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The anti-cancer role of tumor protein p53-inducible nuclear protein 2/nuclear factor-kB/nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway in spinal cord glioma
*Corresponding author: Xingen Zhu, Department of Neurosurgery, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China. ndefy89006@ncu.edu.cn
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Received: ,
Accepted: ,
How to cite this article: Huang J, Pan Z, Yu L, Zhou Y, Zhu X, Wang Y, et al. The anti-cancer role of tumor protein p53-inducible nuclear protein 2/nuclear factor-kB/nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway in spinal cord glioma. CytoJournal. 2025;22:87. doi: 10.25259/Cytojournal_13_2025
Abstract
Objective:
The function of tumor protein p53-inducible nuclear protein 2 (TP53INP2) in numerous cancers has been elucidated, but its role across the development of spinal cord glioma (SCG) remains largely unexplored. This study aims to explore the anti-cancer effect of TP53INP2/nuclear factor-kappa B (NF-kB)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in SCG.
Material and Methods:
Quantitative real-time polymerase chain reaction was employed to determine TP53INP2 messenger RNA expression in vitro. Western blot analysis was conducted to detect TP53INP2, epithelial-tomesenchymal transition (EMT) marker, and NF-kB/Nrf2/HO-1 pathway protein. The proliferative potentials of glioma cells were assessed by 5-ethynyl-2’-deoxyuridine, colony formation, and cell counting kit-8 assays. Transwell assays were used to evaluate migratory and invasive capacities. Apoptotic cells and reactive oxygen species were analyzed using flow cytometer. Enzyme-linked immunosorbent assay was performed to measure superoxide dismutase and glutathione peroxidase levels. A tumor xenograft model in mouse was established.
Results:
High expression of TP53INP2 was observed in glioma cells. TP53INP2 depletion significantly inhibited tumor growth, metastasis, EMT, and oxidative stress and increased the apoptosis rate and number of immune cells. The silenced TP53INP2 hampered the activation of NF-kB and promoted the activation of the Nrf2/HO-1 pathway.
Conclusion:
This work highlights the therapeutic potential of TP53INP2/NF-kB/Nrf2/HO-1 axis in SCG.
Keywords
Nuclear factor-kB/nuclear factor erythroid 2-related factor 2/heme oxygenase-1
Epithelial-tomesenchymal transition
Spinal cord glioma
Tumor protein p53-inducible nuclear protein 2
INTRODUCTION
Spinal cord glioma (SCG) is an exceedingly uncommon tumor and constitutes <5% of all primary central nervous system tumors.[1] Three subtypes of SCG, namely, glioblastoma, astrocytoma, and ependymoma, are frequently identified.[2] In general, ependymoma is the predominant type of spinal glioma, accounting for 50–60%, followed by astrocytoma (20–30%).[3] Other histological types are less common.[3] Gross total resection and/or radiotherapy is advocated as the primary treatment modality for managing low-grade SCG and has achieved a long-term survival rate.[4] However, a standard treatment protocol has not been established for malignant and high-grade SCG. Therefore, the intricate molecular pathogenesis underlying SCG must be comprehensively investigated to develop a new therapeutic approach for this disease.
As a dual-functioning protein, tumor protein p53-inducible nuclear protein 2 (TP53INP2) is reported to be involved in autophagy and gene transcription.[5] In detail, TP53INP2 is found in the nuclei, where it functions as a transcription factor of numerous nuclear hormone receptors, such as peroxisome proliferator-activated receptor gamma, Vitamin D receptor, glucocorticoid receptor, and thyroid hormone receptor alpha.[6] Once stimulated by rapamycin or starvation, TP53INP2 will transfer from the nucleus to the cytoplasm and interact with transmembrane protein VMPI to induce autophagy.[7,8] Increasing lines of evidence indicate that the abnormal expression of TP53INP2 is implicated in epithelialto-mesenchymal transition (EMT), cell invasion, and migration in various solid tumors, including bladder cancer,[9] head-and-neck squamous cell carcinoma,[10] and colorectal cancer.[11] However, the understanding of TP53INP2’s role and function in the development of SCG is still in its infancy.
This study initially explored the function of TP53INP2 in the progression of SCG. Targeting the TP53INP2/nuclear factor-kappa B (NF-Kb)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway could be an effective therapeutic approach for inhibiting SCG in laboratory experiments and animal models. These results highlight the possible importance of TP53INP2 in SCG treatment.
MATERIAL AND METHODS
Cell culture
Four human glioma cell lines including U118 (cat. no. CL-0458), LN229 (cat. no. CL-0578), U87 (cat. no. CL-0238), and U251 (cat. no. CL-0237) and immortalized human astrocytes (cat. no. CP-H122) were obtained from Pricella Biotechnology (Wuhan, China). The cells underwent STR identification and were confirmed to be free of mycoplasma contamination. Dulbecco’s modified Eagle medium (Gibco, Rockville, MD, USA) containing 10% fetal bovine serum (FBS; Gibco) was used to incubate cells under the condition of 5% carbon dioxide at 37°C.
Transfection
Small-interfering (si) RNA-TP53INP2-1/-2 (si-TP53IN P2-1/-2; si-TP53INP2-1: 5’-GCAUGUCCGUUUACGUCAC-3’; si-TP53INP2-1: 5’- CCUGAAAUCUGAAGGGCUU-3’) and its corresponding negative control (si-NC; 5’-UUCUCCG AACGUGUCACGU-3’) were synthesized by General Biol (Chuzhou, China). Cells were collected when grown at logarithmic growth phase and employed for transfection. According to the guidelines of Lipofectamine 3000 transfection reagent (Invitrogen, Karlsruhe, Germany), si-NC or si-TP53INP2-1/-2 were co-cultured with U251 cells for 48 h. In vitro experiments were subsequently conducted using the transfected cells.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
RNA extraction was carried out using Trizol (Invitrogen), followed by the measurement of concentration with Nanodrop2000 (Thermo Fisher Scientific). Complementary DNA synthesis was performed using the PrimeScript RT reagent Kit (Invitrogen). RT-qPCR analysis was carried out with a SYBR Green Premix (Invitrogen). The reaction circumstances included at 95°C for 10 min for predenaturation, denaturation at 95°C for 15 s for 40 cycles, and annealing at 60°C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase was used as the internal reference of TP53INP2 expression. The ratio of target gene expression between control and experimental groups was calculated by 2−ΔΔCt method. The primers of RT-qPCR are listed in Table 1.
| Gene | Sequences | |
|---|---|---|
| TP53INP2 | Forward | 5’-CCTGTTCCCTTATTCTTCATTCC-3’ |
| Reverse | 5’-ATTCCCTCCATCTTCTCCCT-3’ | |
| GAPDH | Forward | 5’-ACCCACTCCTCCACCTTTG-3’ |
| Reverse | 5’-CTCTTGTGCTCTTGCTGGG-3’ | |
PCR: Polymerase chain reaction, TP53INP2: Tumor protein p53-inducible nuclear protein 2, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, A: Adenine, C: Cytosine, G: Guanine, T: Thymine
Apoptosis rate measurement
FACScan flow cytometer purchased from BD Biosciences (Franklin Lakes, NJ, USA) was utilized to assess apoptotic cells in accordance with the instructions in an apoptosis detection kit from Thermo Fisher Scientific (cat.no. BMS500FI-300). Approximately 2 × 105 of U251 cells were re-suspended in phosphate-buffered saline (PBS) and co-incubated with PI (5 μL) as well as annexin V-FITC (10 μL) at 25°C in the dark for 15 min. Following incubation with 500 μL of binding buffer, the apoptosis of U251 cells was analyzed.
Reactive oxygen species (ROS) levels determination
Cellular ROS level was evaluated through flow cytometry utilizing a 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) probe-based ROS Detection Kit (cat.no. S0036S; Beyotime, Beijing, China). FBS-free medium was utilized to dilute and prepare a 10 μM solution of DCFH-DA. Following cell harvesting and PBS washing, 1 × 106 cells were diluted with DCFH-DA (500 μL) for 20 min at 37°C. Subsequently, cellular ROS levels were detected and analyzed.
Determination of cell proliferative potential
First, we assessed the viability of U251 cells via cell counting kit-8 (CCK-8) assay. U251 cells were grown in plates with 96-well (3 × 103 cells/mL) for diverse time intervals before the introduction of the CCK-8 solution (15 μL; cat.no. C0037; Beyotime). With the aid of microplate reader (cat.no. VA000010C; Thermo Fisher Scientific), we evaluated U251 cell viability.
The 5-ethynyl-2’-deoxyuridine (EdU) proliferation assay kit (cat.no. ab219801) was purchased from Abcam (Cambridge, UK). Approximately 50 μM EdU was added to U251 cells and incubated at 37 °C for 2 h. The cells were fixed with 4% formaldehyde and exposed to 0.5% Triton X-100 for permeabilization. A 1× Apollo reaction cocktail was then used to treat the cells for 30 min. We then utilized DAPI (cat. no. C1002; Beyotime) to stain DNA. Half an hour later, EdU-positive cells were assessed under a fluorescence microscope (Scale bar = 50 μm; model name: Axio Imager A2; Carl Zeiss, Oberkochen, Germany).
U251 cells colony numbers were estimated. U251 cells at a density of 3 × 103 were seeded in a 6-well plate. The medium should be replaced twice a week. After 2 weeks of incubation, the cell colonies were immobilized with methanol, and subsequently exposed to 0.1% crystal violet (cat.no.C0121; Beyotime) for staining for 15 min and washed with PBS. The colony number of U251 cells was estimated by an experimental technician under a light microscope (model name: CX33; Olympus, Tokyo, Japan).
Transwell assay
The Transwell chamber from Corning (Cambridge, MA, USA) was utilized for the cell invasion assay. A 2% Matrigel coating (Sigma-Aldrich, St. Louis, MO, USA) was applied before adding U251 cells to serum-free medium to the upper chambers and 10% FBS-containing medium to the lower chambers. After 48 h of culture, non-invaded cells were eliminated, while cells that were invaded were immobilized with paraformaldehyde, followed by staining using crystal violet. Cell observation involved randomly selecting five fields in each well using a light microscope (Olympus). The same experimental procedures were followed for the cell migration assay, but without Matrigel.
Western blot analysis
Proteins were extracted using radioimmunoprecipitation assay lysis buffer (Beyotime). Protein concentrations were detected by a bicinchoninic acid kit (Beyotime). The protein extracts were analyzed by 10% polyacrylamide gel electrophoresis and electro-transferred onto polyvinylidene fluoride membranes. Before the incubation of primary antibodies [Table 2], the membrane was blocked using 5% non-fat milk. The corresponding secondary antibodies (Abcam; 1:10000) were subsequently incubated with the membranes for 1 h and then analyzed by an ECL kit (Beyotime).
| Antibody | Catalog number | Dilution ratio | Source |
|---|---|---|---|
| TP531NP2 | A1169-100 | 1:1,000 | Biovision, San Francisco, CA, USA |
| GAPDH | 60004-1-Ig | 1:50,000 | Proteintech, Wuhan, China |
| β-actin | #4970 | 1:1,000 | Cell Signaling Technology, Boston, MA, USA |
| Lamin B | ab232731 | 1:500 | Abcam, Cambridge, UK |
| Vimentin | #5741 | 1:1,000 | Cell Signaling Technology, Boston, MA, USA |
| E-cadherin | #3195 | 1:1,000 | Cell Signaling Technology, Boston, MA, USA |
| Snail1 | #3895 | 1:1,000 | Cell Signaling Technology, Boston, MA, USA |
| N-cadherin | #13116 | 1:1,000 | Cell Signaling Technology, Boston, MA, USA |
| NF-κB | #8242 | 1:100 | Cell Signaling Technology, Boston, MA, USA |
| Nrf2 | #12721 | 1:100 | Cell Signaling Technology, Boston, MA, USA |
| IκB | #9242 | 1:5,000 | Cell Signaling Technology, Boston, MA, USA |
| HO-1 | #70081 | 1:2,000 | Cell Signaling Technology, Boston, MA, USA |
| Goat Anti-Rabbit IgG H&L (HRP) | ab6721 | 1:10,000 | Abcam, Cambridge, UK |
| Goat Anti-Rabbit IgG H&L (FITC) | ab6717 | 1:500 | Abcam, Cambridge, UK |
TP53INP2: Tumor protein p53-inducible nuclear protein 2, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, NF-κB: Nuclear factor-kappa B, Nrf2: Nuclear factor erythroid 2-related factor 2, IκB: Inhibitor of kappa B, HO-1: Heme oxygenase-1, IgG: Immunoglobulin G, HRP: Horseradish peroxidase, FITC: Fluorescein isothiocyanate
Immunofluorescence
The U251 cells were treated with 4% paraformaldehyde for fixation, followed by PBS washing and permeabilization using 0.1% TritonX-100. The cells underwent blocking with goat serum and incubated with the primary antibody incubation against Nrf2 and NF-kB [Table 2]; (all diluted at 1:100). The cells were then incubated with FITC-labeled secondary antibodies (at a dilution of 1:500). The fluorescence microscope images of the slices were captured using an Olympus microscope (Scale bar = 50 μm).
Tumor xenograft model in mice
Animal testing was conducted in compliance with the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Ethical Committee of the Second Affiliated Hospital of Nanchang University (approval number: NCULAE-20240326001). All mice were housed in specific pathogen-free environments under standard laboratory conditions, including a 12-h light/dark cycle, relative humidity maintained between 40% and 55%, and temperatures ranging from 22°C to 25°C. Food and water were unrestricted access to the animals. BALB/c nude mice (4–5 weeks of age, weighing 20–25 g; Cavens, Changzhou, China) were divided into two groups, si-NC and si-TP53INP2, with six mice in each group included randomly. U251 cells (2 × 105 cells/100 μL) transfected with lentiviral si-TP53INP2-1 or si-NC were subcutaneously injected into mice. Following injection, tumor volume was assessed weekly using the following formula: (A × B2)/2, (A, the longest diameter; B, the shortest diameter). Tumor weight was determined at 4th week after euthanasia by cervical dislocation (preceded by pre-anesthetization with 50 mg/kg pentobarbital sodium). Bronchoalveolar fluid lavage (BALF) was obtained by washing the lungs, and tumor tissues were collected.
BALF analysis
We used Wright-Giemsa Stain Kit (Abcam) to stain the BALF specimens. The cell counts for lymphocytes, macrophages, and eosinophils were documented.
Enzyme-linked immunosorbent assay
The measurements were taken for the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) (Jiancheng Bioengineering Institute, Nanjing, China) which were assessed following the instructions in the respective commercial kits.
Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay
Apoptosis in tumor tissues was assessed using the experimental procedures outlined in the manufacturer’s guidelines for the TUNEL kit (Solarbio Science and Technology, Beijing, China). The Olympus fluorescence microscope was utilized to capture images (Scale bar = 100 μm).
Ki67 immunohistochemical staining
The tumors were mixed with paraformaldehyde (4%) for 2 h at 4 °C, treated with alcohol, and enclosed in paraffin for embedding. The tissue sections were exposed to 3% hydrogen peroxide for 25 min in the absence of light at room temperature. The sections were then rinsed with PBS, and 3% bovine serum albumin (Gibco) was utilized to block the samples. The sections were then subjected to an overnight incubation at 4°C using a primary antibody Ki67 and a secondary antibody (Abcam; 1:1,000) for 50 min at room temperature. Cells positively stained with Ki67 were assessed under a light microscope (Scale bar = 50 μm; Olympus).
Statistical analysis
Differences in data were assessed using one-way analysis of variance plus Tukey’s Honestly Significant Difference test and Student’s t-tests as appropriate. Data analysis was conducted using the Statistical Package for the Social Sciences software v22.0. Data were presented as mean ± standard deviation. A significance level of P < 0.05 was considered.
RESULTS
Knocking-down TP53INP2 induces apoptosis and suppresses proliferative potential in U251 cells
TP53INP2 expression levels in SCG in vitro were initially identified. As illustrated in Figure 1a, four human glioma cell lines, namely, U118, LN229, U87, and U251, were selected, while immortalized human astrocytes were employed as control. In comparison with the control, the pronounced increased levels of TP53INP2 were validated in SCG in vitro, and relatively high expression of TP53INP2 was observed in U251 cells [Figure 1a], (P < 0.0001). Subsequent functional experiments were conducted in U251 cells. As illustrated in Figure 1b, we found that a notable downregulation of TP53INP2 expression was induced when TP53INP2 was silenced by si-TP53INP2-1 or si-TP53INP2-2 (P < 0.001), with the selection of si-TP53INP2-1 based on its superior silencing efficiency by laboratory personnel. We observed a significant increased apoptosis rate of U251 cells when transfected with si-TP53INP2-1 [Figure 1c and d], (P < 0.001). Moreover, the proliferative potential of U251 was identified to be remarkably inhibited following si-TP53INP2-1 transfection, which was reflected by a notable decrease in cell viability [Figure 1e], (P < 0.01), colony number [Figure 1f and g], (P < 0.001), and EdU-positive cells [Figure 1h and i], (P < 0.05) in the si-TP53INP2-1 group compared with those in the si-NC group.
- Knocking-down TP53INP2 induces apoptosis and stimulates proliferative potential in U251 cells. (a) TP53INP2 expression in immortalized human astrocytes (control) and glioma cell lines (U251, LN229, U87, and U118) was detected by qRT-PCR. ✶✶P < 0.01, ✶✶✶P < 0.001, ✶✶✶✶P < 0.0001 versus control. (b) Expression of TP53INP2 in U251 cells transfected with si-TP53INP2-1/-2 or si-NC was detected by qRT-PCR. (c and d) Following transfection of si-TP53INP2-1 or si-NC, the apoptosis of U251 cells was analyzed by flow cytometry. The proliferative capacities of U251 cells transfected with si-TP53INP2-1 or si-NC were assessed through (e) CCK-8, (f-g) colony forming, and (h and i) EdU proliferation assays, respectively. Magnification × 200. Scale bar = 50 μm. ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001 versus siNC. TP53INP2: Tumor protein p53-inducible nuclear protein 2, qRT-PCR: Quantitative real-time polymerase chain reaction, CCK-8: Cell counting kit-8, EdU: 5-ethynyl-2’-deoxyuridine, NC: Negative control, OD: Optical density, DAPI: 4’,6-diamidino-2-phenylindole.
Metastatic capacities of U251 cells and EMT are suppressed by the silenced TP53INP2
The metastatic capacities of U251 cells, including migratory and invasive potentials, were determined. As indicated in Figure 2a-d, U251 cells exhibited a significant decreased migratory (P < 0.001) and invasive abilities (P < 0.001) when TP53INP2 was silenced. EMT is remarkably involved in the metastasis and invasion of numerous types of tumors.[12,13] We therefore further assessed the effects of TP53INP2 on the levels of EMT-related proteins. Knockdown of TP53INP2 markedly inhibited the protein levels of TP53INP2, vimentin, N-cadherin, and snail1 [Figure 2e-j], (P < 0.001) but promoted the levels of E-cadherin protein (P < 0.001).
- Metastatic capacities of U251 cells and EMT process are suppressed by the silenced TP53INP2. (a-d) Transwell assay was performed to detect the changes in U251 cell migrative and invasive capabilities after interference in TP53INP2. Magnification × 200. Scale bar = 50 μm. (e-j) Protein levels of TP53INP2 and EMT markers were detected by Western blot. ✶✶✶P < 0.001 versus si-NC. EMT: Epithelial-to-mesenchymal transition, TP53INP2: Tumor protein p53-inducible nuclear protein 2.
Downregulated TP53INP2 inhibits oxidative stress in U251 cells
Cancer cells commonly display abnormal redox homeostasis.[14] The activities of antioxidant enzymes (GSH-Px and SOD) and ROS are shown in Figure 3a and b. We found that in comparison to the si-NC group, the activities of GSH-Px and SOD were remarkably increased in the si-TP53INP2-1 group (P < 0.01), while si-TP53INP2-1 group exhibited a significant decrease in ROS levels [Figure 3c and d], (P < 0.01).
- Downregulated TP53INP2 inhibits oxidative stress in U251 cells. (a and b) The activities of GSH-Px and SOD were measured using the respective commercial Kits. (c and d) Flow cytometry was employed to measure ROS levels in U251 cells. ✶✶P < 0.01 versus si-NC. TP53INP2: Tumor protein p53-inducible nuclear protein 2, GSH-Px: Glutathione peroxidase, SOD: Superoxide dismutase, ROS: Reactive oxygen species.
Effects of silencing of TP53INP2 on NF-kB/Nrf2/HO-1 pathway in U251 cells
The NF-kB/Nrf2/HO-1 pathway is generally associated with oxidative stress and the pathogenesis of various types of cancers.[15,16] We first explored the relationship between TP53INP2 and this pathway by determining the corresponding proteins in U251 cells. As depicted in Figure 4a-f, the results of Western blot analysis showed a considerable reduction in NF-kB level stimulated by the silenced TP53INP2 (P < 0.001). Moreover, the knockdown of TP53INP2 increased the levels of Nrf2 (P < 0.001), HO-1 (P < 0.001), and inhibitor of kappa B (IkB) (P < 0.001). Immunofluorescence staining of Nrf2 and NF-kB was performed to confirm the role of TP53INP2. Figures 4g and h show that knockdown of TP53INP2 activated Nrf2 signaling (P < 0.001), as evidenced by the increased fluorescence intensity of Nrf2 in U251 cells following transfection of si-TP53INP2. Immunofluorescence staining of NF-kB indicated opposite results. Figures 4i and j show that NF-kB fluorescence intensity was dramatically attenuated after stimulation by silencing TP53INP2 (P < 0.001); that is, silencing TP53INP2 suppressed the activation of NF-kB signaling.
- Silencing of TP53INP2 suppresses the activation of NF-kB/Nrf2/HO-1 in U251 cells. (a-f) Protein levels of NF-kB, Nrf2, IkB and HO-1 in U251 cells transfected with si-TP53INP2-1 were detected by western blot. (g-j) Relative expression of Nrf2 and NF-kB was evaluated through immunofluorescence tests. ✶✶P < 0.01, ✶✶✶P < 0.001 versus si-NC. Magnification × 200. Scale bar = 50 μm. TP53INP2: Tumor protein p53-inducible nuclear protein 2, NF-kB: Nuclear factor-kappa B, Nrf2: Nuclear factor erythroid 2-related factor 2, IkB: Inhibitor of kappa B, HO-1: Heme oxygenase-1, DAPI: 4’,6-diamidino-2-phenylindole, EdU: 5-ethynyl-2’-deoxyuridine
Downregulation of TP53INP2 increases the number of immune cells, inhibits tumor growth and metastasis, and attenuates oxidative stress in vivo
Immune evasion is a characteristic feature of numerous types of cancer. As manifested in Figure 5a-d, the results revealed that the si-TP53INP2-1 group exhibited a substantial elevation in the numbers of eosinophils, lymphocytes, and macrophages, in contrast to the si-NC group (P < 0.001). Tumor volume and weight were reduced remarkably when injected with si-TP53INP2-1 in mice [Figure 5e-g], (P < 0.01). In comparison to the si-NC group, the si-TP53INP2-1 group showed a reduction in the proportion of Ki67-positive cells [Figure 5h and i], (P < 0.01) and an elevation in the level of TUNEL-positive cells [Figure 5j and k], (P < 0.001). On the injection of si-TP53INP2-1, N-cadherin, vimentin, and snail1 levels in tumor tissues were inhibited [Figure 5l-p], (P < 0.01), while that of E-cadherin increased (P < 0.001). Hence, silencing TP53INP2 also promoted the activities of GSH-Px and SOD [Figure 5q and r], (P < 0.001) but suppressed the levels of ROS [Figure 5s and t], (P < 0.01). In addition, the levels of NF-kB/Nrf2/HO-1 pathway-related proteins were found to be reversed when injected with si-TP53INP2-1 in mice [Figure 5u-z], (P < 0.01).
- Downregulation of TP53INP2 increases the number of immune cells, inhibits tumor growth and metastasis, and attenuates oxidative stress in vivo. (a-d) Cell counts in bronchoalveolar lavage fluid were measured by Wright-Giemsa Staining. Magnification × 100. Scale bar = 100 μm. (e-g) Following injection of si-TP53INP2-1 or si-NC in mice, tumor volume and weight were measured. (h-k) Ki67 staining (Scale bar = 100 μm) and TUNEL (Scale bar = 100 μm) were used to assess the proliferation and apoptosis of tumor cells in mice. Magnification × 100. (l-p) Protein levels of EMT markers in tumor tissues were detected by western blot. (q-t) Following injection of si-TP53INP2-1 or si-NC in mice, the levels of GSH-Px, SOD, and ROS were determined. (u-z) Protein levels of NF-kB, Nrf2, IkB, and HO-1 in mice injected with si-TP53INP2-1 were detected by Western blot analysis. ✶✶P < 0.01, ✶✶✶P < 0.001, ✶✶✶✶P < 0.001 versus si-NC. TP53INP2: Tumor protein p53-inducible nuclear protein 2, NF-kB: Nuclear factor-kappa B, Nrf2: Nuclear factor erythroid 2-related factor 2, IkB: Inhibitor of kappa B, HO-1: Heme oxygenase-1, GSH-Px: Glutathione peroxidase, SOD: Superoxide dismutase, ROS: Reactive oxygen species.
DISCUSSION
SCG is an uncommon form of central nervous system tumors and occurs in around 0.22 cases out of every 100,000 individuals annually.[17] Due to its infrequency, treatment approaches rely heavily on retrospective analyses involving limited cases and contradictory findings. Therefore, an urgent clarification is necessary regarding the intricate molecular mechanisms of the disease. In this study, we uncovered the anti-cancer role of the TP53INP2/NF-kB/Nrf2/HO-1 pathway during the progression of SCG.
TP53INP2, which is abnormally expressed in different types of cancerous growths, promotes cancer advancement by stimulating cancer cell proliferation and metastasis. For instance, Cao et al.[10] reported that low expression of TP53INP2 in head-and-neck squamous cell carcinoma individuals is implicated in shorter survival time, while depletion of TP53INP2 exhibited the ability to promote cancer cell proliferation. Similarly, Shi et al.[11] revealed that there is a gradual decrease in the expression level of TP53INP2 as the colorectal cancer advances, and tumor growth and metastasis in mice can be stimulated when TP53INP2 is silenced. However, Zhou et al.[9] proved a completely opposite effect of TP53INP2 in bladder cancer; TP53INP2 was overexpressed in solid tumors, and its silencing suppressed the invasive potential and EMT progression of cancer cells. Interestingly, our findings showed a significant increase in the level of TP53INP2 in glioma cells. Hence, the role TP53INP2 in SCG is similar to its role in bladder cancer, that is, a high level of TP53INP2 may serve as a carcinogenic factor across SCG progression. Our loss-of-function experiments in vitro and in vivo were performed to test this hypothesis. We observed that the downregulation of TP53INP2 resulted in the suppression of various cellular activities, including proliferation, migration, and invasion in glioma cells. Moreover, mice injected with si-TP53INP2 exhibited a reduction in tumor weight and volume. These results confirm that overexpressed TP53INP2 is an oncogene in SCG progression, and silencing it can hamper the development of SCG.
ROS has the potential to cause harm to proteins, nucleic acids, and lipids, resulting in changes in their functions. When the equilibrium between antioxidative defense and ROS production is disrupted, it leads to oxidative stress, which is associated with the development of numerous diseases, including SCG.[18,19] Nrf2 plays a crucial role as a transcription factor to trigger the activation of downstream genes during different responses to oxidative stress. The normal state inhibits the activity of Nrf2 by causing it to bind to Keap 1.[20,21] During oxidative stress, Nrf2 and Keap1 separate and move into the nucleus where they bind to transcription factors. This complex then interacts with ARE, thereby enhancing HO-1 transcription. The development of SCG needs NF-kB activation.[22] In its unstimulated state, NF-kB is typically inactive in the cytoplasm and associates with IkB. When stimulated, IkB undergoes phosphorylation and degradation by its kinase, leading to the release of NF-kB and its translocation to the nucleus, thereby causing temozolomide resistance in SCG.[22] These results imply that under the development of SCG, the Nrf2/HO-1 pathway was inhibited, while NF-kB was activated. We therefore speculated that the NF-kB/Nrf2/HO-1 pathway induced by TP53INP2 may act as a target for SCG treatment. As expected, the group treated with si-NC showed remarkable low levels of Nrf2, IkB, and HO-1, while the level of NFkB was significantly higher than that in the control group. However, the downregulation of TP53INP2 reversed the protein levels of Nrf2, IkB, HO-1, and NF-kB in vitro and in vivo.
SUMMARY
This work preliminarily elucidates the function of TP53INP2 across the development of SCG in vitro and in vivo. The interference of TP53INP2 regulates the NF-kB/Nrf2/HO-1 pathway and could serve as a therapeutic approach for SCG. This study offers supplementary insights and viewpoints regarding potential clinical interventions for SCG.
AVAILABILITY OF DATA AND MATERIALS
The datasets analyzed during the current study are available from the corresponding author on reasonable request.
ABBREVIATIONS
ANOVA: One-way analysis of variance
CCK-8: Cell counting kit-8
DCFH-DA: 2’,7’-dichlorodihydrofluorescein diacetate
EdU: 5-ethynyl-2’-deoxyuridine
EMT: Epithelial-to-mesenchymal transition
GSH-Px: Glutathione peroxidase
HO-1: Heme oxygenase-1
NF-κB: Nuclear factor-κB
Nrf2: Nuclear factor erythroid 2-related factor 2
SOD: Superoxide dismutase
SCG: Spinal cord glioma
TP53INP2: Tumor protein p53-inducible nuclear protein 2
AUTHOR CONTRIBUTIONS
XGZ: Substantial contributions to the conception and design of the work; JH and ZMP: Substantial contributions to the acquisition, analysis and interpretation of data for the work, and drafted the manuscript; LY, YGZ, KH, XZ, and YBW: Conducted the experiments. All authors revised the manuscript critically for important intellectual content. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy and agreed final approval of the version to be published. All authors meet ICMJE authorship requirements.
ACKNOWLEDGMENT
Not applicable.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
All animal testing was conducted in compliance with the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals and received approval from the Ethical Committee of the Second Affiliated Hospital of Nanchang University (approval number: NCULAE-20240326001). Informed consent to participate is not required, as this study does not involve human subjects.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
EDITORIAL/PEER REVIEW
To ensure the integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double-blind model (authors are blinded for reviewers and vice versa) through an automatic online system.
FUNDING: The present study was supported by the National Natural Science Foundation of China (No. 82160455 & No.82172989), the China-Korea young scientist exchange program [2023]9#, the Jiangxi Provincial Department of Education (No. GJJ210152), and the Natural Science Foundation of Jiangxi Province (No. 20224BAB206033).
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