New 5-fluorouracil Anti-tumor Ferric Oxide Nanoparticles Inhibit Cervical Cancer by Regulating the Expression of Nuclear Factor Kappa B

Abstract

Background

5-Fluorouracil (5-FU) is a classic chemotherapy drug. Iron oxide nanocarriers (Fe₃O₄-NPs) significantly increase 5-FU concentration at tumor sites by enhancing targeting and sustained-release properties, while reducing systemic toxicity.

Objectives

Our research aims to explore the mechanism of the activity of novel 5-FU anti-tumor Fe₃O₄ nanoparticles (NPs) in cervical cancer.

Materials and Methods

Prepare novel 5-FU-Fe₃O₄-NPs, analyze the nanomaterial properties through electron microscopy, the effect of 5-FU-Fe₃O₄-NPs on nuclear factor kappa B (NF-κB) expression was determined by reverse transcription polymerase chain reaction (RT-PCR), and cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Flow cytometry and MTT detected cell biological behavior. 5-FU solution (control group) or 5-FU-Fe₃O₄-NPs were injected through the skin to treat skin metastases in nude mice, and the tumor volume was counted, and its size was measured.

Results

The average diameter of the NPs is 310 nm, and 70% of the particles are less than 385 nm, showing strong homogeneity. 5-FU-Fe₃O₄-NPs can inhibit NF-κB signaling molecule, a subunit of NF-κB transcription factor (P65) expression in cervical cancer cells, and the inhibition rate increases with the increase of 5-FU-Fe₃O₄-NPs concentration. 5-FU-Fe₃O₄-NPs can inhibit the increase in cervical cancer cell proliferation. Cell proliferation showed reduced surviving cells in 5-FU-NPs. Mouse cervical cell carcinoma also suggested that 5-FU-Fe₃O₄-NPs can significantly inhibit the growth of tumor cells, and this mechanism is mainly through inhibition of NF-κB expression.

Conclusion

5-FU-Fe₃O₄-NPs, new anti-tumor NPs, can inhibit cell proliferation and promote apoptosis. 5-FU-Fe₃O₄-NPs, new anti-tumor NPs, can inhibit cell activities. It may slow down the proliferation of cervical cancer by regulating NF-κB signaling and have a significant anti-tumor effect.

Introduction

The possible mechanism of cervical cancer is due to the inactivation of tumor cell-induced signal transduction. Some studies have also shown that this mechanism involves the generation of a tumor microenvironment through immune regulatory factors, thus limiting the effective immune response of the host (Hull et al., 2020; Parrondo et al., 2010). In addition, nuclear factor kappa B (NF-κB) is highly expressed in various tumors such as prostate, breast, lung, stomach, colon, esophagus, and uterus. Currently, 5-fluorouracil (5-FU) is the main systemic chemotherapy, especially in the treatment of tumor metastasis (Cai et al., 2020; Liu, Wang, et al., 2022). Direct administration of metastatic lesions can quickly absorb 5-FU into the blood circulation, resulting in insufficient local drug dosage in the body. Therefore, given the current situation, it is required to discover new treatment strategies to achieve better results in the treatment of cervical cancer.

NF-κB signaling regulates cellular functions (Guo et al., 2023; Yu et al., 2018), and its activation has been found in several malignancies, including breast, cutaneous squamous cell carcinoma (cSCC), lung, and female tumors, and is involved in tumor recurrence (Primorac & Musacchio, 2013). In non-small cell lung cancer, signaling through NF-κB has long been considered critical for skin epidermal development and homeostasis, and many studies have confirmed this. Recently, the NF-κB pathway controls the initiation and progression of human papillomavirus (HPV)-driven cSCC (Zhang et al., 2014). The NF-κB signaling pathway regulates cell activities (Hua et al., 2019) and is associated with cervical cancer grade (Chu et al., 2019; Liu, Zhou, et al., 2022). A subunit of the NF-κB transcription factor (P65) is a target of the NF-κB pathway. P65 enters the nucleus after binding and stabilizing to regulate the expression of target genes. P65 is associated with enhanced invasiveness of various cancer types, including cervical cell carcinoma (Grosse et al., 2016; Liu, Fu, et al., 2021).

Nanoparticles (NPs) have received widespread attention from the medical community since the beginning of 1978 (Grigoras, 2017). Polymer microspheres can protect drugs from interference from external factors and reduce the impact on the drug release rate (Rezvantalab et al., 2018). Forming nanospheres by wrapping high molecular polymers can block the interference of hydrophilic groups. Compared with microspheres, NPs have advantages in enhancing target effects and reducing side effects (Klekotko et al., 2015; Lee et al., 2019). New blood vessels within tumors are more permeable to NPs at 400–600 nm (Liu, Wang, et al., 2021). However, the intraperitoneal delivery of NP anti-tumor drugs for the treatment of peritoneal metastasis of cervical cancer has not been extensively studied. In view of this, we first used a new technology to prepare 5-FU NPs and demonstrated the mechanism.

Materials and Methods

5-FU-Fe₃O₄-NPs Preparation

First, add 5-FU-Fe₃O₄-NPs into 80 mL of dichloromethane. Under high shear emulsification conditions, 4 mL of 10% (w/w) NaOH containing 5-FU was mixed to obtain a slightly transparent emulsion. Under strong stirring, drop it into 5-FU solution containing 5% (w/v) polyvinyl alcohol (PVA) for 5 min to acquire a double emulsion (w/o/w) and evaporate using a rotary evaporator. NPs were recovered by centrifugation and then rinsed with the final lyophilized saturated aqueous solution of 5-FU and distilled water.

Evaluate NPs Quality

Evaluate morphological characteristics of NPs. Disperse 1 mg of NPs in 1 mL of water. The NPs suspension was dropped on a glass slide, and the specimen was prepared by fixing it with aldehydes and barium tetroxide (OsO₄) at room temperature, and using 40%, 70%, 90%, and 100% ethanol, the specimen was dehydrated using a dehydrator. Ultrathin sections of biological material are embedded and post-stained with heavy metal salts such as uranium, resulting in the introduction of contrast agents within the sample. After preparation, morphology was studied by scanning electron microscopy (Carl Zeiss [Shanghai] Management Co., Ltd.).

The morphology of exosomes was assessed by transmission electron microscopy (TEM) (JEM-2100, Jeol, Japan).

Encapsulation efficiency was determined by thermogravimetric analysis (STA409 Thermal Analyzer, Germany) at 10 uC/min (under nitrogen) atmosphere.

Disperse 50 mg of 5-FU-Fe₃O₄-NPs in phosphate buffered saline (PBS). Dialyze the solution in a dialysis bag and put it into PBS, seal it, stir and mix well, collect 10 mL samples from the external PBS at specified time intervals, and add an equal amount of fresh PBS. The research mechanism is shown in Figure 1. The average diameter of the NPs determined by dynamic light scattering was 310 nm, and the particle size distribution was concentrated. 70% of the particles were smaller than 385 nm.

Figure 1.

Note: 5-FU-Fe3O4-NPs: 5-Fluorouracil ferric oxide nanoparticles.

Experimental Animals

Mice raised under the same conditions (provided by the Jiangsu University Experimental Animal Center, aged 6–8 weeks) had access to food and water, and were exposed to a 12 h light:12 h dark cycle. Two groups of controls were used. Experimental mice were treated with 5-FU-NPs, and the control group was treated with 5-FU solution. After 8 weeks, skin injection was performed (40 mg/kg). At 14 weeks, collect materials uniformly, use 25 mL of control (50 mg/mL 5-Bru solution) and 800 mL of ethyl acetate, stir for 1 min, centrifuge for 5 min (5,000 rpm), and then filter and precipitate the solution through a filter. Finally, the sample was injected into the high-performance liquid chromatography (HPLC), and the 5-FU concentration was calculated.

Cell Migration Assay

Boyden chambers (8.0 mm pore size) were coated with collagen (10 mg/mL) for migration or Matrigel for invasion, followed by crystal violet staining before observation.

Effect of 5-FU-Fe₃O₄-NPs on Cervical Cancer Cell Transplant Tumors

The principles of animal use follow the guidelines of our hospital’s ethics committee. HeLa cells (1 × 10⁵/mouse)/mL were injected into the skin of mice (n = 5 mice/group). Starting from the 7th day, 5-fluorouracil sol and 5-FU-Fe₃O₄-NPs were intraperitoneally injected (40 mg/kg) every week. The number of peritoneal nodules with a diameter <1.0 mm or >3.0 mm was counted, and the data were from representative experiments.

Cell Apoptosis

Cells were stained with Annexin V-FITC, PI, or isotype control to measure cell apoptosis by flow cytometry.

Cell Cycle

Cells were fixed, followed by the addition of 10 mg/mL RNase A, 400 mg/mL propidium iodide, and 0.1% Triton-X. Labeled cell DNA content was analyzed using FlowJo.

Immunohistochemical Staining

After treatment, primary antibodies (1:100, Abcam) were applied and then followed by secondary antibody, hematoxylin staining, and post-counterstaining with blue reagent.

Immunofluorescence

After transfection, cells were fixed and permeabilized. After serum blocking, the primary antibody was added overnight, and then the secondary antibody, followed by nuclear counterstaining and observation under a fluorescence microscope.

Analysis of the Effect of Cell Proliferation

Cells were collected at the most vigorous period of cell growth, fixed, permeated, and divided into 96-well plates (1 × 10⁵ cells/200 µL). The culture dish was immersed in PBS containing 1 mmol/l MgCl₂, and the non-adherent cells were removed. Then, the adherent cells were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method at 490 nm wavelength. Subsequently, 30 mL MTT (5 mg/mL) was added for 4 h. After incubation, the sample was centrifuged at 2,200× for 5 min. Once the supernatant, 150 mL of dimethyl sulfoxide (DMSO), is added to resolve the crystal, and the optical density (OD) value is measured at 490 nm after 15 min of marking. All experiments were performed four times and repeated twice.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RNA was extracted from miRNeasy FFPE using TransScript Green One-Step qRT-PCR SuperMix (Shanghai Thermo Fischer Co., Ltd.). TM microRNA qPCR quantitative kit. With glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control, the real-time PCR system (Shanghai Thermo Fisher Co., Ltd.) used total cDNA as the starting material for real-time PCR. Software analysis. Table 1 lists the primers and primer sequences.

Table 1.

Primer Sequences.

P65 mRNA	F	5′-CTGCAGACCTTGGAGCTTGT-3′
	R	5′-CAGGTCCCGCTTCTTTACCT-3′
GAPDH	F	5′-GAAGGTGAAGGTCGGAGTC-3′
	R	5′-GAAGATGGTGATGGGATTTC-3′

Note: GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; P65: A subunit of NF-κB transcription factor.

Western Blot

The 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) isolated protein was used. After membrane transfer, it was sealed with 5% skimmed milk. The primary antibody (anti-P65, inhibitor of kappa B alpha [IκBα], diluted at 1:1,000) was incubated at 4°C overnight. The secondary antibody (horseradish peroxidase (HRP)-labeled, diluted at 1:5,000) was incubated for 1 h. After enhanced chemiluminescence (ECL) development, quantitative analysis was performed using Image J.

Statistical Methods

Statistical Package for the Social Sciences (SPSS) processed data, which were displayed as mean ± SD and assessed by Student’s t test. p < .05 refers to a difference.

Results

Effect of 155-FU-Fe₃O₄-NPs on the Expression of NF-κB

5-FU-Fe₃O₄-NPs may affect NF-κB signaling. To further verify 5-FU-Fe₃O₄-NPs’ effect on molecular signaling of cervical cancer cells, NF-κB expression was measured, and it was shown that 5-FU-Fe₃O₄-NPs could inhibit NF-κB signaling molecule P65 expression, and the inhibition rate increased with the increase of 5-FU-Fe₃O₄-NPs concentration (p < .05) (Figure 2A). 5-FU-Fe₃O₄-NPs-treated cervical cancer cells showed reduced proliferation (p < .05) (Figure 2B).

Figure 2.

Notes: Compared with Control Group, *p < .05; Compared with Low Dose Group, #p < .05. 5-FU-Fe₃O₄-NPs: 5-Fluorouracil ferric oxide nanoparticles; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB: Nuclear factor kappa B; P65: A subunit of NF-κB transcription factor; RT-PCR: Reverse transcription polymerase chain reaction.

Silencing of 5-FU-Fe₃O₄-NPs Inhibits Cervical Cancer Cell Activities and Leads to Cell Cycle Arrest

Compared with the control group, 5-FU-Fe₃O₄-NPs inhibited cell growth. Additionally, 5-FU-sol and 5-FU-NPs could reduce the number of colonies. In addition, 5-FU-Fe₃O₄-NPs caused the number of colonies to decrease by more than 35% compared with the 5-FU-sol group (Figure 3A). Both 5-FU solution and 5-FU-Fe₃O₄-NPs inhibited cell migration and slowed down cell motility, but in comparison, 5-FU-Fe₃O₄-NPs inhibited cell motility more significantly (Figure 3B). 5-FU-sol and 5-FU-Fe₃O₄-NPs could promote cell apoptosis compared with mock (Figure 3C). Relative to 5-FU-sol, 5-FU-Fe₃O₄-NPs could partially enhance 5-FU’s effect on cell activities, but the effect was not significant.

Figure 3.

Note: 5-FU-Fe₃O₄-NPs: 5-Fluorouracil ferric oxide nanoparticles.

5-FU-Fe₃O₄-NPs Can Inhibit the Expression Level of NF-κB Signaling Protein

Levels of pP65 and IκBα were downregulated (shown in Figure 4A,B), but P65 expression was increased, as shown in Figure 4C. Immunofluorescence further detected the NF-κB signal, and the results showed that cells entering the nucleus increased significantly (shown in Figure 4D). Therefore, our results show that 5-FU-Fe₃O₄-NPs can inhibit the expression level of NF-κB signaling protein.

Figure 4.

Note: 5-FU-Fe₃O₄-NPs: 5-Fluorouracil ferric oxide nanoparticles; NF-κB: Nuclear factor kappa B; P65: A subunit of NF-κB transcription factor; pP65: Phosphorylated P65.

Anti-tumor Effect of 5-FU-Fe₃O₄-NPs on Cervical Cell Carcinoma

We constructed a cervical cancer mouse model in mice to evaluate the effect of 5-FU-Fe₃O₄-NPs treatment or control treatment in vivo. After constructing cervical cancer by implanting it, the tumor volume was measured after treatment (Figure 5A,B). As treatment progressed, mice receiving 5-FU-Fe₃O₄-NPs had longer survival times (as shown in Figure 5C). As shown in Figure 5D, the histopathological changes in the tumor further illustrate the anti-tumor effect, which was evaluated by horseradish peroxidase (IHC) Ki-67. The staining showed that cell proliferation was significantly weakened after 5-FU-Fe₃O₄-NPs treatment, and the tumor site showed more necrosis. At the same time, cell proliferation was reduced in those treated with 5-FU-Fe₃O₄-NPs.

Figure 5.

Note: 5-FU-Fe₃O₄-NPs: 5-Fluorouracil ferric oxide nanoparticles; IHC: Immunohistochemistry.

Discussion

The dose-dependent toxicity of 5-FU limits intravenous administration, and a lower dose reaches the peritoneal nodules, resulting in a poor anti-tumor therapeutic effect (Ali et al., 2017; Cai et al., 2019; Shi et al., 2018).

The prolonged effect produced by 5-FU-Fe₃O₄-NPs may significantly improve the anti-tumor activity of 5-FU (Zhong et al., 2020). Studies have shown that 5-FU-Fe₃O₄-NPs bind through their target receptors, and colon cells take up more 5-FU-NPs (Li et al., 2019). Similarly, epidermal growth factor (EGF) receptor-mediated uptake of zinc phthalocyanine-loaded silica 5-FU-NPs was significantly more effective against pancreatic cancer cells. This study prepared an anti-tumor NP with 5-FU as the carrier. The high-shear emulsification method replaced the ultrasonic emulsification method to prepare 5-FU-Fe₃O₄-NPs, which increased the emulsification energy of the NPs and obtained 310 nm in diameter of NPs (Xia & Xue, 2012). Compared with previously reported phacoemulsification technology, the experimental process is simplified, and the limitation of relatively low output caused by the application of phacoemulsification technology is overcome (Feng et al., 2023). In addition, 70% of the NPs have a diameter less than 385 nm and a distribution range of 300 ± 10.84 nm. This study shows that this kind of 5-FU NPs can be released slowly and steadily for about 7 days in vitro, simulating the human environment in the abdominal cavity. Meanwhile, in this study, nude mice with breast cancer were treated by skin injection, which enabled a more direct observation of the targeted effect of the drug on skin metastases. Furthermore, intraperitoneal injection may lead to the distribution of the drug throughout the body, while cutaneous injection can better control the local drug concentration, which is consistent with the research goal of the cervical cancer skin metastasis model. Our study also showed a significant decrease in proliferation after 5-FU-Fe₃O₄-NPs treatment. We also found that 5-FU-Fe₃O₄-NPs interfere with NF-κB signaling.

NF-κB activity was upregulated in K-ras-transformed cells. HPV infection (especially the E6/E7 oncoproteins) can promote cervical cancer progression (Garcia-Becerra et al., 2023). In this study, the inhibition of NF-κB by 5-FU-Fe₃O₄-NPs may target both HPV-dependent and HPV-independent pathways simultaneously. Further verification through HPV-positive/negative cell models is needed in the future. Furthermore, deletion of NF-κB p65/RelA subunit reduced the number of K-ras-induced intestinal cancers. Many studies have shown that anti-tumor NPs can inhibit tumor efficacy (Aktepe et al., 2021). 5-FU-Fe₃O₄-NPs treatment was more effective than plain 5-FU control solution, especially after 48 h. Cell scratch experiments also showed the same results. Compared with ordinary 5-FU, 5-FU-Fe₃O₄-NPs significantly enhanced tumor cell apoptosis. Compared to the 5-FU control group, 5-FU-Fe₃O₄-NPs caused changes in morphological characteristics. Integrating multiple signals to regulate tumor suppressor genes of the cell cycle, treatment with synthetic 5-FU NPs also enhanced the expression of Bax, reversed the regulation of p53, and successfully slowed down tumorigenesis (Akbarpour Arsanjani et al., 2022). Some studies have also shown that extracting peritoneal metastatic tumor cells and identifying the dispersed distribution of cells and the spindle or star-shaped morphology of cells may be due to the influence of 5-FU NPs on the regulation of tumor cell functions (Senba et al., 2011). 5-FU-Fe₃O₄-NPs significantly change the polarity of tumor cells and affect tumor cells’ functions through polarity changes (Wu et al., 2020). We have verified that 5-FU-Fe₃O₄-NPs can slow down tumor occurrence in a mouse cervical cancer model, mainly by weakening the NF-κB signaling pathway. Recently, studies (Song et al., 2017) reported that the NF-κB pathway controls the initiation and progression of HPV-driven cSCC. This study shows that P65 is a classic important component involved in NF-κB signaling. Our study shows that 5-FU-Fe₃O₄-NPs can significantly reduce the level of P65 and inhibit the translocation of P65 to the nucleus, suggesting that 5-FU-Fe₃O₄-NPs block the nuclear translocation of P65 by inhibiting the phosphorylation of P65 and reducing its ionization with IκBα, thereby ultimately inhibiting the transcriptional activity of NF-κB target genes. This mechanism directly explains the phenomenon of slowed tumor proliferation.

Conclusion

In conclusion, our study inhibits tumors by constructing tumor NPs. 5-FU-Fe₃O₄-NPs caused a decreased P65 level and inhibited the translocation of P65 to the nucleus, suggesting that 5-FU-Fe₃O₄-NPs significantly delay cervical cancer progression by inhibiting NF-κB signaling (which may involve HPV-dependent and non-dependent mechanisms), providing a new strategy for targeted therapy. However, this study also has certain deficiencies: (a) the long-term toxicity of 5-FU-Fe₃O₄-NPs was not evaluated; (b) the experiment was only based on HPV-positive cell lines (such as HeLa), lacking controls of HPV-negative models; and (c) preclinical data need to be verified through multi-center large-sample studies. In the future, the targeted modification of NPs will be optimized to further enhance the therapeutic effect.