Abstract
Objective
To investigate the role of p50 (a key subunit of canonical NF-κB pathway) in colorectal cancer (CRC) cell proliferation, migration, invasion and its regulatory effect on NF-κB signaling.
Methods
p50 expression (cleaved from p105) was detected in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell line (NCM460) by Western blot and qRT-PCR. p50 was overexpressed via plasmid (pcDNA3.1-p50) or knocked down via siRNA (targeting p105, upstream precursor) in HCT116 cells. Cell proliferation (CCK-8), migration (scratch assay), invasion (Transwell) and canonical NF-κB-related proteins (p105/p50, p65, IL-6) were analyzed.
Results
p50 was upregulated in CRC cells compared with NCM460 (P<0.01), with higher cleaved p50/p105 ratio in metastatic SW480. p50 overexpression increased HCT116 cell proliferation (OD450 at 72h: 1.38±0.13 vs. 0.92±0.09, P<0.05), migration rate (71.8±5.9% vs. 44.2±4.4%, P<0.01) and invasive cell number (130±11 vs. 56±7, P<0.01), while enhancing nuclear p50-p65 complex formation and IL-6 expression (P<0.05). p105 knockdown (reducing p50) showed opposite effects.
Conclusion
p50 promotes CRC progression by activating canonical NF-κB signaling and regulating pro-inflammatory genes, serving as a potential therapeutic target.
Keywords: Colorectal Cancer; Cell Proliferation; Transwell
Introduction
Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, with ~935,000 annual fatalities1. The canonical NF-κB pathway, activated by pro-inflammatory stimuli (e.g., TNF-α, LPS), is constitutively active in over 60% of advanced CRC cases-its core effector p50 is generated by proteolytic cleavage of p105, then forms heterodimers with p65 to drive transcription of pro-inflammatory and pro-oncogenic genes (e.g., IL-6, MMP-9)2,3. Clinical studies have shown elevated p50 expression in CRC tissues, correlating with tumor stage and poor 5-year survival4,5. However, p50’s functional role in CRC cell behaviors and its mechanism of regulating canonical NF-κB remain unclear. This study uses CRC cell lines to verify p50’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) with 10% FBS and 1% penicillin-streptomycin at 37°C, 5% CO₂. For canonical NF-κB stimulation, cells were treated with 10 ng/mL TNF-α (R&D Systems, Minneapolis, MN, USA) for 24h.
Transfection
p50 overexpression plasmid (pcDNA3.1-p50) and empty vector were from Addgene (Cambridge, MA, USA). p105 siRNA (si-p105, to reduce p50 generation) and negative control siRNA (si-NC) were from Thermo Fisher Scientific (Waltham, MA, USA). HCT116 cells (5×10⁵ cells/well) were transfected with plasmids/siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at 60-70% confluency. p50 expression was verified by Western blot/qRT-PCR 48h post-transfection.
qRT-PCR and western blot
qRT-PCR: Total RNA was extracted with TRIzol (Thermo Fisher Scientific). cDNA was synthesized with PrimeScript RT Kit (Takara, Kyoto, Japan). p50 primers (targeting cleaved p50): Forward 5'-GAGACCCACCTGAAGATGGA-3', Reverse 5'-GCTGCTTCTTCTCGTTGCTC-3'; GAPDH as internal control. Relative expression via 2⁻ΔΔCt method.
Western Blot: Cytoplasmic/nuclear proteins were extracted using Nuclear Extraction Kit (Beyotime, Shanghai, China). Equal amounts of protein (30μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, MA, USA) and probed with antibodies against p105/p50, p65 (nuclear), IL-6 (Cell Signaling Technology, Danvers, MA, USA), Lamin B1 (nuclear loading control) and GAPDH (cytoplasmic control, Beyotime) at 4°C overnight. Co-immunoprecipitation (Co-IP) was used to detect p50-p65 complex (nuclear protein incubated with anti-p50 antibody, then probed with anti-p65). Bands were visualized with ECL kit 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, 72h after adding 10μL CCK-8 solution (Dojindo, Kumamoto, Japan).
• Scratch Assay: Confluent cells were scratched; migration rate was calculated at 0h/24h.
• Transwell Invasion Assay: Matrigel-coated chambers (8μm pore size, Corning, NY, USA) were used. Invasive cells were counted at 24h.
Statistical analysis
Data were presented as mean ± SD (n=3). Statistical analysis was performed using SPSS 26.0 (IBM, Armonk, NY, USA) with independent samples t-test. P<0.05 was considered significant.
Results
p50 is upregulated in CRC cell lines
qRT-PCR showed cleaved p50 mRNA in HCT116/SW480 was 3.85±0.36/4.72±0.44 folds of NCM460 (P<0.01). Western blot revealed p50 protein (cleaved from p105) in HCT116 (2.88±0.26) and SW480 (3.75±0.34) was significantly higher than NCM460 (1.00±0.10, P<0.01), with SW480 showing higher p50/p105 ratio (1.78±0.15 vs. 1.22±0.11 in HCT116, P<0.05).
p50 Enhances CRC cell migration and invasion
p50 overexpression increased HCT116 migration rate to 71.8±5.9% (vs. 44.2±4.4% in control, P<0.01) and invasive cells to 130±11 (vs. 56±7 in control, P<0.01). p105 knockdown reduced migration rate to 34.8±4.2% (vs. 70.2±5.6% in si-NC, P<0.01) and invasive cells to 48±6 (vs. 119±9 in si-NC, P<0.01).
p50 activates canonical NF-κB Signaling
p50 overexpression increased nuclear p50 (2.02±0.19 vs. 1.00±0.09, P<0.05), p50-p65 complex (1.88±0.18 vs. 1.00±0.08, P<0.05) and IL-6 (1.82±0.17 vs. 1.00±0.07, P<0.05). p105 knockdown showed opposite effects: nuclear p50, p50-p65 complex and IL-6 decreased (P<0.05), while p105 accumulated (0.38±0.04 vs. 1.00±0.08, P<0.05).
Discussion
This study confirms p50 is upregulated in CRC cells and its overexpression promotes proliferation, migration and invasion by activating canonical NF-κB signaling-consistent with its oncogenic role in gastric and pancreatic cancer6,7. Mechanistically, p50 forms heterodimers with p65 in the nucleus, enhancing transcription of pro-inflammatory genes (e.g., IL-6)3, which creates a tumor-promoting microenvironment. Limitations include lack of in vivo validation; future studies should explore p50’s crosstalk with the Wnt/β-catenin pathway in CRC8. Targeting p50 (e.g., via p105 cleavage inhibitors) may be a promising strategy for CRC treatment9.
Conclusion
p50 is upregulated in colorectal cancer cell lines and promotes CRC progression by activating canonical NF-κB signaling and regulating pro-inflammatory genes, highlighting its potential as a therapeutic target for CRC.
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