Superparamagnetic Iron Oxide Nanoparticles Induce Apoptosis in HT-29 Cells by Increasing ROS and Damaging DNA

Today, research into nanoparticles for diagnostic and therapeutic use in cancer is on the rise. In the present study, the effect of superparamagnetic iron oxide nanoparticles on the formation of apoptosis in HT-29 cells is investigated. In this study, we investigated the mechanisms of apoptosis induced by superparamagnetic iron oxide nanoparticles after MTT assay and determining the appropriate dose of 2.5 µg / mL to induce apoptosis in HT-29 cells. Superparamagnetic iron oxide nanoparticles increased the levels of ROS, Ca2 +, and DNA damage in HT29 cells. Also, at the level of protein and mRNA, they increased the expression of Caspes 3 and 9 and signicantly decreased Bcl-2 compared to the control group (P <0.0001). Fe3O4 causes apoptosis in cancer cells by increasing the level of ROS and intracellular calcium, followed by increasing the expression of caspases 3 and 9 and decreasing the expression of Bcl-2, as well as direct DNA damage.


Introduction
Cancer has always been one of the most fundamental problems of human societies. Despite the vast amount of research and developments over the past decade, cancer remains one of the leading causes of death worldwide. According to recent statistics, cancer is the second leading cause of death in the world after cardiovascular disease [1][2][3]. Current treatments for cancer include chemotherapy, radiation, and surgery. However, these methods are not speci c to cancer. Many researchers have found that a combination of two or three treatments often has better and more effective treatment results than a single treatment. However, in most cases, healthy cells also die and there can be side effects in the patient. Therefore, it is essential to explore new treatments for cancer control [4 and 5].
In recent years, nanotechnology has emerged as one of the major goals in the treatment of cancers. Its bene ts in drug design include increased sensitivity to radiotherapy, protection from the adverse effects of radiotherapy, and increased tumor cell killing. Meanwhile, metal oxide nanoparticles have received a lot of attention for the treatment of cancer. Natural oxides of metals are present in large quantities in nature and their production is not very expensive. Hence, it directs research in this eld toward the production of cost-effective pharmaceutical products and processing and synthesis of these nanoparticles can be one of the least expensive synthesis instructions [6][7]. Therefore, magnetic iron oxide nanoparticles are one of the most important metal nanoparticles that are used in a wide range of biomedical technologies and applications due to their biocompatibility, physicality, low toxicity, stability, cell cycle arrest, and magnetic properties [8,9]. In addition, magnetic iron oxide nanoparticles have been shown to have cytotoxic properties in cancer cells [10,11]. Magnetic iron oxide nanoparticles have been used in chemotherapeutic compounds in the treatment of various cancers such as blood, lung cancer, pancreatic cancer, and prostate cancer cells [12].
Previous studies have shown that magnetic nanoparticles can cause apoptosis in cells due to the production of oxygen free radicals and oxidative stress [15 − 13]. Despite extensive studies on the toxicity of nanomaterials on mitochondrial damage, oxidative stress, genome damage, alteration of cell cycle settings, and denaturation of proteins, the mechanism by which they act on body parts remains unknown.
One of the possible mechanisms associated with nanoparticles is the formation of reactive oxygen species (ROS) and subsequently oxidative stress, which lead to changes in the amount of cellular calcium, occurrence of in ammatory cell responses, activation of transcription factors (p53 is a transcription factor), and increase in the production of cytokines, which eventually cause apoptosis [16][17]. Nanomaterials also play a key role in DNA damage, membrane destruction, and eventual cell death by causing oxidative stress and lipid peroxidation [18]. As such, they can lead to effective therapies resulting in the destruction of the tumor by minimizing treatment side effects [19]. Therefore, the purpose of this study was to investigate the mechanism of apoptosis formation in HT-29 cells by superparamagnetic iron oxide nanoparticles.

Materials And Methods
Synthesis of paramagnetic iron oxide nanoparticles: Superparamagnetic iron oxide nanoparticles were synthesized using the co-precipitation method (20,21). Figure 1A shows the TEM and SEM images of paramagnetic iron oxide nanoparticles synthesized by co-precipitation.
MTT assay: MTT colorimetric method is one of the tests that is performed based on reduction and breaking of yellow tetrazolium crystals by the succinate dehydrogenase enzyme and formation of insoluble blue crystals. Biological evaluation of normal and cancer cells was examined. After cell culture, cell suspension (10 4 cells per ml) was implanted in 96-well microplates and treated for 24 hours in a CO 2 incubator at 37°C. Then, different concentrations of paramagnetic iron oxide nanoparticles were added to the wells containing the cells. A culture medium containing 1% DMSO (dimethyl sulfoxide) without nanotreatment was used as a negative control. Wells containing cells and nanoparticles were incubated for 24 hours. Then 20 µl of solution (MTT Sigma Aldridge, Germany) (5 mg / ml) was added to each well and incubated for another 3 hours. 100 µl of DMSO solution was added to each well and the culture plate was shaken for 10 minutes to dissolve the MTT violet precipitate. Then, by ELISA reading, the light absorption of the samples was measured at 570 nm [22].
Cell culture: In this study, HT-29 colon cancer cell line was used, which was prepared from Ferdowsi University of Mashhad Cell Bank, Iran. RPMI 1640 culture medium supplemented with 10% FBS serum was used for cell culture and treated at 37°C, 5% carbon dioxide, and 95% incubator moisture. The percentage of living cells was determined by cell staining using trypan blue. When the number of cells in the ask reached a density of about 80%, they were passaged. Finally, in the untreated control group and the treated group, 2.5 µg/mL of paramagnetic iron oxide nanoparticles was cultured for 48 hours.
Real-time and analysis: First, speci c primers for 4 apoptosis-related genes (Bcl-2, Bax, Caspase 3, and Caspase9) were designed using the Beacon Designer v8 software (Table 1). Then, RNA was extracted from the two studied groups using an RNA extraction kit (Dena Zist Asia (S-1010-1)), and after converting to cDNA, all biological samples (5 replications in the two studied groups) were placed in a Rotor-Gene Q 2.3.5 RT-PCR and 95°C cDNA denaturation temperature was performed for 10 minutes, then a 40-cycle period including 95°C in 10 seconds, 60°C in 20 seconds, 72°C in 20 seconds was used for (Bax, Bcl-2, Ccaspase3, and Caspase9) and reading. In this experiment, Yekta Tajhiz Azma (YTZ (Cost No: YT2551, Iran)) Master Mix and SYBR Green kits were used. At rst, crew standard was performed for normalization of cDNA. Then, the melting curve and CT were analyzed in each sample. Table 1 List of different RT-qPCR primers used in the study The initial analysis was performed using the GeneX v6.7 software and the results were all calculated based on Delta Delta CT and Log Two. Then they were statistically analyzed with the Graphpad Prism software version 8.

Western Blatting
To determine protein expression, Western blot analysis was performed. Brie y, after 24 hours of treatment with Fe3O4 (10mg/ml) cells were lysed with 70 µL of PhosphosafeTM; then, protein concentration (20 µg) was calculated by BCA protein analysis. Electrophoresis was performed using Nu-PAGE 10% SDSPAGE Bis-Tris gel in SDS-PAGE buffer. Polyvinylidene uoride membrane (PVDF) was used for transfer. Next, membrane was blocked with bovine serum albumin (3%). Afterwards, membranes were washed with tris buffered saline containing tween 20 (TBST) and incubated overnight with primary antibody (procaspase-3, procaspase-9, Bcl-2, Bax and β-actin) diluted 1:1000. After that, membrane was washed three times with TBST and secondary antibody (1:1000) was added to be incubated for 1 hour and washed with TBST. Then, band intensities were detected using chemiluminescent substrate Supersignal Femto kit and band densities were analyzed using ImageJ 1.52a program (Bethesda, Maryland, USA) [23].

Transmission Electron Microscopy (Tem) Analysis
The target HT-29 cells were collected and washed twice with PBS, and after xing the cells with 2.5% glutaraldehyde and 2% paraformaldehyde at 4°C for 2 hours, the cells were coated with 1% osmium tetroxide. The cells were then dewatered with ethanol 30, 50, 70, 90, 100 and acetone 100, and then were placed in a resin for polymerization. Finally, sections of 70 nm were made and before selecting the appropriate section for TEM use, the cut samples (800 microns) were stained with Tluidin Blue and the appropriate area was selected with an optical microscope. Then, the samples were stained with 4% uranyl acetate and 2% lead citrate, and nally, images were taken with a transmission electron microscope in the central laboratory of Ferdowsi University of Mashhad, and 120 kV TEM image analysis was performed.

Flow Cytometry
The Finally, the FLOW J V10 and graph pad version 8 software programs were used for analysis.
Nuclear Staining (DAPI) DAPI (4 ′ 6-Diamidino-2-phenylindole dihydrochloride) is a uorescent dye that binds strongly to adeninethymine-rich parts in DNA. With this dye, cells can be examined morphologically. To investigate the effect of L-phenyl alanine-coated magnetic iron oxide nanoparticles on HT-29 cell line after culturing cells in the presence of L-phenyl alanine-coated magnetic iron oxide nanoparticles after the required time, cells were gently washed with PBS and 1% formalin was added to x the cells and refrigerated for 15 minutes at 4°C. The pellets were then exposed to the laboratory air at 27°C for 5 minutes. After formalin removal, 4% Triton was added to the samples to make the cell membrane permeable to DAPI dye. After 15 minutes and Triton extraction, the cells were washed with PBS and nally 1% DAPI dye of Denazist Asia Company was added to the cells. It should be noted that due to the sensitivity of DAPI dye to light, these steps were performed in the dark. Finally, cell morphology and changes due to the presence of NP Fe 3 O 4 were examined by a blue uorescence microscope. All steps were performed three times.
The inhibitory effect of Fe 3 O 4 NP on HT-29 cell proliferation was examined by determining the degree of dermodoxyuridine participation in the DNA of HT-29 cells using Cell Proliferation ELISA kit, BrdU (colorimetric) and according to the manufacturer's protocol. The optical absorption of samples was read at the wavelength of 405 nm by the ELISA reader.

Number Of Cells
The number of HT-29 cells 24 hours after culturing with Fe 3 O 4 was examined and the cells were photographed with an inverted microscope to record morphological changes and number of cells. The number of cells in the 24-hour group under the in uence of L-phenyl alanine-coated magnetic iron oxide nanoparticles indicated a statistically signi cant decrease compared to the control group (P < 0.05). Also, morphologically, the shape of the cells was similar to apoptosis and degrade (Fig. 2B).

Intracellular And Extracellular Calcium Levels
Ca(2+) levels in HT-29 cells and cell culture medium were assessed. All reagents and solvents were provided from the German Merck company. Each sample was placed in a separate tube. Glass and plastic containers were soaked in 10% HNO3 for 24 hours and rinsed thoroughly with deionized water. All reagents used in the present study were highly puri ed and analytically suitable for trace element analysis. Standard solutions (1000 µg ml-1) of each element were employed using inductively coupled plasma optical emission spectrometry (ICP-OES) in the central laboratory of Ferdowsi University of Mashhad. Deionized water was used during the study to serially dilute the standards.  (Fig. 1b). respectively. Figure 1C shows that Fe 3 O 4 reduces DNA replication and production in HT-29 cells by more than 49%. Figure 1D also  increases the amount of ROS to 75% in treated cells (Fig. 1E). As illustrated in Fig. 1F, the amount of ca inside the treated cells is signi cantly higher than the control group cells (p < 0.0001).  (Fig. 2A). The morphological image of the cell surface also showed that Fe 3 O 4 enters the cell as a vesicle from the membrane surface, causing damage to the cell, which is also evident in the results of DAPI staining (Fig. 2C-E). was not in statistical terms signi cantly different compared to the control group. However, caspase-9 showed a signi cant increase in mRNA and protein levels in the Fe 3 O 4 -treated group compared to the control group ( Fig. 3A2 and B2). As seen in Figs. 3A3 and B3, the expression of caspase 3 at mRNA and protein levels was signi cantly higher in the treated group than in the control group (P < 0.0001). Nevertheless, the expression of Bcl-2 in Fe 3 O 4 -treated group signi cantly decreased at mRNA and protein levels in comparison with the control group (P < 0.0001) (Fig. 3, A4 and B4).

Discussion
This study demonstrated that hours resulted in a signi cant inhibition of cell viability with different inhibitory effects [24]. Various studies have demonstrated that the mechanism of apoptosis by metal nanoparticles pertains to affecting ROS interactions in the treated cells [26 − 25]. Further, a study by Sato et al. (2013) showed that Fe 3 O 4 can cause DNA damage [27].
Cellular uptake of Fe 3 O 4 and cell structure studied in this research by a TEM electron microscope showed that Fe 3 O 4 enters the cell through the cell surface vagules and causes apoptosis by various mechanisms.
The results of various studies have revealed that the amount, manner, and place of Fe 3 O 4 uptake can cause apoptosis [28][29].
The results of the present study indicated that the expression of caspase 3 and 9 in the  the expression of factors such as P53 and subsequently leads the cell to apoptosis [31][32]. Moreover, Fe 3 O 4 increases ROS levels, which in turn causes apoptosis in several ways. Most nano-drugs have been shown to increase cytochrome C by increasing ROS and affecting mitochondria. The increased cytochrome C augments the activation of caspases 3 and 7, which leads to apoptosis [33][34][35]. ROS also releases lysosomes that can play a role in apoptosis [36][37]. In addition, in this study, it was found that Fe 3 O 4 increases intracellular calcium by causing oxidative stress, which following the increase in intracellular calcium levels can open mitochondrial pores in a mechanism similar to ROS, causing cytochrome C to exit and inducing apoptosis [38][39]. Other important results of the present study were the increase in caspase 3 and 9 expression and the decrease in Bcl-2 expression. It seems that Fe 3

Declarations
This article does not contain any studies with human participants. All animals used in this study were purchased from market, and the experimental protocols adhered to the guidelines of the Declaration of Helsinki.
Compliance with ethical standards Con ict of interest all authors declare that they have no con ict of interest.

Data availability
Data and material are available upon reasonable request.