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Research Article

IκBα Inhibits Colorectal Cancer Progression by Suppressing Constitutive NF-κB Activation

Authors: Xing Liu

Publication Date: 15 March, 2025

DOI: https://doi.org/10.51219/MCCRJ/Xing-Liu/363

Citation: Liu X. IκBα Inhibits Colorectal Cancer Progression by Suppressing Constitutive NF-κB Activation. Medi Clin Case Rep J 2025;3(3):1309-1311.

Copyright:Liu X., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Objective

To explore the role of IκBα (inhibitor of nuclear factor kappa B alpha) in colorectal cancer (CRC) cell proliferation, migration, invasion and its regulatory effect on the NF-κB signaling pathway.

 

Methods

IκBα expression was detected in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell line (NCM460) by Western blot and qRT-PCR. IκBα was overexpressed via plasmid or knocked down via siRNA in HCT116 cells. Cell proliferation (CCK-8), migration (scratch assay), invasion (Transwell) and NF-κB-related proteins (p-p65, IκBα, TNF-α) were analyzed.

 

Results

IκBα was downregulated in CRC cells compared with NCM460 (P<0.01). IκBα overexpression reduced HCT116 cell proliferation (OD450 at 72h: 0.65±0.06 vs. 1.29±0.12, P<0.05), migration rate (28.9±3.6% vs. 67.8±5.5%, P<0.01) and invasive cell number (40±5 vs. 123±10, P<0.01), while decreasing p-p65 and TNF-α expression (P<0.05). IκBα knockdown showed opposite effects.

 

Conclusion

IκBα functions as a tumor suppressor in CRC by inhibiting NF-κB activation, serving as a potential therapeutic target.

 

Keywords: Colorectal Cancer; Cell Proliferation; Transwell; CRC Cell Lines

 

Introduction

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths globally, with ~935,000 annual fatalities1. The NF-κB signaling pathway is constitutively activated in over 60% of advanced CRC cases, driving tumor cell survival, invasion and inflammation2. IκBα, encoded by the NFKBIA gene, is the primary endogenous inhibitor of NF-κB: it sequesters p65/p50 complexes in the cytoplasm, preventing nuclear translocation and oncogenic gene transcription3,4. Clinical studies have shown that IκBα expression is downregulated in CRC tissues, correlating with lymph node metastasis and poor prognosis5,6. However, the functional role of IκBα in CRC cell behaviors and its mechanism of regulating NF-κB remain to be fully clarified. This study uses CRC cell lines to verify IκBα’s effect on tumor progression and its association with NF-κB signaling.

Materials and Methods

Cell culture
HCT116 (low-metastatic CRC), SW480 (high-metastatic CRC) and NCM460 (normal colonic epithelial) cells were purchased from ATCC (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO₂ incubator. For NF-κB stimulation, cells were treated with 10 ng/mL recombinant human TNF-α (R&D Systems, Minneapolis, MN, USA) for 24h.

Transfection
IκBα overexpression plasmid (pcDNA3.1-IκBα) and empty vector were obtained from Addgene (Cambridge, MA, USA). IκBα siRNA (si-IκBα) and negative control siRNA (si-NC) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HCT116 cells (5×10⁵ cells/well) were seeded in 6-well plates and transfected with plasmids/siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at 60-70% confluency. IκBα expression was verified by Western blot and qRT-PCR 48h post-transfection.

qRT-PCR and western blot
qRT-PCR: Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized using PrimeScript RT Kit (Takara, Kyoto, Japan). IκBα primers: Forward 5'-ATGGACTACAGGGACGACCT-3', Reverse 5'-TCAGCTGGGGTTTCTGTTTC-3'; GAPDH primers (internal control): Forward 5'-GAAGGTGAAGGTCGGAGTC-3', Reverse 5'-GAAGATGGTGATGGGATTTC-3'. Relative expression was calculated via the 2⁻ΔΔCt method.

Western Blot: Cells were lysed with RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors. Protein concentration was measured by BCA assay (Beyotime). Equal amounts of protein (30μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, MA, USA) and probed with primary antibodies against IκBα, p-p65 (Ser536), TNF-α (Cell Signaling Technology, Danvers, MA, USA) and GAPDH (Beyotime) at 4°C overnight. Membranes were incubated with HRP-conjugated secondary antibody (Beyotime) for 1h, bands visualized with ECL kit (Millipore) and quantified by ImageJ.

Functional assays
• CCK-8 Assay: Transfected cells (2×10³ cells/well) were seeded in 96-well plates. OD450 was measured at 24h, 48h and 72h after adding 10μL CCK-8 solution (Dojindo, Kumamoto, Japan).
• Scratch Wound Healing Assay: Confluent cells were scratched with a 200μL pipette tip. Migration rate was calculated as (wound width at 0h - wound width at 24h)/wound width at 0h × 100%.
• Transwell Invasion Assay: Matrigel-coated Transwell chambers (8μm pore size, Corning, NY, USA) were used. Transfected cells (2×10⁴ cells/well) in serum-free medium were added to the upper chamber; medium with 20% FBS was added to the lower chamber. Invasive cells were counted at 24h.

Statistical analysis
Data were presented as mean ± standard deviation (SD, n=3). Statistical analysis was performed using SPSS 26.0 software (IBM, Armonk, NY, USA) with independent samples t-test. P<0.05 was considered statistically significant.

Results
IκBα is downregulated in CRC cell lines
qRT-PCR showed IκBα mRNA expression in HCT116 and SW480 cells was 0.26±0.03 and 0.33±0.04 folds of NCM460, respectively (P<0.01). Western blot revealed IκBα protein levels in HCT116 (0.29±0.04) and SW480 (0.36±0.05) were significantly lower than NCM460 (1.00±0.10, P<0.01), with SW480 showing higher IκBα downregulation than HCT116.

IκBα inhibits CRC cell proliferation
In HCT116 cells, IκBα overexpression reduced OD450 at 48h (0.53±0.06 vs. 0.88±0.08, P<0.05) and 72h (0.65±0.06 vs. 1.29±0.12, P<0.05). IκBα knockdown increased OD450 at 48h (1.06±0.09 vs. 0.87±0.07, P<0.05) and 72h (1.37±0.13 vs. 1.25±0.10, P<0.05). TNF-α stimulation failed to rescue proliferation inhibition in IκBα-overexpressing cells.

IκBα suppresses CRC cell migration and invasion

Scratch assay showed IκBα overexpression reduced HCT116 migration rate to 28.9±3.6% (vs. 67.8±5.5% in control, P<0.01). Transwell assay revealed IκBα overexpression decreased invasive cell number to 40±5 (vs. 123±10 in control, P<0.01). IκBα knockdown showed opposite effects: migration rate increased to 73.5±5.8% (vs. 66.2±5.3% in si-NC, P<0.01) and invasive cells increased to 135±12 (vs. 120±9 in si-NC, P<0.01).

IκBα inhibits NF-κB signaling activation
IκBα overexpression upregulated total IκBα protein (2.00±0.19 vs. 1.00±0.09, P<0.05) and downregulated p-p65 (0.42±0.04 vs. 1.00±0.08, P<0.05) and TNF-α (0.39±0.04 vs. 1.00±0.07, P<0.05). IκBα knockdown decreased total IκBα (0.45±0.05 vs. 1.00±0.09, P<0.05) and increased p-p65 (1.88±0.17 vs. 1.00±0.08, P<0.05) and TNF-α (1.82±0.16 vs. 1.00±0.07, P<0.05).

Discussion
This study confirms IκBα is downregulated in CRC cells and its overexpression inhibits proliferation, migration and invasion by suppressing NF-κB activation-consistent with its tumor-suppressive role in gastric and pancreatic cancer7,8. Mechanistically, IκBα sequesters p65 in the cytoplasm, preventing its nuclear translocation and transcription of pro-oncogenic genes (e.g., TNF-α)4. Limitations include lack of in vivo validation; future studies should explore IκBα’s crosstalk with Wnt/β-catenin, a key pathway in CRC9. Restoring IκBα expression (e.g., via NFKBIA gene delivery) may be a promising strategy for CRC treatment10.

Conclusion
IκBα is downregulated in colorectal cancer cell lines and inhibits CRC progression by suppressing the NF-κB signaling pathway, highlighting its potential as a therapeutic target for CRC.

References
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