Effect of Ag nanoparticles on viability of MCF-7 and Vero cell lines and gene expression of apoptotic genes

The newly emerged technology, nanotechnology, represents a promising solution for many medical and industrial problems. Random targeting, resistance, and side effects are the main disadvantages of the available cancer chemotherapy which are critical aspects needed to be managed. So the aim of the study was to suggest the nanoparticles as an alternative therapy for the available therapies through detecting the cytotoxic effect of Ag nanoparticles against cancer and normal cell lines and how they affect the apoptotic function and the genes involved. Ag NPs exhibited a killing rate of 40% in MCF-7 cells (the cancer cell model) at a concentration of 100 μg/ml with almost no effect on Vero cells (the normal cell model). Concerning the phenotypic apoptotic changes that were analyzed by Acridine orange and eosin and hematoxylin, Ag NPs caused the apoptosis and Vacuole degeneration as well as cell formation and the emergence of Necrotic cells in MCF-7 cells, whereas in the normal cell line Vero, no change appears in its phenotype. Treating MCF-7 and Vero cells with Ag NPs upregulated the P53 and P21 gene expression in Vero cells, but their expression was downregulated in MCF-7 cells. PTEN was augmented in both MCF-7 and Vero cells compared to the control. The AgNPs displayed selective effect in their cytotoxicity and both induced the apoptosis effect and might be suggested as a potential therapy since an increase in PTEN expression (up to 250-fold more compared to the control) due to the treatment with AgNPs augments the tumor suppressor effects of the PTEN.


Background
Cancer is a fatal disease that is initiated by environmental factors to mutate genes that are involved in regulating the cell growth. It is characterized by uncontrolled growth of cells, where the behavior of abnormal cells destroys the surrounding tissues [1]. The traditional methods of treating cancer are surgery, radiotherapy, immunotherapy, hormonal therapy, and chemotherapy [2], but these treatments randomly target the tumor and cause undesirable side effects [3]. So, efforts were made to find alternatives such as nanoparticles. Nanoparticles or dwarfs " in Latin" are materials that do not exceed 100 nm in their sizes [4]. Nanotechnology offers a new approach to treat cancer since it carries the potential to reduce systemic toxicity by developing functional molecules as directed treatment chemically or biosynthesized such as Fe3O4/Ag nanocomposite. It also provides an alternative strategy to circumvent the resistance to multiple drugs including antibiotics, such as using the biologically synthesized CuFe 2 O 4 @Ag nanocomposite [5][6][7][8][9]. Very small size, large surface area to mass ratio, and high reactivity, these properties make it important in designing high-precision materials and overcoming barriers in diagnostic and therapeutic factors [10]. Silver nanoparticles (Ag NPs) were used due to their unique properties and known effects in cancer treatment [11,12]. Ag NPs have many applications in the field of medicine such as antibacterial agents, drug delivery vectors, and physical treatment agents. They have the ability to induce oxidative stress, mitochondrial membrane change, cell death by apoptosis, DNA damage, and cytokine production [13]. Various kinds of toxicity have been detected after exposure to nanoparticles such as changes associated with oxidative stress such as apoptosis, gene expression changes, and lipid oxidation [14].
So the aim of this study was to analyze the effect of AgNPs on cytotoxicity of cells and effect on genes implicated in apoptosis P53, P21, and PTEN.

Effect of nanoparticles on the growth of cell lines
Nanoparticles were prepared in distilled water using laser beam and were characterized by TEM and UV absorbance. The cytotoxic effect of nanoparticles was performed according to [15]. Confluent Vero (as a model for normal cells) and MCF-7 (as a cancer cell line) monolayers were simultaneously trypsinized as described above, and then, the suspensions were adjusted to a concentration of 10,000 cell/ml in the growth medium. The cells were then seeded in 96-well plates at a volume of 100 μl in each well and i the cells at 37°C were incubated for 24 h then were treated with Ag NPs at serial concentrations of 12.5, 25, and 100 μg/ml.
The plates were incubated for 48 h, after which the media and nanomaterials were removed. MTT staining was performed by adding 10 μg/ml of MTT in each well and 90 μl of the media-free serum; the plates were again incubated for 1.5-3 h. After incubation, MTT was removed and 100 μl of DMSO was added instead for 20 min in the dark; finally, the absorbance was taken at 490 nm using ELISA. Reader and the cytotoxicity was calculated as follows: where A was the mean optical density of control well and B was the optical density of treatment well.

Morphological characterization of cell death Eosin and hematoxylin stain
The confluent monolayer cells were plated in 6-well plate containing coverslips within each well and incubated for 24 h. The coverslip-growing cells were treated with nanoparticles and then incubated for 48 h to allow enough time for the material to inhibit or kill the cells, and then, the staining was achieved according to [16].

Acridine orange stain
Dissolve 1 μg of orange acridine and 1 μg of ethidium bromide with 10 mL of PBS, making the prepared concentration 1%. The dye is prepared under dark conditions and at room temperature. Next, stain cover slid [17].

Agarose gel electrophoresis
DNA was visualized by 1.5% agarose gel electrophoresis stained with ethidium bromide according to SiZer ™ DNA Markers.

Converting RNA to cDNA from cell line
To convert RNA to cDNA, Accupower RocketScript RT Premix from Bioneer, CA, K-2101, was employed relying on reverse transcriptase enzymes.

Protocol
RNA templates were extracted from cell line converted into complementary DNA (cDNA). The kit components were added to the reaction mixture and completed to a final volume of 20 ml according to Accupower Rocket-Script RT Premix from Bioneer instruction as shown in Tables 1 and 2.

qPCR primers
Sequences of PTEN, P21, P53, and β-actin primers that were used in real-time PCR to evaluate gene expression are shown in Table 3.

Preparation of the real-time PCR reaction
To prepare real-time PCR reaction, RealMOD™Green SF 2X qPCR mix from iNtRON kit was used. The reaction was set according to instruction enclosed with RealM-OD™Green SF 2X qPCR mix from iNtRON kit. Then,  the reaction was conducted by PCR reaction condition (Tables 4 and 5).

Ag NPs
AgNPs' characterization results are provided as supplementary 1. In order to evaluate the cytotoxic effect of the prepared AgNps on the viability of MCF-7 and Vero cells, the cells were left to grow in RPMI 1640 media for 24 h and then treated with (12.5, 25, 100 μg/ml) of AgNPs for 48 h. The viability of MCF7 cells was assessed by MTT staining of treated or control samples and the absorbance was detected at 490 nm. MCF-7 viability was clearly reduced in a dose-dependent manner since the viability decreased to 60% when the concentration was increased to 100 μg/ml. In contrast, AgNPs exhibited almost no effect on viability of Vero cells at 100 μg/ml ( Fig. 1). To summarize, AgNPs showed an activity that discriminates between cell lines and the aneuploidy cells and cancer cells.

Effect of nanoparticles on morphology of Vero and MCF-7 cells
The morphological effect of the prepared nanoparticles on Vero and MCF-7 cells was detected by Acridine orange and eosin and hematoxylin staining under fluorescent and light microscope. Vero and MCF-7 cells were grown in RPMI 1640 with 10% FBS media for 24 h and then treated with 100 μg/ml of Ag for 42 h. Acridine orange was used to stain cells, and morphological changes were traced in comparison with the control molecular layer. Treatment with AgNPs made the nuclei smaller (Figs. 2, 3, 4, and 5).

Effect of nanoparticles on genomic DNA (DNA fragmentation test)
Effect of the tested nanoparticles on genomic DNA of different cell lines was analyzed by agarose gel staining. MCF-7 and Vero cells were treated with 100 μg/ml of Ag NPs for 48 h; then, the genomic DNA of each treated sample was loaded on agarose. Obviously, the control MCF-7 genomic DNA showed intense, sharp, and larger band compared to the Ag-treated DNA at 100 μg/ml (Fig. 6).

P53 relative expression in Vero and MCF-7 cells
In order to evaluate the P53 gene expression in Vero and MCF-7 cells, RNA samples that were extracted from cells treated with Ag NPS and were reverse transcribed to make the first strand of DNA were used as the templates. cDNA template was mixed with primers amplifying P53 to measure its gene expression using qPCR SYBR green. AgNPs exhibited a very different outcome on P53 expression; the treated Vero showed the highest expression followed by the control MCF-7, treated MCF-7, and the control Vero respectively (Fig. 7).

P21 gene expression in Vero and MCF-7 cells
Given the important role of P21 in suppressing cancer cells, again cDNA from total RNA was made from cells treated with AgNPs and used as templates for relative gene expression assay normalized to the control genes. P21 expression was upregulated due to treating Vero cells while its level in untreated Vero or both treated and untreated MCF-7 cells was the same (Fig. 8).

PTEN gene expression in Vero and MCF-7
Due to the position of PTEN gene as one of the most tumor suppressor genes missing in cancer cells, PTEN  mRNA expression was evaluated in both treated and untreated. AgNPs treatment enhanced the expression of PTEN in both cell lines compared to its level in the corresponding controls (Fig. 9).

Effect of Ag NPs on cell lines
The cells were treated with a series of concentrations of AgNPs for 48 h. The results showed that the cell survival rate declines with the increasing concentrations of Ag NPs in MCF-7 cell line. This is consistent with [22][23][24] when HeLa cells are treated with plant-manufactured silver nanoparticles. However, MCF-7 shows the lowest sensitivity for treatment with AgNPs compared to other cell lines [25]. Silver nanoparticles show their toxicity through induction of oxidative stress by generating reactive oxygen species [26,27]. Increasing level of ROS creates oxidative stress inside the cells and consequently leads to cell death through programmed cell death [28]. In addition, increased ROS levels may cause mitochondrial membrane damage and consequently leads to programmed cell death [29,30]. In HeLa cells, cells treated with Ag NPs face death due to the decreased glutathione levels and the increase in the level of lipid peroxide, which in turn kills the cells due to the oxidative stress response [31,32]. Another critical effect of Ag NPs is the accumulation of silver nanoparticles in the nucleus of GBM cells which leads to instability of chromosomes and mitosis. Ag NPs interfere with the structure of cellular actin which eventually kills the cells [31]. Mitochondria-dependent apoptosis can also be triggered by treatment with Ag NPs. Ag NPs can dissociate ions, hence disrupting the mitochondria membranes releasing cytochrome to the cytoplasm [31,33,34]. Ag NPs may also exhibit its effect on cancer cells through the physical-chemical interaction of the cellular proteins with the nanoparticles [35].

Morphological analysis of nanoparticle effect on cell lines (apoptosis detection)
Changes in cell morphology of Vero and MCF-7 cells and detection of apoptosis signs were investigated by Acridine orange at 25 μg/ml of Ag NPs for 48 h. The was detected as apoptosis or necrosis compared with the untreated controls. Ag NPs did not show any phenotypic changes in Acridine orange, revealing that these substances do not affect the normal cells, whereas treating MCF-7 cells with the same particles created phenotypic changes such as apoptosis, vacuole degeneration, change in shape of some cells to rod shape, and necrotic cells (enlarged).
Breast cancer cells treated with Ag NPs show several morphological symptoms that can be easily under inverted microscope. Most of the cellular contents are condensed and take a position at one side of the cells, larger cytoplasm and have a granulus appearance. Attached cells tend to look rounded and others become loose since the junctions among them become weaker [36]. Ag NPs-A549-treated cells for 72 h show typical apoptotic features such as condensed nuclei, membrane blabbing, and apoptotic fragments [37,38]. Morphology of HeLa cells is affected with the exposure to AgNPs since they lose their regular shape with shrank edges, loosen adhesions, more dead floating cells, and decreased cellular density which suggests antineoplastic action of Ag NPs through apoptosis induction [39]. More or less the same effect is noticed in Ag NP-treated BEAS-2B cells when Ag NPs is gathered in endocytic vesicles genotoxic effects with increased ROS generation, formation of micronucleus, and enhanced DNA damage [40,41].

Effect of nanoparticles on genomic DNA (DNA fragmentation test)
To further confirm the apoptotic event, a DNA ladder test was conducted in agarose gel. Vero and MCF-7 are treated with 100 μg/ml Ag NPS for 42 h. Genomic DNA is extracted from the treated or control equivalent cultures and the results show that the DNA is not affected and the genomic DNA is detected as one band at the top of the gel. No detectable fragmentation in agarose contradicts with the idea of having apoptosis due to the treatment with the NPS and with the results of Acridine orange staining. This might be due to the need for further processing that the cells need before extracting the genomic DNA such as treating cells with hypotonic solution. Rather, the genomic DNA showed the features of necrosis and spread DNA in the background is detected and this agrees with [42]. Another reason for not being able to detect the small fragments of genomic DNA [4] is that few cell lines including MCF-7 do not follow the DNA ladder pattern when they die [43,44]. Cell death without DNA fragmentation occurs for a reason that it might reduce the risk of transfer of oncogenic genes from an affected cell to an adjacent healthy cell or phagocytic cells [45]. in Vero treated cells but reduced it in MCF-7-treated cells. Ag NPs induce apoptosis in a P53-dependent pathway using the p53 inhibitor pifithrin α in human breast cancer cells [46,47]. Continuous exposure of A549 cells to Ag NPs for 72 h reduces the gene expression of p53  [48]. Starch-covered Ag NPs also increases the P53 gene expression in colon cancer cells. The only explanation for such reduction in MCF-7-treated samples can be the 48 h is a long-term exposure.

Effect of nanoparticles on P21 expression
The data showed that AgNPs treatment causes decrease in p21 expression in cancer cells. When treating normal cells (Vero) with Ag NPs, there is a higher expression in P21 compared to control. Consistent with the use of Ag NPs and Ag/C225 which are approximately 20 nm and which show an inhibitory effect on the proliferation of the human pharyngeal cancer cell line and the throat cancer cell line HEP-2 has been investigated. The inhibitory effect is a reflection of a decrease in the level of P21 in the cellular lines [49]. Continuous exposure of A549 cells reduces the gene expression of p21 [48]. induces the senescence and growth arrest. It inhibits the expression of genes that are implicated in in mitosis and DNA replication and repair [50].

Effect of nanoparticles on PTEN expression
Interestingly, the data show an increase in PTENT expression in AgNP-treated Vero and MCF-7-treated cells but to a lower extent in the MCF-7 cells compared to the control. PTEN is antitumor protein and restoring the function of this protein suppresses the cancer cell proliferation and even the resistance to chemotherapy [40,41]. The PTEN gene functions as a negative regulator of the PI3K/Akt pathway. Therefore, inhibition of this pathway stimulates programmed cell death in different types of cell lines [51]. So, it suggests it as a typical target to develop anticancer treatment strategies for cancer patients [52]. In addition, treatment with polymer lipid hybrid nanoparticles that restores the normal function of PTEN can enhance the apoptosis in prostate cancer [53].

Conclusion
The study was designed in order to evaluate the possibility of using the prepared AgNPs as antitumor therapy.
In cytotoxicity testing, we found that Ag NPS are selective in killing cancerous cells but not the normal ones. Moreover, treating cells with Ag NPs, apoptosis was activated by activating the P21 and PTEN gene. In fact, PTEN increased gene expression (due to the treatment with AgNPs might have anti-tumor effect and suppressing the cell division). Fold in MCF7 or 3 fold in Vero cells compared to the control, PTEN abrogates the tyrosine kinases to control tumor cell invasion and metastasis. More detailed study is needed to use the AgNPs in experimental animals to analyze their effect in an in vivo system or even in other cell lines.