The amounts of propolis in NP-Pro were used to calculate the concentrations applied to the cells. Each XTT test was repeated at least 3 times and averaged. According to these findings, IC₅₀ values (50% of the highest inhibitory concentration) of NP-Pro for each breast cancer were calculated.

2.3.2. Determination of apoptosis/necrosis

Annexin V-FITC/propidium was used to determine the ratios of viable, apoptotic/necrotic cells on MCF-7, MDA-MB-231, and MCF-10A cells by flow cytometry analysis. First, MCF-7, MDA-MB-231 and MCF-10A cells were cultured in a 6-well plate as approximately 3 × 10⁵ cells per well, following13.67 µg/mL NP-Pro were added each well and incubated for 24 h.

The cell group in which NP-Pro was most effective was MCF-7 and the dose that killed 50% of these cells was 13.67. For these reasons, evaluation of apoptosis and necrosis was performed at IC₅₀ doses obtained on MCF-7 for all cells and over 24 h to be compatible with XTT tests and compared to other cells. At the end of this period, the contents of the plate were treated with trypsin-EDTA, PBS was added, and the falcon was transferred to tubes and centrifuged at 800 rpm for 8 min to wash the cells with PBS. Washing with PBS was repeated 3 times. Then, 5 µL each of PI and Annexin V were added. They were held for 15 min at ambient temperature in a dark. Following the incubation, 400 µL of binding buffer was poured over ice and analyzed using flow cytometry [33].

2.3.3. Cell cycle analysis

Flow cytometry can be used to measure cellular DNA content and analyze the cell cycle [34]. MCF-7 cells were cultured at 2 × 10⁵ cells per well in a 6-well plate for 24 h. MCF-7, MDA-MB-231, and MCF-10A cells were treated with IC₅₀ values of Pro-NPs and incubated for 24 h. At the end of 24 h, cells were removed by trypsinization, centrifuged, medium removed, washed three times with PBS (5 ml), and centrifuged. Then, the number of cells in each tube was adjusted to 2 × 10⁵, and the cells were incubated at −20 °C for 3 h. The supernatant on the cells was carefully removed. After addition of 1 ml of PBS, the cells were placed in a 200 µl Muse cell cycle kit and incubated for 30 min at room temperature. The samples examined were measured by flow cytometry.

2.4. Statistical analysis

For statistical analysis, Graph pad Prism 8.0 (GraphPad Software, San Diego, CA) was utilized. The Student’s t-test was used to compare statistical results between groups. When P values were less than 0.05, the findings were regarded as statistical significance.

3. Results

According to Figure 2 (a), the size distribution of propolis in the nanoscale is from 43.82 nm up to 91.28 nm, above 100 nm has not been found. The Derived Count Rate was as high as 271.136 while the average particle size was 59.28 nm (PDI: 0.507). This suggests that there is a significant amount of NP-Pro present. The surface potential distribution of propolis at the nanoscale is shown in Figure 2(b). The conductivity (mS/cm) of NP-Pro was measured to be 0.0756 and the surface potential to be −4.21 mV. The PDI value is indicative of stable matured particles with uniform distribution of nanoparticles. When this value is >0.725, the correlation curve is not a smooth parabolic curve, which is due to the constantly changing dimensions of the nanoparticles as they have not matured sufficiently and become stable. Therefore, the PDI value of 0.507 in this study indicates that high-quality, stable nanoparticles are produced [35, 36]. DLS measurements of raw propolis could not be taken because the particle sizes of raw propolis are very large.

Figure 2. (a) Zetasizer Measurement of NP-Pro (b) Zeta Potential Distribution of NP-Pro.

FTIR spectrum of propolis is also given in Figure 3. Vibration peaks belonging to -OH and -NH groups are seen at 3269 cm⁻¹. At 2916-2848 cm⁻¹, peaks of stress vibrations belonging to the –CH aliphatic group were observed. While the peaks of –C = O stretching vibrations are seen at 1736cm⁻¹; 1603–1462 736 cm⁻¹ again –C = O stretching and –NH vibration signals belonging to the amide or amine group were found. -C–O–C vibration signals were observed at 1162 cm⁻¹. At 718 cm⁻¹, –N-H vibration peaks were detected. According to these results, especially 3269-1736-1603-1462 cm⁻¹ vibration signals are characteristic for propolis and have been previously confirmed in the literature [37]. Vibration signals are also very difficult to distinguish in raw propolis. Although no vibrational signals of the hydroxyl and amine functional groups were visible, a sharp peak of the aliphatic groups was detected at 2906 cm-1. Signals of -C = O stretching and -NH vibrations were observed at 1597 cm-1, and signals, albeit weak, of -C–O–C vibrations were detected at 1270-1163-1064 cm-1. In contrast to NP -Pro, C = C = C stretching vibrations were also detected in raw propolis at 2000cm-1.

Figure 3. FTIR Spectrum of Raw Propolis and Nano Propolis.

Figure 4 (a) shows the SEM image of nanoscale propolis. NP-Pro is generally seen below 100 nm. Figure 4 (b) shows SEM images of raw propolis dissolved in water. Although the nanoparticles have a size in the micrometer range (> 10-20 µm), they are in the form of spherical particles. This is because propolis does not dissolve well in water. Propolis generally has sticky properties. Therefore, it is normal for it to ball up and disperse in water to form spherical particles. This result SEM is evidence that we cannot measure raw propolis with the DLS instrument. Figure 4 (c) shows the EDX analysis of NP-Pro. As the sample solution is dropped onto the glass substrate, the atomic ratios of Ca, Si, Mg, and Na from the base material can be observed. The percentage abundance of carbon and oxygen in the sample is remarkable. This shows the presence of organic species in the structure of propolis. However, in Figure 4(d), raw propolis was found to contain oxygen, silicon, and chlorine with a very high percentage of carbon before it becomes nanoscale. This is due to the fact that propolis loses some of its carbon content during the transition to nanoform. Therefore, metals such as magnesium, calcium and sodium, which were previously undetectable, could be detected thanks to the reduction of the carbon content by 70%.

Figure 4. (a) SEM image of NP-Pro (b) SEM image of Raw-Pro (c) EDX analysis of NP-Pro (d) EDX analysis of Raw-Pro.

Recently, the determination of cytotoxic and antiproliferative properties of compounds has become the most important technique for the development of new anticancer drugs. In this work, we tested different doses of Propolis and NP-Pro at MCF-7, MDA-MB-231, and MCF-10A after 24 h. Table 1 shows the effects of 24 h application of NP -Pro on MCF-7 and MDA-MB-231 and the control group MCF-10A cell lines.

Table 1. IC₅₀ values of NP-Pro on breast cancer cell lines for 24 h.

IC50 (µg/mL)	MCF-7	MDA-MB-231	MCF-10A
NP-Pro	13.67 ± 0.89	17.89 ± 0.6	29.90 ± 0.56
Propolis	43.34 ± 2.1	51.18 ± 2.8	58.05 ± 1.87

When the viability of breast cancer and healthy cells was examined, it was found that the viability of healthy cells (MCF −10A) was considerably higher compared to MCF −7 and MDA-MB −231 (p < 0.0001).

The IC₅₀ values of NP-Pro were 13.67 ± 0.89 µg/mL for MCF-7, 17.89 ± 0.6 µg/mL for MDA-MB-231. In addition, raw propolis was also studied in the same cell groups as a control. The IC₅₀ values of Propolis were 59.34 ± 2.1 µg/mL for MCF-7, 63.18 ± 2.8 µg/mL for MDA-MB-231. While the IC₅₀ value of propolis in healthy MCF-10A cells was 67.05 ± 1.87, the IC₅₀ value of NP-Pro was 29.90 ± 0.56. According to our findings, it is clearly seen that the NP-Pro form of propolis is more effective on breast cancer cells. This is a promising result for the treatment of breast cancer. In addition, it was determined that NP-Pro showed the highest cytotoxic effect on MCF-7 cell lines, and also showed a highly selective effect on MDA-MB-231 compared to healthy cells (MCF-10A). The anticancer activity of Np-Pro was higher than Propolis on MCF-7 and MDA-MB-231 cells (Table 1).

Early apoptosis, late apoptosis, necrosis, and viable cell numbers were determined by flow cytometric analysis using a Muse AnnexinV and Cell Dead Kit (MCH100105, Millipore). (Table 2, Figure 5). It is clearly seen that NP-Pro causes cell death through apoptosis in breast cancer cells. In apoptotic evaluation, we compared 3 cell lines, MCF-7, MDA-MB-231 and MCF-10A. According to the IC₅₀ results, it was determined that NP-Pro showed the highest cytotoxic effect on MCF-7 cell lines, and also showed a highly selective effect on MCF-7 compared to healthy cells (MCF-10A). For these reasons, we used the IC₅₀ dose (13.67 ± 0.89 µg/mL) that was effective on MCF-7 for all cell lines and compared the apoptotic activities with other cells. In conclusion, the apoptosis results were in good agreement with the XTT results. When 13.67 ± 0.89 µg/mL of NP-Pro was administered, a dose that killed 50% of MCF-7 cells, 52.4% of MCF-7 cells were viable and 46.5% were apoptotic. In MDA-MB-231 cells, on the other hand, under the same conditions, 65.60% of the cells remained viable and 35% of apoptosis was observed. As for MCF-10A, while 84.26% of the cells were alive, only 15.07 percent of the cells were apoptotic. Furthermore, no remarkable rate of necrosis was observed in all cell groups, suggesting that NP-Pro induces cell death via apoptosis. Moreover, Np-Pro had no significant damage to healthy cells.

Figure 5. Apoptosis analysis of NP-Pro on MCF-7, MDA-MB-231 breast cancer, and MCF-10A human breast epithelial cell lines after 24 h incubation by flow cytometry.

Table 2. Evaluation for apoptosis of MCF-7, MDA-MB-231 and MCF-10A cells applied NP-Pro for 24 h.

Cell Lines	Viability %	Early Apoptosis %	Late Apoptosis %	Total Apoptosis %	Necrosis %
MCF-7	52.40	12.00	34.50	46.5	1.10
MDA-MB-231	65.60	7.30	26.70	34	0.40
MCF-10A	84.26	6.98	8.09	15.07	0.67

In apoptotic evaluation, there was a considerable difference between MCF-7 and MCF-10A regarding total apoptosis (p < 0.0001). There was also a considerable difference between MDA-MB-231 and MCF-10A (p < 0.001). It also seems that apoptotic activation is highest in MCF-7 cells.

In addition, cell cycle analyses were studied to determine the phase in which NP-Pro stopped the cell in the cell cycle using Muse Cell Cycle Assay Kit (MCH100106, Millipore) by flow cytometer on MCF-7 cells (Figure 6, Table 3). We studied cell cycle analysis on MCF-7 cells, the cell group in which NP-Pro was most effective and selective. Cell cycle analysis was performed on MCF-7 cells with the IC₅₀ value obtained from the XTT assay. As a result of the 24 h application of NP-Pro on MCF-7; Values of 45.2 were determined in G0 / G1 phase, 26.8 in S phase, and 27.4 in G2 / M phase (Figure 6, Table 3). Accordingly, it is seen that NP-Pro increased the G0/G1 phase from 39.6% to 45.2% on MCF-7 cells, decreased the S phase from 30.7% to 28.8%, and reduced the G2/M phase from 32.1% to 27.4%.

Figure 6. Cell cycle analysis on MCF-7 cells treated with NP-Pro for 24 h. Flow cytometric analysis was used to determine the percentage of cells in the G0/G1, S, and G2/M phases.

Table 3. Cell cycle analysis of MCF-7 applied NP-Pro by flow cytometry for 24 h.

	G0/G1	S	G2 /M
Control (%)	39.6	30.7	32.1
NP-Pro (%)	45.2	26.8	27.4

4. Discussion

In the present study, NP-Pro was successfully obtained for the first time with a different method, and NP-Pro reduced the proliferation of breast cancer cells in a dose and time-dependent manner, while it did not significantly damage healthy cells.

According to a study by Amalia et al, the IC₅₀ values of water soluble propolis and bee pollen were 10.8 ± 0.06 and 18.6 ± 0.03 mg/mL on MCF-7, respectively [38]. Hugo Melo de Lima et al. determined that IC₅₀ values ranged from 84.12-129.40 µg/mL in a study they conducted with propolis at MCF-7 cells [39]. A study by Motomura et al., reported that propolis induced cell cycle arrest (blocking G2/M) and apoptosis in leukemic U937 cells via Bcl-2/Bax end of the 24 h incubation [40]. Nascimento et al. synthesized 200–800 nm nanoparticles with propolis [41]. The study by Motomura et al. reported that free propolis was effective in leukemic U937 cells at 300 µg/mL and above [40]. In comparison, in our study, nano propolis seems to be much more effective in MCF-7 cells. In a study by Mısır et al., ethanolic propolis extract (EEP) was incubated with MCF-7 cells for 72 h and the IC₅₀ value of EEP was reported to be 61 mg/mL. [42]. On this basis, the nanopropolis we synthesized appears to be significantly more effective and selective against breast cancer cells (Table 1). The NP-Pro version of propolis is more efficient against breast cancer cells according to our findings. This is an encouraging finding in the treatment of breast cancer. Moreover, it is shown that the NPs-Pro we synthesized (Table 1)  were more effective than many propolis-loaded nanoparticles (Table 4).

Table 4. Comparison of the effects of different propolis-loaded nanoparticles on cancer cells.

Nanostructure	Cell Line	IC50 value	Ref.
Brazilian green propolis	A549	69.17 ± 11.28 µg L^–1	[43]
Phytosomal propolis	Rats prostate hyperplasia cell line	50.46 µg/mL	[44]
FP1 (Propolis extract loaded- NPs1)	HepG2	58.6 µg/ml	[27]
FP3 (Propolis extract loaded- NPs3)		91.8 µg/ml
Silver NPs-propolis	A549	38 µg/mL	[45]
Nano-propolis	PANC-1	26.4 ± 2.1 µg/mL	[46]
NP-Pro	MCF-7	13.67 ± 0.89	This Study
	MDA-MB-231	17.89 ± 0.6

In one study, different dosages of propolis NP were tried in different types of cancer to reduce the side effects of cisplatin, a drug used in cancer treatment, and it was found that propolis in the form of nanoparticles had reduced the side effects of cisplatin [47]. In another study, chitosan-propolis nanoparticles were characterized in terms of their physical properties and in vitro release profile, and altered the expression of genes involved in biofilm formation in vitro by E. faecalis. The results of the study showed that chitosan-propolis nanoformulation can be considered as a potential anti-biofilm agent in the defense against biofilm-forming infections, such as chronic wounds and surgical site infections [48].

Apoptosis is controlled cell death and is important for the balance between cell proliferation and cell death. When this balance is disturbed, the inability of the cell to undergo apoptosis can lead to cancer formation. For these reasons, anticancer drugs that target in cell death by apoptosis are gaining importance [49]. In our study, it was observed that NP-Pro significantly increased apoptosis in breast cancer cells, especially in MCF-7 cells (Figure 5, Table 2).

In a study by Luo et al., the dose-dependent (0.5, 0.25, 0.125, and 0.063 mg/mL) apoptotic and antitumorigenic effects of propolis obtained from different geographical regions on MCF-7 cell line were investigated and it was shown that apoptosis induction increased with increasing dose rate [50]. In another study, the active substance PM-3 (3- [2-dimethyl-8- (3-methyl-2-butyl) benzofuran] −6-propenoic acid) of propolis was significantly cytotoxic on MCF-7 cells. This effect has been associated with the induction of apoptosis and inhibition of cell cycle development, and proplis has been shown to inhibit the expression of cyclin D1 at the transcriptional level [51].

Cell cycle disruption plays an important role in the development of cancer. The G0 phase is a resting phase in which the cell has left the cycle and is no longer dividing. G1 is the initial phase of interphase, which extends from the end of the preceding M phase to the beginning of DNA synthesis. S phase begins after DNA synthesis begins and ends after all chromosomes have been replicated. G2 phase is a phase of protein synthesis and rapid cell development that begins after DNA replication to prepare the cell for mitosis. M phase is also referred to as the chromosome segregation phase [52–54]. Studies on the effects of anticancer drugs on the cell cycle will also shed light on the mechanisms of apoptosis [55, 56]. The G0/G1 and G2/M phases are the cell cycle checkpoints. Cells may temporarily stop at cell cycle checkpoints to allow the repair of cell damage. If cellular damage cannot be repaired properly, cell death can be induced at this point by signaling apoptosis [57, 58]. In our study, NP-Pro increased G0/G1 phase from 39.6% to 45.2% in MCF −7 cells. These results suggest that NP-Pro inhibits the survival of MCF −7 cells by causing cell arrest in G0/G1 phase (Figure 6, Table 3).

5. Conclusion

In this study, NP-Pro was successfully produced using a simple and low-cost method. The NP-Pro version of propolis is more effective against breast cancer cells according to our findings. NP-Pro first reduced the proliferation of breast cancer cells in a dose – and time-dependent manner. Secondly, using AnnexinV/ PI staining, Nanopropolis was found to increase the apoptosis rate in cancer cells, thirdly, it arrested the cell cycle in G0/G1 phase. According to these results, Nanopropolis synthesized by us has a remarkable antiproliferative effect on cancer cells and is also a promising agent with its selective properties on breast cancer cells.

Disclosure statement

No potential conflict of interest was reported by the author(s).