Cerium(IV) oxide:silver/graphene oxide (CeO₂:Ag/GO) nanoparticles modulate gene expression and inhibit colorectal cancer cell growth: a pathway-centric therapeutic approach

Abstract

Colorectal cancer (CRC) represents a substantial global health burden, necessitating advancements in diagnostic and therapeutic modalities. This study aimed to investigate the potential of CeO₂/GO nanoparticles (NPs) in managing CRC and to compare their efficacy with Ag-doped CeO₂ (CeO₂:Ag) NPs. The synthesis of cerium oxide (CeO₂) and graphene oxide (GO) was meticulously performed, followed by a comprehensive evaluation of NPs' toxicity and their impact on apoptosis-related genes in CRC cells. Characterization techniques, including XRD, EDX, SEM, TEM, FT-IR, and DLS, validated the successful synthesis and unique properties of CeO₂:Ag/GO NPs. XRD confirmed the CeO₂ and GO structures, while EDX analysis confirmed high purity in the synthesized NPs and uniform element distribution in CeO2/GO NPs. SEM and TEM micrographs illustrated CeO₂ NPs attached to graphene sheets, showcasing reduced size post-attachment. FT-IR revealed characteristic peaks for CeO₂ and GO, confirming their composite structure. DLS showed an average NP size of 20 nm in solution. Notably, MTT assays demonstrated that CeO₂:Ag/GO NPs exhibited enhanced cytotoxicity against CRC cells (C-26 cell line) compared to CeO₂:Ag NPs, with higher doses showing heightened efficacy. CeO₂:Ag/GO NPs induced stronger growth inhibition and apoptosis in CRC cells, which was linked to their improved cellular uptake and ability to target multiple cancer-related pathways. In contrast, GO NPs alone lacked cytotoxic effects. Gene expression analysis via qPCR revealed CeO₂:Ag/GO NPs significantly downregulated Zeb-1, VEGF, Cyclin-D1, and Twist compared to controls, altering cancer-related pathways more effectively than CeO₂:Ag NPs. Additionally, CeO₂:Ag/GO NPs significantly upregulated BAX and Caspase-1 while downregulating Bcl-2, implicating a more potent apoptotic response. This study highlights the advantage of CeO₂:Ag/GO NPs over CeO₂:Ag NPs in impeding CRC cell growth and inducing apoptosis, offering a promising foundation for innovative therapeutic strategies.

Introduction

Cancer, a prominent contributor to mortality, poses a substantial challenge to enhancing global life expectancy (Bray et al. 2021). Colorectal cancer (CRC) stands as the third most prevalent cancer and the second most widespread cause of death across multiple nations (Sung et al. 2021). Clear evidence demonstrates a global rise in early colorectal cancer incidence over the past 30 years (Burnett-Hartman et al. 2021). In the United States, CRC is regarded as the third highest cause of cancer-related mortality among both males and females (Siegel et al. 2020). Identifying individuals likely to have CRC through diagnosis and optimizing their screening procedures can significantly decrease both the morbidity and mortality associated with CRC. Conventional treatments include tumor resection, radiation therapy, and chemotherapy (Wang et al. 2021). While these approaches are effective, they often come with notable side effects. For instance, conventional chemotherapy primarily targets cancer cells by impeding mitosis and hindering DNA synthesis. Tissues in the gastrointestinal tract and hair follicles exhibit a rapid reproduction rate, which can occasionally lead to severe and fatal side effects (Hossen et al. 2019). Consequently, scientists are actively exploring novel methods to selectively eradicate cancer cells. Over the past 20 years, the utilization of nanoparticles (NPs) has become a crucial approach in cancer research and therapy. Nanomedicine, a rapidly advancing domain, employs NPs in various methodologies for diagnosing and treating cancer. Due to their distinctive physical traits such as electrical conductivity, stability, and optical properties, these particles are optimal for biotechnological applications. As a result, they find widespread use in medicine, pharmaceuticals, tissue engineering, and other fields (Khan et al. 2019).

NPs offer a more precise delivery mechanism for insoluble drugs to tumors, minimizing systemic side effects typically associated with conventional drug treatments (Sun et al. 2021). Moreover, certain NPs are employed in cancer therapy and tumor inhibition due to their cytotoxic and anticancer effects on cells (Raj et al. 2021). Metal NPs have garnered significant interest because of their versatility and multifunctional capabilities, setting them apart from other NP types. Barium, bismuth, calcium, cerium oxide, copper, gold, iron, magnesium, nickel, silver, titanium, and zinc metallic NPs have been recognized for their potential as anticancer treatments (Jeevanandam et al. 2022). Developing novel therapies for cancers, particularly CRC, is imperative. Prioritizing the creation of drugs with minimal side effects and heightened efficacy remains a crucial goal in this endeavor (Nowak-Sliwinska et al. 2019). The groundbreaking combination of cerium oxide (CeO₂) and silver NPs (Ag) with graphene oxide (GO) offers fresh insights into the design of NP-based therapies, potentially paving the way for innovative approaches in this field. The objective of this study was to investigate the impact of CeO₂:Ag/GO NPs on a colorectal cancer cell line. Initially, CeO₂:Ag NPs were produced using the hydrothermal synthesis technique, followed by the attachment of GO to enhance the cytotoxic effects of CeO₂:Ag NPs using the Hummers’ technique. Subsequently, the toxicity of these NPs was evaluated. NPs influence cancer cells through various pathways, with apoptosis being a significant mechanism that plays a pivotal role in this process. Numerous genes participate in apoptosis, among which caspase-3, Bax, and Bcl-2 are some of the most crucial. The impact of CeO₂:Ag/GO NPs on apoptosis was explored by targeting these genes using the qPCR technique.

Materials and methods

Materials

The chemicals utilized in the current study include cerium(IV) nitrate [CeNO₃, 99%], silver nitrate [AgNO₃, 99%], polyvinylpyrrolidone, ethanol, ammonia, hydrogen peroxide, potassium permanganate, graphite, hydrogen peroxide (30%), sulfuric acid (98%), ethanol, dimethyl sulfoxide (DMSO), and MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide), all sourced from Merck, were utilized without additional purification in this study.

Synthesis of CeO₂:Ag NPs

CeO₂:Ag NPs were produced using the hydrothermal synthesis technique. Initially, a solution of cerium(IV) with a concentration of 0.188 g was prepared. Subsequently, 0.1 g of the surfactant polyvinylpyrrolidone (PVP) was introduced to 10 ml of distilled water. Following that, 0.003 g of the obtained substance was dissolved in 5 ml of distilled water. The resulting mixture was then positioned on a magnetic stirrer and homogenized for 30 min at room temperature (25 °C). Afterward, the silver solution was gradually introduced drop by drop to the cerium solution while continuously mixing. After a 20-min incubation period, PVP was slowly added to the mixture while maintaining mixing for an additional 20 min. Subsequently, a mixture comprising 20 ml of water and ammonia in a 1:1 volume ratio was added drop by drop to the solution and mixed for 20 min. Following these steps, the solution underwent incubation for 24 h at 170 °C. The resulting product was divided into multiple tubes and centrifuged for 5 min at 4000 rpm. This procedure was repeated triplicate using deionized water and ethanol for washing purposes. Subsequently, the precipitate was placed on a watch glass and incubated for 5 h at 90 °C. Finally, the product was heated in laboratory furnaces for 4 h at 600 °C.

Synthesis of graphene oxide

Graphene oxide was produced using Hummers' technique with the following steps: initially, a combination of 1.0 g of graphite and 23 ml of sulfuric acid was stirred on a magnetic stirrer for 24 h at ambient temperature. Afterward, 0.1 g of sodium nitrate was introduced into the solution and stirred for 30 min. An ice bath was used to regulate the temperature, and after 30 min, the temperature was lowered to below 5 °C (specifically 2 °C). Subsequently, 3.0 g of potassium permanganate, hydrogen peroxide (30%), sulfuric acid (98%), and ethanol were gently incorporated into the mixture. Following the addition of potassium permanganate, the temperature was maintained between 35 and 45 °C for 30 min. Then, 46 ml of water was added to the mixture and stirred for 25 min. Afterward, the mixture received an additional 140 ml of water and 10 ml of hydrogen peroxide. Finally, the product underwent washing three times with water and ethanol at 4000 rpm for 5 min each time (Hummers and Offeman 1958; Hansora et al. 2015).

Characteristics of nanoparticles

The crystalline structures and formed phases of the NPs were identified using X-ray diffraction (XRD) analysis performed with an X-Pert-MPD X-ray diffraction device (Philips X'pert Pro MPP model). This analysis employed Cu-Kα1 (1.54060 Å) radiation, operating at a voltage of 40 kV and a current of 30 mA. The study involved diffraction over the 2θ range of 10°–80°, using a scanning increment of 0.05° and a stop time of 1 s. Fourier-transform infrared (FT-IR) spectroscopy was conducted with a Nicolet Magna–IR550 spectrophotometer, examining the functional groups present in KBr pellets over the 400–4000 cm⁻¹ spectrum with a resolution of 1 cm⁻¹. Investigation of the dimensions and morphology of submicron powders was carried out using a scanning electron microscope (SEM) from TESCAN (TESCAN Mira 3–XMU model, Czech Republic) with energy-dispersive X-ray spectroscopy (EDX) capabilities. The chemical composition of the NPs was analyzed using EDX. A transmission electron microscope (TEM) with a Zeiss EM900 model was used to thoroughly examine the morphological structure of the NPs. Additionally, the dynamic light scattering (DLS) method, facilitated by Vasco/Cordouan Technologies, France, was used to measure the particle diameter and size distribution of the NPs.

In vitro anticancer activity of CeO₂:Ag/GO

Procurement and maintenance of cell lines

The colorectal cancer cell (C-26 cell line) and the HFF-2 (Human Foreskin Fibroblast 2) cell line as a normal cell were obtained from the Pasteur Institute (Iran). The cells were cultured in a growth medium containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) and maintained at 37 °C in a humidified environment with 5% CO₂.

Assessment of cytotoxicity

The cytotoxicity of GO, CeO₂:Ag, and CeO₂:Ag/GO NPs was assessed using the MTT assay in C-26 colorectal carcinoma cells. Initially, 5 × 10³ cells were seeded per well in a microtiter plate and incubated for 24 h. The cells were then treated with varying doses (0–100 µg/mL) of different NPs, followed by an additional 24-h incubation in a CO₂ incubator. After washing, the cells were cultured in a complete culture medium for an additional 24 h at 37 °C. After this incubation period, 20 µL of MTT solution was added to the cells, and the mixture was incubated at 37 °C for 4 h. Following this, 100 µL of DMSO was added, and after dissolving the crystals formed with DMSO, the absorbance was measured at 570 nm using a spectrophotometer.

Gene expression

Quantitative polymerase chain reaction (qPCR) was employed to assess the transcriptional expression of the Bax, Bcl-2, Caspase-1, ZEB-1, VEGF, Cyclin-D1, and Twist genes, with GAPDH used as the reference gene. This allowed for the evaluation of mRNA levels in treated cells across various experimental groups. Total RNA extraction was performed using the Yekta Tajhiz kit, and the concentration was determined using a NanoDrop device. In each sample, 200 ng of total RNA was converted into cDNA using Oligo-dT and random primers, with the Parstous cDNA kit. The subsequent step involved real-time PCR utilizing the generated cDNAs, and specific primers for Bax, Bcl-2, Caspase-1, ZEB-1, VEGF, Cyclin-D1, Twist, and SYBR Green dye. The device's heating schedule consisted of three stages. The initial stage involved a 5-min duration at 95 °C to denature the cDNA molecules. The second stage consisted of 45 cycles at 95 °C for 15 s for denaturation, followed by 10 s at 60 °C for annealing, and 9 s at 72 °C for extension. The reactions were conducted in triplicate, and all analyses were performed using relative quantification methods, normalized to GAPDH as the housekeeping gene.

Results

Nanoparticle characteristics

XRD patterns

The XRD spectra of the NPs, as shown in Fig. 1, correspond to CeO₂, CeO₂OH, GO, and CeO₂:GO, confirming the successful formation of all samples and displaying pronounced crystallinity. To eliminate redundant peaks observed in Fig. 1b, CeO₂ was calcined for 4 h at 600 °C. The XRD pattern of calcined CeO₂ NPs spans the 2θ range of 10–80° (Fig. 1a). All identifiable peaks correspond to a fully cubic configuration of CeO₂ (space group: Fm3̅m) with a lattice constant of a = 5.411 Å, in agreement with the JCPDS (Joint Committee on Powder Diffraction Standards) file for CeO₂ (JCPDS 00–034–394) (Wang et al. 2018). As clearly evident in the XRD pattern of CeO₂, the most intense diffraction peak occurs at 2θ = 28.660°, originating from the (111) lattice plane within the face-centered cubic structure of CeO2. The GO XRD spectra display three prominent peaks at 2θ = 12, 26, and 43, primarily indexed as 001, confirming the layered structure of GO (Fig. 1c). Upon combining CeO₂ with GO (Fig. 1d), the XRD pattern and crystalline structure remained unchanged. However, a slight shift in peak positions was observed compared to Fig. 1a. The XRD pattern of CeO₂:GO NPs shows a combination of peaks from both CeO₂ and GO. The presence of sharp peaks indicates the retention of the cubic structure of CeO₂, while the broad peak around 2θ = 12° corresponds to GO, confirming the successful formation of the CeO₂:GO nanocomposite. The slight shift in the CeO₂ peaks suggests an interaction between CeO₂ and GO, potentially due to the attachment of CeO₂ NPs to the GO sheets. This combination of peaks indicates that the CeO₂:GO NPs retain the crystallinity of CeO₂ while integrating the layered structure of GO.

Fig. 1

The XRD patterns of different nanoparticles: a CeO₂, b CeO₂OH, c GO, and d CeO₂:GO

EDX analysis

EDX analysis complements the XRD pattern by providing precise details regarding NPs quantity and elemental composition, indicated in both weight and atomic percentages. In Fig. 2, it is evident that the synthesized NPs exhibit high purity with minimal impurities. Additionally, the figure elucidates the percentage composition of each element present in the samples. In Fig. 2a, it is shown that 66.38% of GO is composed of carbon, while 32.62% consists of oxygen. Meanwhile, Fig. 2b demonstrates that CeO₂:Ag NPs contain 44.17% oxygen, 54.96% cerium, and 0.87% silver. Finally, the carbon, oxygen, cerium, and silver contents of CeO2:Ag/GO NPs were 73.49%, 16.13%, 1.01%, and 0.37%, respectively.

Fig. 2

EDX analysis of synthesized a GO, b CeO₂:Ag and c CeO₂:Ag/GO nanoparticles

FT-IR spectral analysis

Figure 3 displays the FT-IR spectra for analyzing the adsorption species on the surfaces of the as-synthesized CeO₂, CeO₂:Ag, and CeO₂:Ag/GO NPs. The spectral profile of CeO₂:Ag NPs exhibits a subtle band around 3473 cm⁻¹, attributed to the O–H stretching vibration mode of adsorbed water from moisture. The intricate bands observed at approximately 1513 cm⁻¹, 1436 cm⁻¹, and 1067 cm⁻¹ arise from undesirable residues in the sample, associated with CH₂ bending, C–H bending vibration, and C–O stretching vibration, respectively. The absorption bands at 711 cm⁻¹ and 781 cm⁻¹ indicate metal–oxygen bonds (Lin et al. 2006). Additionally, the faint absorption band observed at 860 cm⁻¹ is attributed to the stretching vibration of Ce–O (Zhang et al. 2005; Sharma et al. 2020; Kumar et al. 2010). In the GO spectrum, the broad peak at 3445.39 cm⁻¹ is ascribed to OH stretching vibrations, while the peak at 1644 cm⁻¹ is assigned to the bending vibration of absorbed water molecules and contributions from the sp² characteristics (Dezfuli et al. 2015). The peak at 2921 cm⁻¹ indicates sp³ C–H bonding, signifying that the product was generated in an acidic phase, confirming the presence of carboxylic acid in its structure (Spitalsky et al. 2011). The significant band at 3445 cm⁻¹ is linked to the vibration of the OH group, corresponding to residual water and/or hydroxyl groups. The peaks observed at 1573 cm⁻¹ correspond to the C = C stretching mode of the quinoid rings and C = OH, indicating the presence of partial functional groups in GO (Duan et al. 2016). The presence of peaks at 1207 cm⁻¹ is attributed to the stretching vibration modes of C–O–C, confirming the coexistence of both CeO₂ and GO-Ag NPs in the CeO₂:Ag/GO nanocomposite (Sun et al. 2023).

Fig. 3

FT-IR analysis of GO, CeO₂:Ag, and CeO₂:Ag/GO nanoparticles

X-ray mapping analysis

The X-ray mapping analysis illustrates the distribution of individual elements within the GO, CeO₂:Ag, and CeO₂:Ag/GO NPs. Figure 4a shows the uniform distribution of carbon and oxygen within the GO sample, indicating a homogenous elemental spread in the graphene oxide. However, Fig. 4b displays a much weaker X-ray mapping signal in the center of the CeO₂:Ag sample compared to the background. This unusual observation may be due to the aggregation of silver NPs in localized regions, leading to inconsistent distribution and, consequently, signal intensity variations. Aggregation of silver could result in diminished signal density in specific areas where the concentration of Ag particles is higher, creating apparent discrepancies in the mapping signal. In contrast, Fig. 4c confirms the successful synthesis of the CeO₂:Ag/GO NPs, demonstrating a more uniform and even distribution of all constituent elements within the structure, attributed to the interaction between CeO₂ and GO, which helps to reduce the aggregation effect seen in CeO₂:Ag.

Fig. 4

X-ray mapping analysis of fabricated nanoparticles: a GO, b CeO₂:Ag, and c CeO₂:Ag/GO

Microstructure characteristics of nanoparticles

The microstructural properties of CeO₂:Ag, pure GO, and CeO2:Ag/GO NPs were studied using the SEM technique. In Fig. 5a, b, CeO₂:Ag/GO NPs are visible, where silver NPs are decorated onto CeO₂ particles and are attached to the graphene oxide sheets. The size of the CeO2:Ag NPs has notably decreased after the composite formation with GO compared to the observations of CeO₂:Ag NPs alone, as depicted in Fig. 5c. In this image, the agglomeration of CeO₂:Ag NPs is evident, with particle sizes distinctly within the nanoscale range. Figure 5c shows the structure of CeO₂:Ag NPs, where silver atoms are doped into the CeO₂ lattice. These NPs exhibit a spherical shape, and the majority measure less than 200 nm in size. The agglomerated particles in this figure highlight the structural and morphological characteristics of the CeO₂:Ag NPs without any graphene oxide present. Regarding the reduced size of CeO₂:Ag NPs, while SEM images were taken at different magnifications, the scale bars in the images indicate a reduction in size following their combination with graphene oxide, as shown in Fig. 5a, b. Additionally, Fig. 5d presents CeO₂:Ag/GO NPs, where the CeO₂:Ag NPs are uniformly dispersed over graphene oxide sheets. The presence of graphene in the composite aids in the reduction of agglomeration and results in a more homogeneous distribution of CeO₂:Ag particles on the graphene surface. Figure 5e displays pure GO sheets without the presence of any NPs, providing a contrast to the nanocomposites shown in Fig. 5a, b.

Fig. 5

SEM analysis of nanoparticles: a, b, and d CeO₂:Ag/GO, c CeO₂:Ag, and e graphene oxide

TEM imaging

The dimensions and morphology of the synthesized NPs were examined through TEM analysis. TEM micrographs of CeO₂:Ag/GO NPs are shown in Fig. 6c, d. From these micrographs, the measured particle size of CeO₂:Ag NPs appears to be approximately 150–200 nm. The images clearly depict the placement of CeO₂:Ag NPs onto GO sheets, with an apparent accumulation of CeO₂:Ag NPs, as shown in Fig. 6c, d. Meanwhile, graphene sheets are visible in both Fig. 6a, b.

Fig. 6

TEM micrograph of nanoparticles: a and b graphene oxide, c and d CeO₂:Ag/GO

Particle size

Dynamic light scattering (DLS) is a method used to determine the hydrodynamic diameter of NPs in a solution. It provides insights into the state of NPs aggregation within the solution. The DLS analysis conducted on CeO₂:Ag/GO NPs revealed an average particle diameter of approximately 20 nm (Fig. 7).

Fig. 7

DLS analysis of CeO₂:Ag/GO nanoparticles

Evaluating cytotoxic effects of fabricated nanoparticles through MTT assay

The MTT method is a colorimetric assay designed to measure the reduction of MTT by the succinate dehydrogenase enzyme present in the mitochondria of living cells. This assay is widely used to assess cytotoxicity and determine the viability of cells treated with various compounds by measuring the metabolic activity of viable cells. The MTT reagent permeates the cell and enters the mitochondria, where it is reduced to form an insoluble, purple-colored formazan. This conversion occurs due to mitochondrial enzyme activity in viable cells. The purple formazan is then dissolved in DMSO, and its absorbance is measured at a wavelength of 570 nm using a spectrophotometer. The reduction of MTT and similar tetrazolium dyes correlates with cell metabolism: reduced metabolic activity leads to lower MTT reduction, while higher activity results in increased reduction.

This investigation involved applying varied doses of CeO₂:Ag/GO, CeO₂:Ag, and pure GO NPs to CRC cells (Fig. 8). After 24 h, cell viability percentages were assessed using the MTT assay. The findings indicated that both CeO₂:Ag and CeO₂:Ag/GO NPs significantly inhibited the growth and proliferation of C26 cells, with IC50 values of 13.76 and 6.8 µg/mL, respectively (Fig. 8a, b). The more pronounced inhibitory effect of CeO₂:Ag/GO can be attributed to the role of GO in enhancing the interaction of the NPs with cancer cells. GO acts as a carrier platform that improves the dispersity and stability of CeO2 and Ag NPs, which in turn facilitates a more efficient delivery of the active components to the cancer cells. While CeO₂:Ag NPs alone displayed significant cytotoxic effects, the integration of GO into the CeO₂:Ag/GO nanocomposite likely enhances the overall efficacy of the NPs. GO's layered structure allows for better attachment and distribution of CeO₂ and Ag NPs on the cell surface, leading to improved cellular uptake and interaction. This synergistic effect results in a lower IC50 value for CeO₂:Ag/GO, indicating greater cytotoxicity compared to CeO₂:Ag NPs alone. Conversely, GO NPs alone did not exhibit cytotoxic effects on the cells (Fig. 8c), underscoring the importance of its role as a facilitator for the combined therapeutic action of CeO₂ and Ag in the CeO₂:Ag/GO composite rather than as a standalone therapeutic agent. We addressed the sensitivity of normal cells in the context of our study. We observed no cytotoxic effects on HFF-2 cells in the treatment groups (Fig. 9). We emphasized that this does not necessarily imply a complete lack of sensitivity in all normal cells. We clarified that differential toxicity can occur and discuss the implications of our findings regarding normal cell responses.

Fig. 8

Cell viability assay of a CeO₂:Ag/GO, b CeO₂:Ag, and c graphene oxide against C-26 cells

Fig. 9

Cell viability assay of a CeO₂:Ag/GO, b CeO₂:Ag, and c graphene oxide against HFF-2 cell line

Gene expression

A multi-analyte real-time PCR was conducted to discern the activated pathways triggered by NPs in cells pre-treated with CeO₂:Ag/GO, CeO₂:Ag, and pure GO NPs. The aim was to evaluate gene expression levels in crucial pathways linked to human cancer cell survival in C-26 colorectal cancer cells exposed to these NPs. The expression levels of Bax, Bcl-2, Caspase-1, Cyclin-D1, Twist, VEGF, and ZEB-1 in human colorectal cancer cells were investigated using RT-PCR (Figs. 10, 11). The analysis indicated a notable reduction in the mRNA expression of the VEGF gene in the CeO₂:Ag/GO group (p = 0.02 compared to the CeO₂:Ag group), emphasizing the impact of the combined therapy involving GO and CeO₂:Ag. The treatment with CeO₂:Ag/GO notably diminished the gene expression of ZEB-1, VEGF, Cyclin-D1, and Twist (p < 0.0001, p = 0.0008, p = 0.003, and p = 0.001, respectively) compared to the control group. Ag-doped CeO₂ reduced the levels of ZEB-1 (p = 0.001) and Twist (p = 0.04), but did not significantly impact VEGF and Cyclin-D1 expression. Interestingly, GO NPs alone did not significantly affect gene expression in any of the groups.

Fig. 10

Using graphene oxide (GO), CeO₂:Ag, and CeO₂:Ag/GO nanoparticles, gene expression and functional assays were conducted against C-26 cells, evaluating the fold difference in mRNA expression of a Zeb, b VEGF, c Cyclin-D1, and d twist across colorectal cancer cell lines via quantitative real-time polymerase chain reaction (qRT-PCR). The data are presented as mean values with standard deviation (mean ± SD), and "ns" denotes insignificance in the observed results

Fig. 11

Assessing graphene oxide (GO), CeO2:Ag, and CeO2:Ag/GO nanoparticles, gene expression and functional tests were performed on C-26 cells, analyzing the mRNA expression fold changes of a Bax, b Caspase-3, and c Bcl-2 across colorectal cancer cell lines using qRT-PCR. Results are expressed as mean values ± standard deviation (mean ± SD), with 'ns' indicating statistical insignificance in the findings

Discussion

CeO2 NPs have demonstrated potential in modulating oxidative stress in various conditions such as retinal degeneration, neurological disorders, ischemia, heart disease, diabetes, gastrointestinal inflammation, liver disease, and cancer. This versatile behavior suggests that CeO₂ could serve as a promising candidate for the targeted production of anti-tumor drugs (Inbaraj and Chen 2020). Research has shown that cerium oxide induces considerable oxidative stress in human bronchial and hepatocellular carcinomas (Marzi et al. 2013; Lin et al. 2006). In adenocarcinoma cells (A549), CeO2 has been observed to increase the generation of reactive oxygen species (ROS), consequently reducing the cell's antioxidant levels and leading to apoptotic cell death (Mittal and Pandey 2018). There is evidence that CeO2 NPs can induce apoptosis by activating caspases and causing chromosomal DNA fragmentation. Additionally, they elevate ROS levels in colorectal cancer cells without causing harm to normal cells (Datta et al. 2020). Silver NPs (Ag NPs) also stand out as highly effective NPs, renowned for their antibacterial and anticancer properties (Kovács et al. 2022). The primary distinction between silver NPs and small-molecule drugs lies in their structure, which helps mitigate the severity of undesirable side effects (Maeda et al. 2013).

Research has established that the optimization effects of Ag NPs rely on several factors, including shape, size, capping material (Danhier et al. 2010), and the utilization of folic acid-targeted Ag NPs (Kovács et al. 2022). The activation of various intercellular molecular pathways has been suggested for Ag NPs, encompassing Ag NP-induced anti-proliferative signaling, classical apoptotic pathways, DNA damage response, MAP kinase stimulation, oxidative stress, sustained autophagy induction, and ER stress (Simard et al. 2016; Chen et al. 2020). In research conducted by Valenzuela-Salas, Girón-Vázquez (Valenzuela-Salas et al. 2019), the AgNP-TAT combination was applied in both in vivo and in vitro methods within a melanoma cancer model. The observed effects demonstrated reduced tumor growth and size. They also employed silver NPs in melanoma cancer, leading to apoptosis and subsequent reduction in tumor growth. Furthermore, the silver NPs prompted an overproduction of ROS, eventually leading to cancer cell death (Valenzuela-Salas et al. 2019). In a separate study by Zhang, Choi (Zhang et al. 2015), the inhibitory impact of silver NPs was observed, resulting in a decrease in the proliferation and self-renewal of spermatogonial stem cells (SSCs) (Zhang et al. 2015). In another study by Li, Sun (2012), uniform silver NPs were examined for a quantitative investigation of cytotoxicity. The impact of silver NPs on cell viability, apoptosis, necrosis, and ROS production in human lung fibroblasts was thoroughly explored using silver NPs of varying sizes and doses. The research revealed a notable escalation in cytotoxicity correlating with a reduction in the size of silver NPs and an increase in their dosage. These outcomes were linked to the creation of monodispersed silver NPs of varying sizes (Li et al. 2012).

In a study by Hadrup, Loeschner (2012), it was concluded that both silver ions and silver NPs influence neurotransmitters in the brains of rats. Silver ions and silver NPs also trigger apoptosis in vitro through the death receptor and mitochondrial pathways, exerting a neurotoxic effect. In a study conducted by Jeyaraj, Sathishkumar (2013), the effects of Ag NPs on breast cancer were investigated. The findings indicated that silver NPs induce DNA damage by generating ROS, suggesting that the synthesized silver NPs might serve as a potential alternative chemotherapy agent (Jeyaraj et al. 2013). In separate research by Govender, Phulukdaree (2013), the effects of AgNPs were observed, including heightened activity of caspases 3/7/9, escalated mitochondrial depolarization, and elevated levels of Bax and Smac/Biablo in lung carcinoma cells. The study by Mata, Nakkala and Sadras (2015) explored the impact of silver NPs on colon cancer. The findings revealed that AIAg NPs triggered apoptotic cell death at a remarkably low concentration in colon cancer cells by elevating intracellular ROS production and reducing mitochondrial membrane potential, resulting in DNA fragmentation and cell cycle arrest (Mata et al. 2015). Additionally, there are promising results regarding the anticancer and antioxidant effects of GO composites. For instance, Ag NPs decorated with graphene oxide NPs diminished colon cancer cell growth through a radiosensitizing mechanism (Habiba et al. 2019).

Graphene oxide (GO) is a chemical similar to graphene, but it incorporates oxygen-based functional groups into its structure. GO has garnered significant attention due to its distinctive electrical, mechanical, and thermal characteristics (Liao et al. 2011). There is controversy regarding the anticancer effects of GO. Findings from one study suggest that GO does not exhibit cytotoxic effects on its own (Inbaraj and Chen 2020). However, when combined with certain drugs, such as doxorubicin, it significantly enhances the drug's cytotoxicity compared to the effects of pure doxorubicin (Sun et al. 2023). On the other hand, multiple research studies have demonstrated GO's ability to trigger oxidative stress and elevate ROS levels (Duan et al. 2016). Moreover, GO is recognized for its involvement in activating diverse intracellular pathways, such as AMPK/mTOR/ULK-1, which are associated with autophagy and apoptosis (Shen et al. 2022). While debates persist regarding graphene oxide's anticancer effects, its efficacy as a co-drug to enhance cytotoxicity remains unquestionable. In a particular study, both Ag NPs and Ag-GO NPs elevated the ratio of Bax to Bcl-2 and increased intracellular ROS. However, Ag NPs demonstrated the highest efficacy in increasing the Bax/Bcl-2 ratio, whereas Ag-GO further enhanced ROS formation (Ganjouzadeh et al. 2022). Recent findings suggest that CeO₂ NPs impact oxidative stress in conditions such as retinal degeneration, neurological disorders, ischemia, heart disease, diabetes, gastrointestinal inflammation, liver disease, and cancer. Thus, CeO₂ emerges as a viable candidate for the development of targeted anti-tumor medications (Casals et al. 2020). In 2020, research conducted by Pandiyan Nithya et al. employed silver and gold NPs doped with CeO₂ in the context of cervical cancer. The findings revealed a notable impact of these NPs, indicating a dose-dependent effect. Silver and gold NPs doped with CeO₂ triggered ROS generation and impeded tumor cell growth. Therefore, Ag–Au/CeO2 NPs possess both antibacterial and anticancer attributes (Nithya and Sundrarajan 2020). In research conducted by Melissa S. Wason and her team, CeO₂ NPs were found to induce JNK activation, facilitating apoptosis in pancreatic cancer cells (Wason et al. 2018). In a study by Singh, Gupta (2018), combining GO with doxorubicin revealed enhanced effectiveness compared to using pure doxorubicin alone. According to the study by Jafarinejad-Farsangi, Hashemi (2021), GO exhibits no toxic effect on its own, but in combination with a drug, it can augment the drug's toxicity. However, controversy surrounds the antibacterial and anticancer properties attributed to GO. Graphene-based materials like GO have garnered significant attention in biosensor applications due to their robust signal output and immense potential for rapid industrial growth. The exceptional conductivity and mechanical resilience of graphene, coupled with its heightened reactivity to chemical molecules, make it particularly appealing. The existence of surface waves, whether inherent or induced, presents another variable that, if effectively utilized, holds immense potential (Yildiz et al. 2021).

The Wnt/β-catenin signaling axis plays a crucial role in the development of CRC (Ji et al. 2022). Cyclin D1 and β-catenin are primary components involved in both the cell cycle and carcinogenesis. The Wnt/β-catenin signaling pathway regulates the transcriptional programs essential for the growth and development of various tissues. Multicellularity is vital for proper biological function, and dysregulated activity within the Wnt/β-catenin complex can lead to cancer, particularly colorectal cancer (Jung et al. 2015). Wnt signaling is essential for governing stem cell growth and maintaining tissue homeostasis (Clevers et al. 2014). Additionally, this pathway is fundamental in cell proliferation, cell fate determination, cell migration, and establishing cell polarity during embryonic development. Wnt ligands bind to Frizzled receptors and 5/6 protein receptors, which are linked to low-density lipoprotein receptors. By inhibiting the degradation complex involving adenomatous polyposis coli (APC), axin, casein kinase 1, and glycogen synthase kinase, Wnt signaling stabilizes the β-catenin protein, consequently initiating the activation of β-catenin-targeted gene transcription (Clevers and Nusse 2012). Common mutations in the genetic components of the Wnt signaling pathway are strongly linked to human colon cancer and CRC. Research in mouse models indicates that genetic mutations causing heightened Wnt signaling activity result in mammary tumors and intestinal adenomas. This disruption in Wnt signaling significantly influences the onset of intestinal tumors. Mutations in key components of Wnt signaling contribute to the hyperactivation of the Wnt/β-catenin signaling pathway. For instance, over 70% of colorectal cancer cells exhibit significant mutations in elements of the Wnt signaling pathway, such as APC, AXIN1, or β-catenin/CTNNB1 (Kasprzak 2020). It has also been demonstrated that hyperactivation of Wnt signaling in CRC cells is triggered by silencing Wnt signaling antagonists at the transcriptional level. Of the 19 Wnt ligands in mammals, Wnt1, Wnt3A, Wnt8, and Wnt10 activate the canonical Wnt signaling pathway through β-catenin, which is recognized as a canonical Wnt ligand. Studies have revealed that heightened expression of Wnt3A is linked to the development of intestinal tumors (Kasprzak 2020). Thus, β-catenin serves as the central mediator of this pathway, undergoing proteasomal degradation in the absence of Wnt (Roche et al. 2008). In various malignancies, β-catenin has been identified as an oncogene with a significant role. One of the molecules regulated by β-catenin, Cyclin D1, stands out as a potential stimulator of the carcinogenic pathway.

Clinical samples of colon cancers have been examined to explore the correlation between β-catenin and Cyclin D1 (Utsunomiya et al. 2001). Cyclin D1 is a key regulator in the cell cycle, particularly in the G1 phase, serving as an oncogene known for promoting malignant transformations. In human cancers, elevated levels of Cyclin D1 have been frequently observed in clinical studies, correlating with the tumor's malignant potential (Utsunomiya et al. 2001). Furthermore, the impact of epithelial–mesenchymal transition (EMT) programs on cell proliferation and survival has been associated with the development of tumors. During EMT, epithelial cells gradually transform in morphology and adopt biochemical traits characteristic of a more mesenchymal state. Transcription factors play a crucial role in promoting cell plasticity, aiding tumor advancement. EMT transcription factors (EMT-TFs) have been linked to unfavorable clinical outcomes in various cancers, regardless of their origin in epithelial or non-epithelial tissues. EMT does not follow a singular pathway; it involves EMT-TF families such as SNAIL, SLUG, TWIST, and ZEB, which are tissue-specific and contribute to its diverse biological nature. Various tumor events associated with EMT can converge toward similar phenotypic outcomes in clinical samples (Umar 2014). TWIST and ZEB1, both members of the ZEB family derived from EMT-TFs, serve as consistent markers in the progression of tissue malignancies, including colon cancer (Wu et al. 2019). Cyclin D1, encoded by the CCND1 gene, is a cell cycle regulatory protein associated with multiple cancers, including colon cancer. Increased expression of this gene has been shown to be related to metastasis and proliferation of CRC cells (Albasri et al. 2019).

Conclusion

This study highlights the promising potential of CeO₂:Ag/GO nanoparticles (NPs) as a novel therapeutic approach for colorectal cancer (CRC). The synthesized NPs demonstrated notable cytotoxicity against CRC cells while exhibiting minimal toxicity to normal cells, making them a compelling option for targeted cancer therapy. The presence of graphene oxide (GO) enhanced the anticancer efficacy of the CeO₂:Ag NPs, as evidenced by improved cell viability assays and significant modulation of gene expression related to cancer progression and apoptosis pathways. Specifically, CeO₂:Ag/GO NPs downregulated key cancer-related genes and upregulated pro-apoptotic genes, indicating their role in disrupting tumor growth mechanisms. While the results are encouraging, further research is needed to delve deeper into the precise molecular mechanisms and pathways affected by these NPs. Additionally, exploring the potential combination of CeO₂:Ag/GO NPs with conventional therapies, such as chemotherapy or radiation, could enhance treatment outcomes. These findings lay the groundwork for future investigations and the development of CeO₂:Ag/GO-based therapeutics for more effective CRC management.