Copper Nanoparticles Induce Apoptosis and Oxidative Stress in SW480 Human Colon Cancer Cell Line

Cu nanoparticles (CuNPs) have various applications in biomedicine, owing to their unique properties. As the effect of CuNPs on the induction of oxidative stress and apoptosis in the human colorectal cancer cell line SW480 has not yet been studied, we investigated the toxicity and mechanism of action of these NPs in SW480 cells. MTT assay was performed to assess the effect of the particles on the viability of SW480 cells. The levels of oxidative stress were assessed after 24 h of treatment with CuNPs by evaluating the Reactive Oxygen Specious (ROS) production. The antioxidant enzyme activity was assessed using a colorimetric method. To investigate the effect of NPs on cellular apoptosis, Hoechst33258 staining was performed, and the expression of Bax, Bcl-2, and p53 was evaluated by qRT-PCR. The MTT assay results showed that CuNPs inhibited the viability of SW480 cells. Moreover, the increase in ROS production at all three concentrations (31, 68, and 100 μg/ml) was significant. It has been observed that CuNPs lead to increased expression of Bax and p53, and decreased expression of Bcl-2. Hoechst staining was performed to confirm apoptosis. In conclusion, the induction of apoptosis demonstrated the anticancer potential of the CuNPs.


Introduction
Colorectal cancer (CRC) is the second most lethal cancer in both male and female [1]. Epidemiological studies have successfully identified many environmental factors, such as lifestyle, diet, anthropometrics, and pharmacological factors, associated with CRC risk. The identified risk factors include family history of CRC, inflammatory bowel disease, cigarette smoking, physical activity, BMI, consumption of processed and red meat, alcohol consumption, height, obesity, and diabetes [2,3]. Some factors reduce the risk of CRC like, physical activity [4], use of aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) [5] and postmenopausal hormone therapy [6]. Depending on the stage and severity of the disease, treatments, such as surgery, radiotherapy, chemotherapy, and immunotherapy, are used [7].
Currently, extensive nanotechnology studies have been conducted. Rapid growth in nanotechnology has led to the development of cancer treatments [8]. Nanoparticles are 1-100 nm [9]. The properties of nanoparticles include submicron size, high surface-to-volume ratios, targeting rates, and dissimilarities [10]. Contact between nanomaterials and cell lines can lead to a better response in cancer cells and reduce the concentration of the desired compound [11]. Over the last few years, metal nanoparticles have been used for pharmaceutical purposes [12]. Moreover, to their various applications metal nanoparticles can provide electrons to molecular oxygen (O 2 ) and produce superoxide radical anions (O 2 − ), leading to an increase in ROS. Some studies have shown that cells exposed to nanoparticles and stress conditions can activate antioxidant defense and inflammation by increasing the production of pro-inflammatory cytokines and immune cells, such as macrophages, which can increase the production of ROS and ultimately, cell death [13].
Cu is an important mineral that plays a vital role in the human body. The role of copper as a co-factor in some important enzymatic systems such as oxidoreductases has been identified and copper-containing proteins have several roles in electron and oxygen transport [14,15]. Cu Parvin Ghasemi and Gholamreza Shafiee are Equal first author. nanoparticles (CuNPs) are highly practical because of their unique properties. Several studies have shown that Cubased nanoparticles can induce apoptosis via ROS overproduction and DNA injury [16]. Moreover, studies have shown that copper nanoparticles can damage mitochondrial membrane potential and induce apoptosis through the mitochondria [17].
Bax is a member of the Bcl-2 family that causes apoptosis. Bax gene expression is enhanced by the p53 tumor suppressor gene [21]. The Bcl-2 family of proteins is antiapoptotic and plays an important role in mitochondrial apoptosis. Stimulants of apoptosis can reduce the protein expression in this family [22]. This study aimed to explore the anticancer effects of CuNPs in SW480 cells by examining the molecular mechanisms associated with apoptosis.

Materials and Methods
CuNPs with 99.9% purity were purchased from NANOSANY (Mashhad, Iran). The purchased CuNPs were 40 nm in size and were spherical. The mean particle sizes (diameter + SD) and size distribution (PI) of CuNPs were determined using particle sizer (HPP5001, Malvern Instruments, UK). Figure 1 represents Dynamic light scattering images of CuNPs and Fig. 2 shows micrograph and size distribution data of CuNPs by transmission electron microscope (TEM).

Cell Culture
The SW480 human colorectal adenocarcinoma cell line was purchased from the Pasteur Institute (Tehran, Iran) and maintained in RPMI-1640 (KRT100) medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were then incubated at 37 °C in 5% CO 2 and 95% air. The cells were then treated with 0.25% trypsin-EDTA. Cells from the harvested passage 2 were used in the experiments.

Determination of SW480 Cells Viability using MTT Assay
To assess the effect of the nanoparticles on the viability of the SW480 cell line, an MTT assay was performed, which is based on the reduction of MTT to a purple formazan product by mitochondrial dehydrogenase in intact cells. SW480 cells in logarithmic growth phase were seeded in a 96-well plate at 1.1 × 10 4 cells/well and incubated overnight [23]. After 24 h of incubation, to ensure the adhesion of cells to the plate bed, diluted CuNPs in RPMI1640 with different concentrations (0, 4, 7, 15, 31, 62, 100, 125 and 200 μg/ml) [24] were added to each well in triplicate. After 24 h, the MTT reagent (5 mg/ml in PBS) was added to each well, and the cells were incubated for 4 h. The contents of the wells were carefully drained and replaced with 100 μl of dimethyl sulfoxide (DMSO). After 30 min of incubation, to ensure that the paint particles were dissolved, the light absorption of the wells at 570 nm was measured using an ELISA plate reader (RT-2100C Microplate Reader, China). The viability of the SW480 cells was calculated using the following formula: (OD of CuNP-treated cells)/ (OD of untreated cells) × 100. The 50% inhibitory concentration (IC50) was calculated using the GraphPad Prism 9 software.

Intracellular ROS Measurement
ROS release was determined using a microplate fluorometric assay, according to the manufacturer's instructions (Kiazist, Iran). Briefly, SW480 cells were cultured in a 96-well black plate at a density of 22 × 10 3 cells per well. The next day, the cells were treated with various concentrations of CuNPs (0, 31, 68, and 100 μg/ml). After 24-h incubation, the medium was removed and the cells were exposed to DCFDA (2′, 7′-dichlorofluorescein diacetate) solution (100 μl). The cells were incubated for 45 min at 37 °C. The fluorescence intensity was measured at Ex/Em = 485/528 nm (excitation/ emission) using a fluorescence microplate reader (Bio-Tek Instruments, Winooski, USA).

Apoptosis assay by Hoechst 33,258
Hoechst 33,258, a DNA-specific fluorescent dye, was used to assess nuclear morphology during apoptosis. The SW480 cells were seeded in a 6-well plate. After 24 h, they were treated with defined concentrations (0, 31, 68, and 100 μg/ ml) of CuNPs and were incubated overnight. The samples were fixed in methanol for 15 min. The cells were stained with Hoechst 33,258 at 37 °C for 30 min. Stained cells were visualized using a fluorescence microscope (BEL, Italy).

RNA Extraction and cDNA Synthesis
Total RNA from treated and untreated SW480 cells was isolated using RNX-plus™, according to the manufacturer's protocol. The quantity and purity of the extracted RNAs were checked using a NanoDrop One UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and %1 gel agarose electrophoresis. cDNA was synthesized using the Pars Tous Company Kit (Iran), according to the manufacturer's instructions. qRT-PCR was conducted using Syber Green qPCR Master Mix (Amplicon, Denmark), forward and reverse primers (Table 1), a cDNA template, and RNase-free dH 2 O on a LightCycler 96 System (Roche, Germany). All gene sequences were designed using Primer3 software. β-actin was used as an internal housekeeping gene. The expression levels of Bax, Bcl-2, and p53 were calculated based on the cycle threshold (Ct) number and 2 −ΔΔCt method.

Antioxidant Markers Measurement
CAT (Kiazist, KCAT-96, Hamedan, Iran), SOD (Kiazist, KSOD-96, Hamedan, Iran), and GPx (Kiazist, KGPx-96, Hamedan, Iran) activities were evaluated according to the manufacturer's protocol. SOD activity was measured using a colorimetric method in which resazurin (blue to purple) in the presence of O 2 − was converted to resorufine and changed to pink. The GPX kit uses a coupling reaction between glutathione reductase and its coenzyme NADPH. CAT enzyme has peroxidase activity in the presence of methanol but stops in the presence of its inhibitor, and its formaldehyde reacts with purpald to produce a purple color. The dye absorbed light at a wavelength of 540 nm. In the total antioxidant capacity (TAC) experiment, Cu +2 was reduced to Cu +1 in the presence of antioxidants and produced dye in the presence of a chromogen. In the total oxidant status (TOS) assay, ferrous metal is oxidized to ferric metal in the presence of oxidants and produces color in the presence of a chromogen.

Statistical Analysis
Data are expressed as mean ± standard deviation of triplicate experiments (n = 3). Statistical analyses were performed using GraphPad Prism version 9. The MTT and enzymatic assay data were analyzed using one-way ANOVA, followed by Tukey's test for the comparison of means among the different groups. Statistical significance was set at P < 0.05.

Determination of Intracellular ROS
The capacity of CuNPs to generate ROS was studied using the DCFH-DA assay. As shown in Fig. 4, intracellular ROS production after 24 h in SW480 cells was observed at a CuNP concentration of 31 μg/ml (p < 0.05). However, the increase was statistically significant at 68 μg/ml and 100 μg/ ml CuNPs compared to the control group (untreated cells) (p < 0.001).

Hoechst 33,258 Staining Assay
Hoechst 33,258 stains DNA and is commonly used to visualize nuclei and mitochondria. Nuclear density and fragmentation of DNA in apoptotic cells were detected using a fluorescence microscope (Fig. 5). In this study, the effect of the desired concentrations of CuNPs after 24 h on the rate of apoptosis in SW480 cells showed that CuNPs increased apoptosis in SW480 cells compared to that in the control group.

Discussion
Colorectal cancer (CRC) is a perilous disease in developed countries, with limited treatment options. Therefore, efforts are being made to develop new approaches to scrutinize and treat this disease [25].
The most important application of nanotechnology is to treat diseases and improve health in a new way. Nanoparticles have a variety of nanoscale sizes with specific chemical and physical properties. Small nanoparticles can be absorbed by cells, causing cell toxicity [26,27]. Studies have suggested that nanoparticles interact with the membrane to enter the cell and are then transported inside through endocytosis [28].
This study demonstrated the cytotoxicity of CuNPs in a colorectal adenocarcinoma cell line (SW480) and the possible mechanisms related to these NPs. Our results showed that increasing the dose of CuNPs decreased the viability of cells, with an IC50 of 68 μg/ml. In addition, the present findings showed that SW480 cells exposed to CuNPs exhibited increased ROS production. Oxidative stress, caused by ROS overproduction, can induce apoptosis. Following treatment with CuNPs, qRT-PCR analysis revealed that Bcl-2 gene expression was reduced, whereas p53 and Bax gene expressions were increased. Furthermore, apoptosis induced by CuNPs was analyzed by Hoechst33258 staining and apoptotic cells were observed under a fluorescence microscope.
Previous studies have shown that CuNPs exert cytotoxic effects and induce apoptosis by increasing cellular oxidative stress [29][30][31][32] which is consistent with the results of the present study.
According to Saranya et al. [33], CuNPs inhibited the growth of MCF7 cells, and the IC50 value based on the MTT assay was 250 μg/ml. Mehdizadeh et al. evaluated the anticancer properties of CuNPs in K562 cells and reported an IC50 value of 25.24 μg/ml [34]. Furthermore, Sharma Purnima et al. investigated the induction of oxidative stress by CuNP sizes of 11-14 nm, and MTT assay results showed that these NPs induced cytotoxicity in RAW 246.7 macrophage cells through oxidative injury [35]. However, the IC50 of the synthesized green CuNPs against the HepG2 cancer cell line was significantly higher (IC50 = 500 μg/ml) [36]. In addition, Shilapa et al. investigated the toxicity of biologically synthesized CuNPs (5-20 nm) in SK-MEL-3 cells. According to the MTT test results, after 48 h of incubation with 16 μg/ml CuNPs, SK-MEL-3 cell proliferation was significantly reduced [37]. Song et al. studied the cytotoxicity of CuNPs with four different sizes (25,50,78, and 100 nm). Four different cell lines from two species were selected: mammalian (H4IIE, HepG2) and piscine (PLHC-1, RTH-149) cell lines. The results showed that the piscine cell lines were more resistant than mammalian cell lines. In addition, the IC50 values of NPs of the selected sizes were significantly different. 25 nm CuNPs exhibited the highest cytotoxicity in all cell lines [38]. Moreover, the green synthesized CuNPs in the sizes of 39.09 to 18.9 nm showed the cytotoxicity effects on MCF7 cells and T3T normal cells, and IC50 values were reported in concentrations of 37.02 and 262 μg/ml, respectively. In addition, the AO/EtBr assay showed that CuNPs induced apoptosis in MCF7 cells [39]. The toxicity of CuNPs synthesized from Quisqualis indica Fig. 8 Bax, Bcl-2, and P53 genes expression in SW480 cell line following CuNPs treatment for 24 h. Results are the mean ± SD (n = 3) expressed as fold changes in mRNA expression. β-actin was used as the reference gene. *p < 0.05, **p < 0.01, and ***p < 0.00 plant extract was evaluated using MTT and LDH assays in B16F10 melanoma cells and NiH3T3 (embryonic fibroblast cells). The IC50 value for B16F10 cells was reported to be 102 μg/ml, whereas the toxicity was not high in normal cells. In contrast, the amount of intracellular ROS in treated B16F10 cells increased in a dose-dependent manner, and Annexin V-FITC staining showed that apoptotic damage was inflicted on these cells [24]. According to these studies, it seems different IC50s have been reported based on the type of synthesis, particle size, and different cell lines.
In accordance with our results, the cytotoxicity of the green synthesized CuNPs in A2780-CP cells showed that ROS production caused apoptosis in A2780-CP cells. However, they are not toxic to normal foreskin fibroblasts [40]. Moreover, Zou et al. showed that exposure to CuNPs increased the production of ROS and induced the expression of heme oxidase protein in COV434 cells. In addition, CuNP treatment led to changes in mitochondrial membrane potential and increased the apoptosis rate in COV434 cells [41]. In another study, treatment of primary hepatocytes of E. coioides with CuNPs caused a significant increase in ROS levels. In addition, the induction of apoptosis was observed because of the increase in the concentration of cytochrome c in the cytosol and the increase in the activity of caspases 3, 8, and 9. The expression of apoptosis-related genes also increases after treatment with CuNPs(42). Sharma et al. (2020) investigated the effect of colloidal CuNPs on MCF7 cells. The results showed cell growth inhibition and dosedependent cytotoxicity caused by ROS production in MCF7 cells [43]. ROS production by cells after exposure to nanoparticles in response to an alien species is completely normal [44]. The levels of ROS production caused by nanoparticles are related to certain factors, such as the physical properties (shape, size, etc.) and particle chemistry (such as free radicals on the surface) [45]. The structural change in metal nanoparticles leads to different biological functions, which in turn leads to different capacities for ROS production [46]. Metal nanoparticles, such as copper, can affect ROS production and DNA damage via Fenton reactions [47].
The interaction of nanoparticles with the mitochondrial outer membrane and the resulting instability disrupts the potential of the mitochondrial membrane, causing damage to the electron transport chain and induction of ROS production. In addition, ROS production can be induced by NADPH oxidase activation [44]. Moreover, ROS production in cells is closely related to the concentration of nanoparticles used. Cells strengthen their antioxidant defense against low concentrations of nanoparticles; however, high concentrations of nanoparticles cause cytotoxicity [46]. Oxidative stress via ROS overproduction can damage biological macromolecules, such as DNA, lipids, and proteins, eventually causing apoptosis [48]. Antioxidants also attempt to minimize the cytotoxic effects of ROS [49]. Consistent with our data, previous studies have shown that the activity of antioxidant enzymes increases after treatment with nanoparticles to maintain a redox state [50,51].
In addition, evidence suggests that nanoparticles may induce cellular apoptosis by targeting the mitochondrial pathway, reducing Bcl-2 protein expression, and increasing Bax and p53 expression (52)(53)(54). Similar to our results, some studies have shown that treatment with CuNPs alters Bax, p53, and Bcl-2 gene expression (55,56). Overall, it seems that CuNPs can be considered a potential candidate for further studies on the treatment of colorectal cancer.

Conclusion
Our results showed that exposure to CuNPs caused cytotoxicity and ROS-mediated apoptosis in SW480 colorectal adenocarcinoma. The increased expression of Bax and p53 and reduced expression of Bcl-2 confirmed that CuNPs induced apoptosis in SW480 cells. In addition, the activities of the antioxidant enzymes GPX, SOD, and CAT increased; however, they could not completely counteract the overproduction of ROS.