Journal: Oncology Reports

Article Title: NF-κB-driven LUZP1 promotes metastasis and chemoresistance in head and neck squamous cell carcinoma

doi: 10.3892/or.2026.9115

Figure Lengend Snippet: NF-κB signaling activation promotes LUZP1 expression in HNSCC cells. (A) The expression of LUZP1 in FaDu, OECM-1 and SAS cells treated with SB431542 (10 µM), LY294002 (10 µM), Rapamycin (10 µM), BAY 11–7085 (5 µM) or YC-1 (30 µM) was determined by western blot assay. Signal quantification was measured using ImageJ 1.54 g software (National Institutes of Health) and the relative intensity was normalized to untreated control. The red dashed line represents the normalized value as 1. (B) The expression of LUZP1 in FaDu, OECM-1 and SAS cells with or without BAY 11–7085 was validated by western blot assay. (C) Spearman's monotonic correlation between LUZP1 and NFKB1 or NFKB2 expression in HNSCC was analyzed using The Cancer Genome Atlas RNA-Sequencing database on the GEPIA server. (D) Protein expression of NF-κB p65 and LUZP1 in OECM-1 and SAS cells with NF-κB p65 knockdown (+) or control shRNA -), as determined by western blot analysis. β-actin, loading control. (E) IC 50 values of docetaxel in OECM-1 and SAS cells with or without NF-κB p65 knockdown. (F) IC 50 values of cisplatin in OECM-1 and SAS cells with or without NF-κB p65 knockdown. The expression of LUZP1 in different HNSCC cells treated with (G) IL-1β (3 ng/ml) or (H) TNFα (10 ng/ml) for the indicated times was determined by western blot assay. β-actin, loading control. (I) Transwell cell migration assay was conducted using OECM-1 cells with or without LUZP1 knockdown in the presence or absence of TNFα (10 ng/ml) treatment. Signal quantification using crystal violet extract was measured by colorimetric analysis at 570 nm, and the relative signal intensities were normalized to untreated shControl (shLUZP1, -; TNFα, -) (n=3). (J) Genomic visualization of the human LUZP1 locus (GRCh38/hg38) showing RefSeq-curated exon annotations, NF-κB RelA ChIP-seq binding signals in FaDu cells (ReMap), layered H3K27ac ChIP-seq profiles from ENCODE cell lines, and GeneHancer regulatory element annotations. Red boxes denote promoter regions and gray boxes indicate putative enhancers. Blue vertical bars mark the locations of ChIP-qPCR primer sets designed for experimental validation. (K) ChIP-qPCR analysis showing increased NF-κB (RelA) occupancy at the LUZP1 promoter in response to TNF-α treatment. For statistical analyses, (E and F) a 2-tailed unpaired Student's t -test; (I) a factorial two-way ANOVA, followed by Tukey's Honestly Significant Difference post hoc test. **P<0.01. TPM, transcripts per million; ChIP-seq, chromatin immunoprecipitation sequencing; LUZP1, leucine zipper protein 1; sh, short hairpin.

Article Snippet: The human pharyngeal squamous cell carcinoma cell line FaDu (cat. no. HTB-43) was obtained from the American Type Culture Collection.

Techniques: Activation Assay, Expressing, Western Blot, Software, Control, RNA Sequencing, Knockdown, shRNA, Cell Migration Assay, ChIP-sequencing, Binding Assay, ChIP-qPCR, Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Andrographis paniculata Inhibits Tongue Squamous Cell Carcinoma via Regulating Wnt/β-Catenin Signaling and Epithelial-Mesenchymal Transition

doi: 10.3390/ijms27093772

Figure Lengend Snippet: APW inhibited colony formation and induced apoptosis in Cal-27 and SCC25 cells. ( A ) Cal-27 and SCC25 cells were treated with increasing concentrations of APW (25, 50, 100 μg/mL) for 48 h. Representative images of colonies are shown on the left panel, and quantification of colony numbers relative to control is shown on the right panel. ( B ) Cal-27 and SCC25 cells treated with APW (62.5, 125, 250, 500 μg/mL) for 48 h were subjected to flow cytometry analysis of apoptosis using Annexin V-PE/7-AAD staining. Representative dot plots are shown on the left panel, and the percentage of apoptotic cells (early + late apoptosis) is quantified on the right panel. Data are presented as mean ± SD ( n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control.

Article Snippet: Human tongue squamous cell carcinoma (TSCC) Cal-27 and SCC25 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Control, Flow Cytometry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Andrographis paniculata Inhibits Tongue Squamous Cell Carcinoma via Regulating Wnt/β-Catenin Signaling and Epithelial-Mesenchymal Transition

doi: 10.3390/ijms27093772

Figure Lengend Snippet: APW induced apoptosis in Cal-27 and SCC25 cells through activation of the caspase-dependent pathway. ( A ) Cal-27 and ( B ) SCC25 cells treated with various concentrations of APW (62.5, 125, and 250 μg/mL) for 24 h and subjected to RT–qPCR analysis of apoptosis-related genes (BCL-2, CASP3, CASP9, and BAX). Cisplatin (12 μM in Cal-27 and 15 μM in SCC25 cells) was used as a positive control. ( C ) Cal-27 and ( D ) SCC25 cells treated with APW (62.5, 125, and 250 μg/mL) for 24 h or 48 h and then subjected to Western blot analysis of apoptosis-associated proteins. ( C , D ) Representative blots of proteins and ( E , F ) quantification of protein band intensities were shown. Data are expressed as mean ± SD ( n = 3). Tumor tissues of mice treated with APW (160 or 320 mg/kg) or cisplatin (2.5 mg/kg) were collected for RNA and protein extraction. ( G ) Relative mRNA expressions of apoptosis-related genes (BCL-2, CASP3, CASP9, and BAX) in tumor tissues was determined using RT-qPCR. ( H , I ) Protein expression of cleaved caspase-3, BAX, and BCL-2 in tumor tissues were examined using Western blot analysis. ( H ) Representative blots of proteins and ( I ) quantification of protein band intensities were shown. Data were presented as mean ± SEM, n = 12–14 in each group, and statistical significance was determined using one-way ANOVA, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control.

Article Snippet: Human tongue squamous cell carcinoma (TSCC) Cal-27 and SCC25 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Activation Assay, Quantitative RT-PCR, Positive Control, Western Blot, Protein Extraction, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Andrographis paniculata Inhibits Tongue Squamous Cell Carcinoma via Regulating Wnt/β-Catenin Signaling and Epithelial-Mesenchymal Transition

doi: 10.3390/ijms27093772

Figure Lengend Snippet: APW down-regulated the Wnt/β-catenin signaling pathway in Cal-27 and SCC25 cells. Cal-27 and SCC25 cells were treated with APW (62.5, 125, or 250 μg/mL) or cisplatin (12 μM in Cal-27 cells or 15 μM in SCC25 cells) for 48 h and subjected to RT–qPCR analysis. Expressions of ( A , B ) Wnt signaling components (AXIN1, LRP6, DVL2, WNT5A, NKD2, and DVL3) and ( C , D ) downstream target genes (CTNB1, JUN, MMP-7, MET, CD44, CCND1, MYC, and TCF1) were normalized to GAPDH and presented as mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control.

Article Snippet: Human tongue squamous cell carcinoma (TSCC) Cal-27 and SCC25 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: Andrographis paniculata Inhibits Tongue Squamous Cell Carcinoma via Regulating Wnt/β-Catenin Signaling and Epithelial-Mesenchymal Transition

doi: 10.3390/ijms27093772

Figure Lengend Snippet: APW suppressed the expressions of proteins in Wnt/β-catenin signaling pathway in Cal-27 and SCC25 cells. Cal-27 and SCC25 cells were treated with APW (62.5, 125, 250 μg/mL) for 24 h or 48 h and subjected to Western blot analysis. Expressions of Wnt pathway components (LRP6, DVL3, DVL2, Naked1, and Wnt5a) and downstream targets (Met, CCND1, and β-catenin) was shown in representative blots of ( A , E ) Cal-27 cells and ( B , F ) SCC25 cells. Quantification of band intensities were shown in ( C , D , G , H ). Data are expressed as mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control.

Article Snippet: Human tongue squamous cell carcinoma (TSCC) Cal-27 and SCC25 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Andrographis paniculata Inhibits Tongue Squamous Cell Carcinoma via Regulating Wnt/β-Catenin Signaling and Epithelial-Mesenchymal Transition

doi: 10.3390/ijms27093772

Figure Lengend Snippet: APW triggered mitochondrial dysfunction in Cal-27 and SCC25 cells. ( A ) STRING-based protein–protein interaction network showing the known/predicted associations between β-catenin and apoptosis-related proteins relevant to APW treatment. Confocal microscopy images showing mitochondrial morphology in ( B ) Cal-27 and ( C ) SCC25 cells treated with APW (62.5, 125, 250 μg/mL) for 48 h, stained with DAPI (nuclei, blue) and MitoTracker Red CMXRos (mitochondria, red). Scale bar = 10 μm.

Article Snippet: Human tongue squamous cell carcinoma (TSCC) Cal-27 and SCC25 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Confocal Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Andrographis paniculata Inhibits Tongue Squamous Cell Carcinoma via Regulating Wnt/β-Catenin Signaling and Epithelial-Mesenchymal Transition

doi: 10.3390/ijms27093772

Figure Lengend Snippet: APW promoted cytochrome c release in Cal-27 and SCC25 cells. Cal-27 and SCC25 cells treated with APW (62.5, 125, and 250 μg/mL) for 24 and 48 h were subjected to Western blot analysis for cytosolic cytochrome c. ( A ) Representative blots of Cal-27 and SCC25 cells and ( B ) quantification of band intensities were shown. ( C ) Flow cytometry analysis of mitochondrial membrane potential (ΔΨm) of Cal-27 and SCC25 cells after APW treatment was performed using JC-1 staining. Representative JC-1 dot plots were shown on left panel. Percentage of cells with decreased mitochondrial membrane potential (in Q4-4) was shown on right panel. Data are presented as mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control.

Article Snippet: Human tongue squamous cell carcinoma (TSCC) Cal-27 and SCC25 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Flow Cytometry, Membrane, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Andrographis paniculata Inhibits Tongue Squamous Cell Carcinoma via Regulating Wnt/β-Catenin Signaling and Epithelial-Mesenchymal Transition

doi: 10.3390/ijms27093772

Figure Lengend Snippet: APW reduced migration and modulated EMT-related protein expressions in Cal-27 and SCC25 cells. ( A ) In transwell migration assay, Cal-27 and SCC25 cells were treated with increasing concentrations of APW (62.5, 125, and 250 μg/mL) for 24 h, and the number of migrated cells was quantified after crystal violet staining. Data are presented as mean ± SD ( n = 3). ( B ) In wound healing assay, Cal-27 and SCC25 cells were treated with APW at indicated concentrations for 16 h, and wound area was photographed at 0 and 24 h. The wound closure area was calculated and normalized to control wells. Data are presented as mean fold of control ± SD ( n = 3). Representative photographs of transwell migration and scratch wound healing assays were shown on the left panel (magnification 400×). The quantified data were shown on the right panel. Cal-27 and SCC25 cells were treated with APW (62.5, 125, 250 μg/mL) for 24 h or 48 h and subjected to Western blot analysis of EMT-related proteins. Representative blots was shown in ( C , D ). Quantification of band intensities were shown in ( E , F ). Statistical significance was determined using one-way ANOVA, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control.

Article Snippet: Human tongue squamous cell carcinoma (TSCC) Cal-27 and SCC25 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Transwell Migration Assay, Staining, Wound Healing Assay, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Andrographis paniculata Inhibits Tongue Squamous Cell Carcinoma via Regulating Wnt/β-Catenin Signaling and Epithelial-Mesenchymal Transition

doi: 10.3390/ijms27093772

Figure Lengend Snippet: APW suppressed tumor growth, angiogenesis, and EMT in vivo. Cal-27 tumor-bearing mice were treated with APW (160, 320, or 960 mg/kg), cisplatin (2.5 mg/kg), or vehicle (Control) for 5 weeks. ( A ) Body weight changes of mice during the treatment period. ( B ) Tumor growth curves and ( C ) final tumor weights of mice in different treatment groups. ( D ) Tumors of mice from different treatment groups were subjected to IHC staining with Ki67 and CD31 antibodies. Representative immunohistochemical images of Ki67- and CD31-stained tumor sections. Scale bars = 100 μm. ( E ) Quantification of Ki67- and CD31-positive cells in tumor sections. ( F ) Tumor tissues of APW (160 and 320 mg/kg) or cisplatin (2.5 mg/kg)-treated mice were subjected to Western blot analysis of EMT-related proteins (E-cadherin, N-cadherin, and vimentin). Representative blots were on the left panel, and quantification of band intensities was shown on the right panel. ( G ) Bar chart showing the plasma levels of ALT, AST, ALP, CREA, UREA, and LDH in plasma collected from mice treated with APW (320 mg/kg) or cisplatin (2.5 mg/kg). Data were presented as mean ± SEM. Number of mice: control = 13; APW-160 mg/kg = 12; APW-320 mg/kg = 13; APW-960 mg/kg = 14; cisplatin-2.5 mg/kg = 13. Statistical significance was determined using one-way ANOVA, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control.

Article Snippet: Human tongue squamous cell carcinoma (TSCC) Cal-27 and SCC25 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vivo, Control, Immunohistochemistry, Immunohistochemical staining, Staining, Western Blot, Clinical Proteomics