Nano Research & Applications

Keywords

Iron oxide nanoparticles; Cell viability; Oxidative stress; DNA damage; Kegg analysis; Biological pathways

Introduction

According to the International Agency for Research on Cancer, breast cancer is the second most frequently occurring cancer in females and widely diagnosed malignancy in males [1]. With approximately 1.45 million new cases reported in 2012, breast cancer is the third most frequent malignancy and the fourth major cause of cancer related deaths globally. It has been estimated that 2.4 million breast cancer cases will rise to 1.35 million for men and 1.07 million for women by 2035 [2]. Therefore, it is desirable to minimize the breast cancer [1,3]. For researchers, gastroenterologists, and oncologists, treating breast cancer remains a major challenge. 5-fluorouracil is the only drug accepted for the treatment of advanced breast cancer. [4]. Even though chemotherapy is still a conventional treatment, it has some adverse effects, including nausea, loss of appetite, vomiting, constipation, and alopecia. As a result, new therapeutic approaches are required [5].

Iron Oxide Nanoparticles (IONPs) have recently been used as strong broad spectrum antibacterial agents in textiles, food storage containers, antiseptic sprays, bandages, and anti-cancer medicines due to their unique qualities. [6,7]. IONPs are effective anticancer agents. Low concentrations of IONPs cause DNA damage and chromosomal disorder without substantial toxicity [8,9]. Lima et al. found no evidence of genotoxicity in human culture cells treated with 10 mg/mL of capped IONPs with an average size of 6-80 nm. IONPs are known to associate with cells and to manage different cellular responses in both active and passive ways [10]. By suppressing the phosphorylation of protein kinase B/Akt, Gurunathan et al. revealed the antiangiogenic properties of IONPs in vascular endothelial cells, which depicts equal efficacy to a natural antiangiogenic molecule called as pigment epithelium-derived factor [11].

SriRam et al. revealed exceptional cytotoxicity capacity against Dalton's Lymphoma Ascites (DLA) and DLA-induced tumours in mice, with considerably better survival rates in the tumour mouse model as compared to the untreated group [12]. After penetrating the cells through the endocytic route endosomes of low concentrations of poly (N-vinyl-2-pyrrolidone)-coated IONPs reduces the viability of acute myeloid leukemia cells and K562 cells in a dose-dependent manner [13]. Importantly, IONPs have anti-cancer and cytotoxic effects on cancer cells via various mechanisms, such as inducing cellular apoptosis via mitochondrial dependent and mitochondrial independent pathways against various types of cancer cells, such as human breast cancer cell lines (MCF-7, MDA-MB-231), liver (HepG2), lung (A549), and skin and oral cancer cell lines (HT144), via the leakage of lactate dehydrogenase, impairment of mitochondrial dysfunction, and the Reactive Oxygen Species (ROS) generation [14-19].

However, the anticancer activity of IONPs is affected by a variety of factors, including their shape, size, and surface coatings, as well as surface charge, cell types, and reducing agent utilised to synthesize the IONPs. While conventional, physical, and chemical procedures are straightforward, the processes consume energy and need the use of harmful compounds [15]. Synthetic capping agents, like polyethylene glycol, polyvinyl pyrrolidone, and polyvinyl alcohol, are also toxic and poisonous. As a result, interest in utilising biomolecules as capping agents for IONPs has elevated [20].

With the emergence of next generation sequencing technologies, such as RNA sequencing (RNA-Seqe), which potentially substitute complementary DNA (cDNA) microarrays as the preferred approach for gene expression profiling of cells and tissues, the transcriptomics field has advanced fast in recent years [21,22]. Transcriptome analysis, in conjunction with bioinformatics datamining techniques, could be utilised concurrently for studying several genes/targets and determine action mechanism following treatments. RNA-Seqe is a helpful technique for detecting differentially expressed genes after treatment with several drugs [23]. When compared to whole-genome sequencing, RNA-Seqe has several advantages: it focuses on transcribed regions of genomes, it is free of probe-specific hybridization of microarrays, and it has broad coverage, allowing for unbiased identification of both coding and noncoding novel transcripts, as well as lowabundance transcripts [23-25]. To overcome the restrictions of conventional approaches, we developed IONPs with well controlled morphological and physicochemical properties for physiological application in humans, thus, used biological method to synthesise IONPs utilising a pure aqueous solution of NAR.

Few studies have investigated into the synthesis of IONPs using pure flavonoid reduction of Iron Oxide. In this study, we used MCF 7 cell lines to systematically examine the anticancer potential of NAR stabilised IONPs, as well as the mechanism of IONPs in regulating the development of breast cancer cells using the RNA-Seqe technique. Although IONPs have been proven to reduce cell viability and proliferation in various cancer cells, the method by which they do so remains unknown. As a result, gene expression profiling could be employed as a new method for detecting molecular pathways in interactions with nanoparticles and biological systems. Furthermore, our methodology can predict IONP toxicity mechanisms in cancer cells.

Materials and Methods

Material

MCF 7 breast cell lines were purchased from National Centre for Cell Science (NCCS), Pune, India. Life Technologies/Gibco (Grand Island, NY, USA) supplied trypsin-EDTA, penicillin-streptomycin, Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibioticantimycotic. Sigma-Aldrich (St. Louis, MO, USA) provided in vitro toxicity assay kit and foetal bovine serum. Unless otherwise specified, all chemicals including iron oxide nitrate were bought from Sigma-Aldrich.

Synthesis and characterization of IONPSs

The synthesis and characterization of IONPs were carried out as described previously [17]. The IONPs were synthesized using NAR, dissolved in DMSO. IONPs were synthesized by incubating 50 M NAR in 100 mL of water containing 2 mM Fe(NO₃)₃, for 1 hr at 37°C. The development of IONPs in the reaction mixture was attributed to the colour change from pale yellow to dark yellowish brown.

Cell viability and cell proliferation

A Cell Counting Kit-8 was used to determine cell viability (CCK- 8, CK04-01, Dojindo Laboratories, Kumamoto and Japan). Cell proliferation was measured in accordance with the manufacturer's instructions (Roche, Basel, Switzerland). MCF 7 cells was placed on 96-well plates with varying concentrations of IONPs. After 24 hrs of culture at 37°C and 5% CO₂ in a humidified incubator, CCK-8 solution (10 μL) were added into each well, and the plate was incubated for next 2 hr at 37°C. At 450 nm, absorbance is recorded with a microplate reader (Multiskan FC, Thermo Fisher Scientific, Inc, Waltham, MA, USA)

Membrane integrity

Using an LDH Cytotoxicity Detection kit, the membrane integrity of MCF 7 cell lines was evaluated. Cells were exposed for 24 hrs to varying concentrations of IONPs. Following that, 100 μL of cell free supernatant from each well was transferred in triplicate to the wells of a 96-well plate, followed by 100 μL of LDH reaction mixture. After 3 hr, 100 μL of cell free supernatant was transferred in triplicate from each well to the well of 490 nm microplate reader.

Assessment of dead-cell protease activity

As previously stated, dead-cell protease activity was measured. In MCF 7 cells, the cytotoxicity test was used to determine the cytotoxicity of IONPs. An intracellular protease reaction using a luminogenic peptide substrate was used to assess cytotoxicity (alanyl-alanylphenylalanyl-aminoluciferin). Luminescence is evaluated with a Luminescence Counter (Perkin Elmer, Waltham, MA, USA).

Determination of intracellular ROS

MCF 7 cells were exposed to IONPs for 24hr. ROS was measured using previously model based on the intracellular peroxide dependent oxidation of 20, 70-Dichlorodihydrofluoresceindiacetate (DCFHDA, Molecular Probes, Eugene, OR, USA) to generate the fluorescent molecule 20, 70-Dichlorofluorescein (DCF).

Measurement of MDA

Oxidative stress indicators like Malondialdehyde (MDA) was assessed in accordance with the manufacturer instructions. The cells were cultivated in 75cm² culture flasks and exposed to different concentrations of IONPs for 24 hrs. The cells were extracted in chilled Phosphate-Buffered Saline (PBS) by scraping and washing twice with saline, followed by centrifugation at 4°C for 6 minutes at 1500 rpm. To obtain the cell lysate, the cell pellet was sonicated at 15 W for 10 sec (thrice). The generated supernatant was kept at -70°C until evaluation.

Jc-1 assay

MCF 7 cells undergoes treatment with IONPs for 24 hrs. JC-1, a cationic fluorescent dye, is used for measuring the changes in Mitochondrial Membrane Potential (MMP) (Molecular Probes). The fluorescence of JC-1 accumulates and JC-1 monomers was measured using a Gemini EM fluorescent microplate reader at excitation wavelengths of 488 nm and emission wavelengths of 583 and 525 nm (Molecular Devices, Sunnyvale, CA, USA).

Measurement of ATP

ATP levels in MCF 7 cells subjected to various concentrations of IONPs for 24 hrs were measured according to the manufacturer instructions (Sigma-Aldrich, Catalog Number MAK135).

Tunel analysis

The terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method was used with an insitu detection kit (Promega, Madison, WI, USA) to detect apoptotic cells in groups undergoes treatment with IONPs (5 μg/mL). MCF 7 cells were plated in 6 well plates and incubated with IONPs (5 μg/mL) for 24 hrs and TUNEL analysis is performed to determine cell apoptosis. Nikon Eclipse E400 fluorescent microscope (Nikon 40 Plan 40/0.65, Tokyo, Japan) was used to examine the samples. The difference in the no. of TUNEL-positive cells between the control and experimental samples was statistically analysed.

RNA-seqe and downstream bioinformatics analysis

Total RNA was extracted using the TRIzol reagent according to the manufacturer instructions (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA quality was determined utilizing the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and the RNA 6000 Nano LabChip kit. RNA-Seqe libraries was created with the Illumina^® TruSeq stranded total RNA Library Prep Kit v2 (San Diego, CA, USA) and sequenced with an Illumina^® Hiseq2500 to yield 150-base paired end reads. Each sample had a sequencing depth of more than 20 million reads. The readings were aligned using TopHat 2.0.13 default settings to GRCh37, and then assembled using Ensembl v75 annotations by Cufflink 2.2.1. The abundance of transcripts were measured in fragments per kb of exon per million fragments mapped (FPKM). Differentially expressed genes were found when the FPKM >2 and fold change >2. The RNA-Seqeuence data can be found on the GEO website (GSE100687). The DAVID (v6.8) approach was used to enrich biological processes and KEGG pathways. Scatter plots and Gene Ontology terms were created using the R package (v3.3.2) and the GO plot programme (version 1.0.2, respectively). To predict the expression patterns of representative genes, the integrative genomics viewer was employed.

Statistical methods

At the least 3-independent experiments were carried out, and the data is expressed as the mean ± standard deviation for all duplicates within an individual experiment. Data were analysed using the Graph-Pad Prism analysis programme (GraphPad, Inc, La Jolla, CA, USA) to discover differences between groups, denoted by an asterisk, using a t-test, multivariate analysis, or one-way analysis of variance, and for multiple comparisons the Tukey test.

Results and Discussion

Synthesis and characterization of IONPs using NAR:

Pure aqueous solution of NAR (50 M) were combined with 2 mM Iron Oxide nitrate and incubated at 37°C for 1 hour to synthesize IONPs. NAR is a flavonoid with effective antibiotic, anticancer, and antiinflammatory properties that act against different types of carcinogenic cells [26-28]. Colour generation occurs quickly in this mixture, most likely due to the interaction between iron oxide ions and NAR [12,28]. UV-visible spectroscopy (UV-vis) was used to confirm the synthesis of IONPs. At 442 nm a single absorption peak was detected in the UV-visible spectrum, indicating that the synthesized IONPs were pure (Figure 1A). IONPs exhibit strong surface plasmon resonance in aqueous solution and their absorbance spectra is affected by their size, shape, and morphology. Pure flavonoids are used in the synthesis of IONPs, which is quicker than using the whole plant extract and it prevents the incorporation of contamination [28-30].

Figure 1: Synthesis and characterization of Iron Oxide Nanoparticles (IONPs). (A) UV–Vis spectra of IONPs, (B) XRD pattern of IONPs, (C) FTIR spectra of IONPs, (D) Size distribution analysis of IONPs using DLS, E) TEM images of IONPs and (F) Particle size distributions from TEM images. The diameter of IONPs was determined from more than 200 particles.

X-ray Diffraction (XRD) analysis was used to analyze the crystalline structure & surface morphology of the synthesized IONPs and it was analyzed between the range of 20° to 80°. The XRD pattern of IONPs produced by reacting aqueous Iron Oxide salt with NAR is depicted in Figure 1B. The XRD pattern revealed typical IONP peaks, indicating that the IONPs are crystalline in nature. Highintensity peaks of IONPs synthesised utilising NAR was detected at approx 39°, 45°, and 77°, corresponding to (111), (200), and (311) Bragg reflections, respectively, and are the precise peak positions for Iron Oxide's face centered cubic lattice structure. For IONPs synthesised using NAR, the average particle size obtained from the XRD pattern using the Scherrer equation is around 7 nm. Our findings are consistent with previous reports of IONPs production employing plant extracts [14], different flavonoids such as hesperidin, naringenin, and diosmin [28], and Ocimum Sanctum leaf extracts and its derivative, quercetin [30].

We performed Fourier-Transform Infrared (FTIR) spectroscopy to analyze whether NAR was responsible for reducing Iron Oxide ions to IONPs. The synthesized IONPs displayed peaks at approx 1641, 2111, and 3269 cm^-1, which corresponds to the groups C=C, C≡C, and amine N-H/O-H stretching vibrations, respectively, as shown in Figure 1C. This denotes that NAR was responsible for reducing Iron Oxide ions to IONPs, which strongly corresponds to the same functional groups found in quercetin is important for reducing Iron Oxide into IONPs [30,31]. Also, IR spectra shows a significant stretching of the O-H bond as a strong signal peak between 3000 and 3500 cm^-1 [14,15,17]. In a previous study, flavonoids was utilised as reducing agents to synthesize IONPs indicated a strong signal for an O-H bond [32].

Although Transmission Electron Microscopy (TEM) can be used to evaluate particle size and morphology, it is essential to determine particle size in solution before analysing toxicity in cells. To analyse a large number of particles in a single solution, the Dynamic Light Scattering (DLS) approach is used [15,17]. Figure 1D depicts the particle size distribution measured by DLS for the IONP combination. According to the particle-size histogram, IONPs ranged in size from 1 to 10 nm, with a mean diameter of 7 nm. The DLS intensity analysis indicated a single broad and sharp peak with an average size of 7 ± 1 nm. We utilized TEM, to analyse the consistency of particle sizes and morphologies. Figure 1E shows a TEM picture of IONPs in the micrograph with spherical shape and homogeneous particle size distribution, with sizes similar to those analysed using DLS. The results of size measurement of IONPs from TEM images are summarised in Figure 1F. Both TEM and DLS investigations revealed that the produced IONPs were 7 nm in size. The sizes of nanoparticles synthesised from hesperidin, diosmin, and NAR was determined by Sahu et al. to be around 5 to 50 nm, 5 to 40 nm, and 20 to 80 nm, respectively [28]. IONPs formed from hesperidin and NAR were oval-shaped and polydispersed, whereas diosmin-derived IONPs were hexagonalshaped. Using citrus plant extract, Prathna et al. generated IONPs with an average size of 50 nm [33]. Tulsi extract- and quercetinmediated IONP production, according to Jain and Mehata, had an average size of 14.6 nm and 11.35 nm, respectively [30]. Our outcomes indicate that NAR produces smaller particles, which easily permeate cells and releases Iron Oxide ions more quickly than larger particles.

Effect of IONPs on cell viability and proliferation of MCF 7 Cells

MCF 7 cells were treated with several IONP concentrations and their cell viability was assessed based on mitochondrial activity to determine the toxic effects of IONPs. After 24hrs of exposure, mitochondrial activity was reduced in response to a concentration of 2 μg/mL, and cell viability was quickly reduced when IONP concentrations were elevated from 2 to 10 μg/mL. (Figure 2A). At 4 to 10 μg/mL IONPs, mitochondrial activity is significantly reduced to 50% in MCF 7 cells treated to 5 and 4 μg/mL IONPs. Mitochondrial activity in IONP-exposed cells was significantly reduced at this time and dose. Miethling-Graff et al. [34] investigated the size-dependent (10, 20, 40, 60, and 100 nm) effects of IONPs in the human LoVo cell line and discovered that cellular uptake and toxicity were size-dependent, with small particles easily penetrating the cells, whereas larger particles such as 100-nm particles did not. Likewise, in the human breast cancer cell line MCF 7, para-hydroxybenzoatetetrahydrate (SPHT)-assisted IONP production inhibited cell viability in a dose dependent and time dependent manner. After 24hrs of treatment, cells that were exposed to SPHT-IONPs (8 μg/mL) showed a 50% suppression of cell proliferation [35]. When compared to synthetic IONPs and Fe(NO₃)₃, plant-assisted IONPs exhibited greater antiproliferative activities against MCF 7 cells at a minimal concentration of about 450 nM [36]. The anti-proliferative effects of Abutilon indicum-assisted IONPs on COLO 205 (human colon cancer) and MDCK (normal) cells were dose-dependent [37].

Figure 2: Cell viability and proliferation assessment of IONPs in MCF 7 and HT-29 cells. (A and B) cell viability of MCF 7 and HT-29 cells, (C and D) Cell proliferation of MCF 7 and HT-29 using a BrdU cell proliferation assay. The findings are expressed as the mean ± standard deviation of three separate independent experiments. There was a significant difference is determined by a Student’s t-test (* p <0.05).

Then, using BrdU incorporation during DNA synthesis, we evaluated at the dose-dependent effects of different IONP concentrations on cell proliferation. MCF 7 cells subjected to various concentrations of IONPs exhibited considerably lower proliferation rates as compared to control cells, which is consistent with the cell viability analysis results (Figure 2B). After 24 hrs exposure to various concentrations of IONPs, the proliferation rate was measured in comparison to non-exposed control cells. The results indicate that as the doses increased, the rate of proliferation reduced. Breast cancer cells, lung cancer cells, ovarian cancer cells, neuroblastoma cells all showed antiproliferative effects in the presence of IONPs. HT-29 appears to be more sensitive than MCF 7 cells among the two cell lines studied. As a result, additional research focused on MCF 7 cells [8,38,17,15,39].

IONPs increase cytotoxicity in MCF 7 Cells

Lactate Dehydrogenase (LDH) leakage assay is a cytotoxicity assay for determining cytotoxicity based on the leakage of intracellular molecules through damaged plasma membranes. LDH is a soluble cytoplasmic enzyme found in almost all cells that is released into the extracellular space when the plasma membrane is damaged [16,40,41]. To identify LDH leakage into culture medium, cells were treated for 24 hrs with various concentrations of IONPs (2-10 μg/mL), and leakage was evaluated as the quantity of formazan product generated by standard spectroscopy. LDH activity was measured at 495 nm using an LDH cytotoxicity kit. As expected, increasing IONP doses resulted in LDH leakage that was directly proportional to IONP dosage and improved cytotoxicity (Figure 3A), denoting that cells experiencing accidental cell death due to IONPs, swelling and losing membrane integrity before switching down and releasing their intracellular contents into the surrounding environment. These findings suggest that elevated IONP concentrations disrupt cellular membrane integrity. Our findings support the previous reports that IONPs lead to LDH leakage in many cancer-type cell and Non-Cancerous Cell types, including human breast cancer cells, lung cancer cells, ovarian cancer cells, neuroblastoma cells, microvascular endothelial cells, male somatic cells, spermatogonial stem cells, and neural stem cells [14,38,17,16,39,42,8,43]. The results indicate collectively that the low viability in IONP-exposed MCF 7 cells coincides with increased LDH leakage and significant cytotoxicity. While a number of assays for measurement of cell viability and toxicity have been developed, proteolistic actions associated to cellular death are truly sensitive and are cytotoxic and dependent on membrane integrity.

Figure 3: (A) Measurement of LDH leakage activity and (B) measurement of cell-death protease activity using the CytoTox-Glo cytotoxicity assay. The results are expressed as the mean ± standard deviation of three independent experiments. There was a significant difference is determined by a Student’s t-test (* p <0.05).

Consequently, in MCF 7 cells exposed to various doses of IONPs, we assessed their impact on dead-cell protease activity, their cell viability was measured according to manufacturer instructions (Proméga Corp, G9292, WI, USA), and the method reported previously [44]. With increasing IONP concentrations MCF 7 cells treated with IONPs showed a lower viability (Figure 3B). The results reveal a substantially reduction in the viability of MCF 7 cells.

Effect of IONPs on ROS generation and Malondialdehyde (MDA)

In the presence of oxygen species, iron oxide ions play a significant role in catalysing ROS generation, and IONPs can induce oxidative stress in a range of cellular systems by producing ROS, including human ovarian cancer cells [16], lung cancer (A549) cells [45,46], and human neuroblastoma cells [39]. MCF 7 cells were treated with different concentrations of IONPs for 24 hrs to know the influence of IONPs on ROS formation, and ROS generation was evaluated using an H2DCF-DA assay. When compared to the control, increasing IONP concentrations (2-10 μg/mL) significantly increased ROS levels at concentrations over 6 μg/mL after 24 hrs of exposure (Figure 4A). The amount of ROS produced in response to IONPs treatment was substantially higher than in control cells. Greater concentrations, up to 2-3-fold higher ROS levels, were the most significant effects at all concentrations examined. Miethling-Graff, et al. [34] found that ROS formation was size-dependent in the human LoVo cell line, with small particles producing excess ROS than bigger ones. COLO205 cells subjected to IONPs produced much more ROS than untreated control cells, according to Mata, et al. [37].

Figure 4: Effect of IONPs on Reactive Oxygen Species (ROS) generation. A) MCF 7- ROS generation measured using (DCFH-DA) and B) MDA levels in MCF 7 cells. The results are expressed as the mean ± standard deviation of three independent experiments. There was a significant difference is determined by a Student’s t-test (* p <0.05).

The primary anticancer effect of IONPs is the induction of apoptosis and autophagy via a variety of molecular mechanisms, including a significant decrease in cell viability and motility, impairment of matrix metalloproteinase-2 and -9 activities, and the promotion of ROS production, which induces cell death via apoptosis and autophagy [47]. The potential toxicity of IONPs is determined by the formation of Reactive Oxygen Species (ROS), the depletion of antioxidant defence systems, and the loss of mitochondrial membrane potential [44]. ROS formation in human breast cancer cells is influenced by the surface coating of IONPs by different biological reducing agents, such as bacterial cellular extracts and fungal cellular extracts. For example, IONPs made with fungal extracts generated more ROS than IONPs made with bacterial cellular extracts, implying that coating materials influence ROS formation and, ultimately, cell death [14]. IONPs coated with chitosan-derived polysaccharide, on the other hand, displayed antibacterial action while causing no toxicity in eukaryotic cells [48]. Our investigation imply that IONPs produce significant amounts of ROS, which may induce apoptosis by disrupting the balance between the oxidant and antioxidant enzyme systems, causing cellular redox to change [44].

ROS-mediated oxidative stress is a common cause of cell death, and it is used to control the cellular processes like proliferation, apoptosis, and necrosis based on the amounts of oxidant and antioxidant enzymes present [49]. The disturbance between pro oxidants and antioxidants occurs when cancer cells are exposed to chemotherapeutic drugs, cytotoxic agents, and nanoparticles. As a result, we measured MDA levels in MCF 7 cells that had been exposed to various amounts of IONPs for 24hrs. Lipid peroxidation (LPO) was determined via the reaction of Malondialdehyde (MDA) with thiobarbituric acid to form a colorimetric (533 nm)/ fluorometric (excitation and emission wavelengths of 533 and 554 nm, respectively) product, whose quantity was proportional to that of MDA. The findings showed that IONPs enhanced MDA levels in human prostate cancer cells, and that increasing IONP concentrations dramatically increased MDA levels (Figure 4B). Long-term exposure to IONPs in lung cells up-regulated the gene expression of antioxidant enzymes such as glutathione-Stransferase enzymes i.e., involved in clearing lipid peroxidation products, according to Gliga et al [50]. Ferroptosis, which is emerging as a form of controlled cell death, is characterised by lipid peroxidation, which is a source of free radical generation and also characteristic of ferroptosis. Overall, our data indicate that IONPs cause oxidative stress by oxidising lipid biomolecules.

IONPs induce mitochondrial dysfunction and reduce ATP generation in MCF 7 Cells

The loss of mitochondrial function is an important factor and predictor of cell apoptosis that may be evaluated by monitoring changes in Mitochondrial Membrane Potential (MMP). Using cationic fluorescent dyes, we evaluated MMP in IONP-treated MCF 7 cells [47]. MCF 7 cells were treated with different concentrations of IONPs for 24 hrs to determine effect of IONPs on MMP, and then the MMP status was measured. When compared to control cells, the treated cells showed significant changes (Figure 5A). These findings show that incubation with IONP for 24 hrs significantly decrease MMP, and the reductions in cell viability and toxicity were linked to MMP loss and enhanced ROS accumulation. Increased ROS production leads to mitochondrial-dependent cell death pathways via the formation of mitochondrial permeability transition holes in previous studies [51,52].

Figure 5: Measurement of Mitochondrial Membrane Potential (MMP) and ATP. (A) Change in MMP using the cationic fluorescent dye, JC-1 (Molecular Probes, Eugene, OR, USA), (B) Flow cytometric analysis of the MMP using the JC-1 dye in MCF 7 cells and (C) Measurement of ATP. The results are expressed as the mean ± standard deviation of three independent experiments. There was a significant difference is determined by a Student’s t-test (* p < 0.05).

ROS production increases due to mitochondrial membrane depolarization, which is an important element in activating intrinsic cell-death pathways [16,46]. As a result, MMP deficiency suggests that mitochondrial depolarization is a key mechanism of IONP-induced toxicity, involving one or more signalling cascades with crosstalk between the mitochondrion and other cellular components [45,53,54]. The cytotoxicity of IONPs was elevated due to cellular uptake by MCF 7 cells via an MMP alteration. Flow cytometry examination of IONP-treated MCF 7 cells using JC-1 (Molecular Probes, Eugene, OR, USA) dye validated the change in MMP. In healthy cells, the mitochondrial stain JC-1 can aggregate and emit red fluorescence at high membrane potential, whereas in apoptotic cells, JC-1 remains in the form of green fluorescent monomers due to mitochondrial membrane depolarization. Figure 5B shows the change in MMP in MCF 7 IONP-induced cells.

This signifies that apoptosis induction by IONPs is linked to the mitochondrial tract.

We next assessed ATP levels in both IONP-treated and untreated cells to see if the loss of MMP affects ATP synthesis in IONP-treated cells. The ATP level was measured according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA, Catalog Number MAK135) in MCF 7 cells exposed to IONPs for 24 h. Damage to mitochondria causes reduced or inefficient energy generation, potentially affecting ATP production or ATP-dependent cellular functions [55]. When compared to controls, IONP-treated cells produced considerably less ATP, as expected (Figure 5C). Interestingly, higher IONP concentrations resulted in lower ATP synthesis. The findings revealed that IONP concentration and ATP generation had a direct linear connection. As a result, the cells seems to be vulnerable to mitochondrial toxicants like IONPs that is analogous to drug-induced mitochondrial poisoning and mitochondrial inherited disorders [56].

IONPs induce apoptosis

Oxidative stress and cell death are caused by high ROS levels [57]. Our lab and other research groups previously demonstrated that IONP-induced cytotoxicity is primarily caused by oxidative stress, which induces cell apoptosis by activating the intrinsic apoptosis, autophagy, or both pathways [15,39,46,53]. We used terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) analysis to see if IONP-induced cell death is caused by apoptosis. Through numerous processes generating macromolecular damage, such as lipid peroxidation, DNA fragmentation, protein denaturation, and mitochondrial malfunction, oxidative stress is a crucial component in the activation of apoptosis in cancer cells [53]. ROS acts as signalling molecules, promoting cell division and generating oxidative DNA damage [51,58].

The best method for evaluating IONP-induced apoptosis tends to be measuring DNA fragmentation. MCF 7 cells was analysed with the half maximum inhibitory concentration (IC50) of IONPs and a DNA-fragmentation test was performed to detect apoptotic characteristics generated by IONPs. Figure 6 shows the results, which show a remarkable number of positively marked cells, indicating apoptotic DNA fragmentation. Few or no apoptotic cells were found in control cultures. Cell shrinkage, widespread plasma membrane bleb, chromatin condensation, and the dissociation of cell fragments into apoptotic bodies are all characteristics of apoptosis [59]. Cancer cell lines analysed with IONPs displayed the similar "laddering" pattern as suggested by DNA fragmentation in this study [14]. The accumulation of Iron Oxide particles inside the nucleus during DNA fragmentation may alter DNA and cell division, and nanoparticles may produce dosedependent DNA damage, chromosomal abnormalities, and errors in chromosomal segregation, as well as the development of sister chromatic exchanges [60]. Our findings are consistent with prior research indicating that cancer cells treated with IONPs produce micronuclei [51]. As a result, we discovered that IONPs cause DNA fragmentation and, subsequently apoptosis in MCF 7 cells.

Figure 6: IONP-induced apoptosis in MCF 7 cells. The terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) test was then used to determine cell apoptosis, nuclei were counterstained with 40, 6-Diamidino-2-Phenylindole (DAPI) apoptotic DNA (red staining) and cell nuclei (blue staining). Original magnification is × 100, scale bar is 100 μm.

IONP treatment impairs expression of genes involved in mitochondrial function and cell apoptosis

Cells with the 50% inhibitory concentration of IONPs (5 μg/mL) to discover which genes were affected by IONP treatment, and then we did RNA-Seqeuence analysis, generating 27 million reads of RNA-Seqe data in each sample. In IONP-treated MCF 7 cells, 257 up regulated and 175 down regulated genes were denoted with cut-off values of fragments per kilobase of transcript per million mapped reads (FPKM) >2, and fold changes >2 (Figure 7A). Figure 7B depicts examples of up-regulated (CYP1A1 and CYP1B1) and down-regulated (CCNB1 and CCNB2) genes after IONP treatment. To discover biological processes, both up and down regulated genes were subjected to Gene Ontology (GO) term analysis. The most important biological processes were drug reactions, oxidation-reduction processes, cellular responses to cadmium ions, and cell cycle regulation (Figure 7C). Following that, each up- or down-regulated gene was submitted to a GO term analysis. The results of the cellular and molecular analyses are supported by the findings of this study. The most highly rated biological process with up regulated genes was oxidationreduction, as depicted in Figures 7D and 7E, while the most highly ranked biological process with down regulated genes was cell cycle regulation.

Figure 7: Impact of IONPs on gene expression. (A) Scatter plot showing up-regulated and down-regulated genes in IONPtreated MCF 7 cells. Red dots indicate down-regulated and blue dots indicate up-regulated genes in IONPtreated MCF 7 cells, (B) IGV image of down-regulated gene (CCNB1 and CCNB2) and up-regulated gene (CYP1A1 and CYP1B1), (C) Gene Ontology term analysis using both up-regulated and down-regulated genes. Each dot i.e., red dot (down-regulated) and blue dot (up-regulated) represents one gene belonging to each term and (D and E) Graphs showing Gene Ontology terms of individual upregulated and down-regulated genes.

The oxidation-reduction related genes, in particular, code for cytochrome P450 monooxygenases such as CYP1A1 and CYP1B1. Cytochrome P450 enzymes are found largely in the endoplasmic reticulum and mitochondria and catalyse many of the processes involved in steroid and cholesterol production as well as drug metabolism. Previous research has linked mitochondrial CYP1B1 to melatonin-induced apoptosis in the SH-SY5Y neuroblastoma cell line. Melatonin is known widely for its double role in apoptosis, antitumorigenic effects have been seen in some cancer cells by inducing apoptosis, although little or no effect has been seen in normal cells. Forced expression of CYP1A1 in cancer cells resulted in cell death, which was consistent with our findings. Asik et al. [61] have found that giving a high dose of cobalt ferrite magnetic nanoparticles to human breast cancer cell lines causes cell death and increases CYP1A1 and CYP1B1 expression. In IONP-treated cells, genes associated with ageing were preferentially upregulated. In comparison, G2/M check-point regulators such as CCNB1 and CCNB2 have been discovered in the biological process of cell cycle control with down-regulated genes.

IONP treatment dysregulates multiple biological pathways

To investigate the biological pathways linked with the differentially expressed genes, a KEGG pathway assay was performed. Down regulated genes were found to have p53 signalling and cell-cycle pathways, whereas up regulated genes had mineral absorption and metabolic pathways, as depicted in Figures 8A and 8B. Surprisingly, the p53 pathway has been found in both up- regulated and down-regulated genes. GTSE1, an S/G2 phase-specific gene in the p53 pathway that is an IONPs-mediated repressed gene, promotes p53 degradation by generating a protein complex. Furthermore, increased GTSE1 gene expression has been seen in numerous types of cancer, including lung cancer, myeloma cells, and gastric cancer. GADD45G, a p53 down-stream regulator, has been demonstrated to be down-regulated in a variety of cancers. Hsu et al. [62,63] stated that treatment with a natural substance, cucurbitacin E, triggers G2/M arrest in GBM 8401 malignant glioma cells. GADD45G was identified in the p53 pathway, along with genes up-regulated by IONPs, according to our KEGG study. Our findings are consistent with that of Gurunathan et al. [15], who found p53 involvement and a key function for p53 upregulation in IONP-mediated cell death in human breast cancer cells. As a result, our genome-wide investigation reveals that IONPs' tumor-suppressive action is mediated by a combination of cell apoptosis caused by DNA damage and mitochondrial dysfunction and cell cycle arrest caused by aberrant regulation of p53 effector proteins.

Figure 8: KEGG analysis pathway. (A) Biological pathways associated with down-regulated genes following IONP administration and (B) Biological pathways associated with Up-regulated genes following IONP administration.

Conclusion

Despite increasing attention to the anti-cancer properties of IONPs, research into the possible impacts and molecular mechanisms of IONPs in cancer is limited. As a result, the main aim and objective of this investigation was to analyse the molecular mechanism of IONP exposure in human breast cancer cells using RNA-Seqe. The human MCF 7 cell line was treated for 24 hours with 2-10 μg/mL IONPs (7 nm), followed by several cellular assays and RNA-Seqeuence analysis. To evaluate the effective anticancer activity of IONPs, we synthesised them with NAR, a flavonoid found primarily in grapefruit. MCF7 cells were treated to IONPs for 24hrs and showed a dose-dependent decrease of viability and proliferation. The IC₅₀ of 5 g/mL IONPs significantly increases LDH leakage, ROS production, and MDA levels while significantly lowering dead-cell protease activity and ATP production. By disrupting mitochondrial activity and inducing DNA damage, these events led cell death. IONPs also up-regulate and downregulate the most highly ranked biological processes of oxidationreduction and cell cycle regulation, respectively, according to our findings. GADD45G was found in the p53 pathway, with genes up-regulated by IONPs, according to our KEGG study.

As a result, our findings imply that IONPs' tumor-suppressive action is mediated by a combination of DNA damage-induced cell apoptosis, mitochondrial malfunction, and cell cycle arrest caused by aberrant regulation of p53 effector proteins. Following IONPs exposure, transcriptomic analysis revealed that a large number of genes, 257 up regulated and 175 down regulated, were differently expressed, significant impacts were detected in genes related to oxidation-reduction and cell cycle regulation. The p53 pathway was identified as one of the damaged pathways after down-stream analysis of the transcriptomics data. In conclusion, our analysis revealed that IONP exposure causes cell death via DNA damage, mitochondrial dysfunction, and cell-cycle arrest via aberrant regulation of p53 effector proteins, utilising a combination of RNA-Seqe and functional assays. Furthermore, gene-expression profiling technologies like RNA-Seqe can be utilised to predict IONP toxicity pathways. The molecular mechanism of IONPs' anti-cancer activity was discovered by GO studies. The study revealed that transcriptome analysis can reveal the molecular mechanism of biogenic IONPs' anti-cancer effect.

Funding

This work was supported by a grant from the DST-INSPIRE MST New-Delhi.

Acknowledgments

This study was supported by the UDPS, Utkal University.

Conflicts of Interest

The authors declare no conflict of interest.

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